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Zeiss software guide

1. oo o a 131 Activating and Deactivating the TimMer aaa aaa a ea 132 FIUOrescence RIOT a o is a A ee ad de a A Oe Soe A 132 Information About the Installed Filter Wheel ooo e 132 Specifying Names for the Fluorescence Excitation Filters lt lt o 133 Selecting a Fluorescence Excitation Filter o o e es 133 Editing a Fluorescence Settings Record auaa aaa a a a 134 Creating a New Settings Record y 0er ke hed a Aw EN eR ee DARE Le 134 Ghanging a Settings Record it abe in a BRK Bea Cod dane eed Cain eae ee amp 136 Deleting a Settings Record 1 es 136 Changing the Sequence of Entries inthe List naaa aaa a a 136 Adjusting the Reflector Offset escauta oi i d i E A e aa a E E a E A A 136 Control Microscope Zeiss Axiovert 200M 0 0 lt lt lt lt o 139 TR MICOSCODE TOOS 5 ant ii Bee dos dd ARA es bse o ok we ARA ad 139 The Microscope WInNdow 44 4 444 a E A A AA AA A A Ba A 140 Setting Objective Reflector Condenser and Filter o o ee ee 140 Setting ING FOCUS s eiia balear a bh a ee ee le o do eee 140 Defining and Setting Fixed Positions for the FOCUS 0 00 eee ee ee 141 Setting the Aperture Diaphragm 1 aa a a a a a a 142 Switching the Lamp On and Offi es a 4 4 4 dai id a e en eb wks Cie bes 142 Setting the Intensity of the LaMp aa 142 Opening a
2. PALM RoboSoftware 3 1 0106 EN You can set the speeds of both types of move ments in the PALM Robo Speed window and with the Speed Tools tool Speed changes with keyboard or menu commands always refer to the type of movement that is currently selected in the Speed Tools tool Note The settings that you enter always apply to the magnification that you have set in the program As soon as you have set another magnification the values that were last used or the factory defaults are automatically ap plied Therefore prior to setting the speed first select the magnification that you want to work with Before you initiate a movement of the IR laser beam all three setting options are available for both movement types The speed can be varied using keyboard commands during movement To select the unit for speed setting Setup PALM Robo e Inthe Setup menu click on menu item PALM Robo The Preferences and Configuration window with the Configuration tab opens e Click in Radio Button Logic units percent or Micrometer sec units um sec in section Metric e Close the window To change the speed using the PALM RoboSpeed window Move Setup Speed Fa e Click on Setup Speed inthe Move menu or press F8 on the keyboard 125 The 3D Laser Beam Positioning PALM Robo Speed ES la Cancel Values for Leng Dx Stage Arnon Scroll Position C
3. in the File menu in the PALM RoboSoftware main window or in the Element List window The Print Elements window will open Print Elements Filter criteria Elements t all i selected prints only selected Elements from Screen or Element List Types Freehands Lines Rectangles Circles bd Attributes Nr Color Type z Sorted by no sorting I print Summary lw print Totals Print Eo f Printer co output to default print device co output to File cow Format cane e Choose Elements if you want to print all ele ments or the selected ones only the option selected is available if only certain elements are selected only e Inthe Types list select the element types that should be contained in your file You can select which kind of element properties the file should contain Attributes Nr Color Type Select Atributes and Define Columns default like Element List like Export File e Inthe Attributes list select the desired entry As soon as you select the first entry the Select Attributes and Define Columns window opens Select Attributes and Define Columns cancel Columns Drag amp drop a Column to the right place Add a new Column Select Attribute v Click in Column Header to remove Column 86 e Select the desired columns and column order and click on OK to close the window e Inthe list Sorted b
4. S Color Palette Show Hide View Menus d Color Palette Ctrl P e Incase the Color Palette is hidden Click on the Color Palette icon button on the Tool Bar or choose Color Palette from View gt Menus Change Figure Color e Click on the Change Figure Color icon button e Click on the desired color in the Color Palette e Click on the element whose color you want to change e Click on any other tool on the graphics toolbar to turn off the Change Color function again To change the color of the comment background for text elements Info If you follow the procedure described below you will change the background color of all the existing text elements It is not possible to change the background color of one individual text element e Select the Text Tool in the Graphic Tools e Choose the desired background color from the Color Palette The background color will be changed 17 Working with Element Groups 7 9 Working with Element Groups You can merge multiple elements into a group For example you need element groups for the Serial Sections function refer to chapter 10 9 The Se rial Sections Function An element group can contain any element types In the Element List window groups are processed as elements for each group a separate entry is displayed You can edit an element group similar to editing single elements You can move stretch straighten and rotate or copy
5. The selected elements are merged into a group A new entry for the group appears in the Element List window For the elements belonging to the group the name of the group is entered in the Grp column the group name Is assigned auto matically by the PALM RoboSoftware Grp04 in the example shown above Grpo Grpo4 1 rpo4 3 To the element number of each element contained in a group the group number is prefixed All ele ments of the group are displayed with a surround ing dashed rectangle PALM RoboSoftware 3 1 0106 EN Working with Element Groups To add other elements to a group Edit Add to Group e Select the elements you want to add to a group either on the monitor on in the Element List window e Click on menu item Add to Group in the Edit menu in the main window or in the Element List window If you have created only one element group so far the elements will be added to this group If you have created more than one group the Groups window will open e Click on the desired group to which the ele ments should be added and close the window by clicking on OK To remove elements from a group Edit Remove from Group e Select the elements you want to remove from the group either on the monitor or in the Ele ment List window e Click on menu item Remove from Group in the Edit menu in the main window or in the Element List window To ungroup elements
6. Cut Speed for automatic laser function Cut LPC Speed for automatic laser function AutoLPC the defined dots for this function are targeted sequentially at the set speed You can save the speed settings together with oth er parameters in a settings file Refer to chapter 3 4 Saving and Loading Settings for instructions on how to do this You have the following options for setting the movement speed of the stage via the PALM Robo Speed window which you open with a menu command or a keyboard shortcut with the Speed Tools tool with keyboard or menu commands Speed slower Speed faster These setting options are intended particularly to allow changes with a movement operation in progress For speed changes entered with keyboard or menu commands you can define an increment Every time the corresponding keyboard or menu command is entered the speed is increased or decreased by this value You can set the speeds of all types of movements in the PALM Robo Speed window and with the Speed Tools tool PALM RoboSoftware 3 1 0106 EN Speed changes with keyboard or menu commands always refer to the type of movement that is cur rently selected in the Speed Tools tool Note The settings that you enter always apply to the magnification that you have set in the program As soon as you have set another magnification the values that were last used or the factory defaults are automatically ap plie
7. Database Mode is available only when an appropriate soft ware license has been purchased The standard version of the Information Center program file based File Mode can be used for or ganizing the stored microscope images You can thus view the stored images display them in the form of a slideshow save them tn a different for mat as well as display and edit information that was saved with the images In the upgraded version of the program file based and database based File Mode and Database Mode you can access even more powerful func tions for documenting your work than those in cluded in the standard version If you are working with the PALM RoboSoftware in Database Mode refer to chapter 1 7 Launching and Exiting the Program not only the images you are saving are stored in a database but also the elements you draw information on samples experiments and microdissection operations You can also enter additional information to be attached to the imag es or you can archive all images and elements for the same project together In the upgraded version of the Information Center you can switch at any time between Database Mode and File Mode PALM RoboSoftware 3 1 0106 EN 1 4 About this User Manual 1 4 1 Contents This User Manual includes instructions on how to move your stage with PALM RoboSoftware a de scription of the steps that are essential before us ing the laser functions and instructions for
8. Database mode with pre selected DB Database User ID Password e Inthe Operating Mode section select the mode for PALM RoboSoftware to operate in when launched 23 Setting Slideholder If you have chosen Database Mode with pre se lected DB e Choose the database you wish to work with using either the selection list or the button mj Note You can protect your database with a pass word and user ID see Information Center Manual If you enter this information here then the PALM RoboSoftware will be automatically linked with the selected data base every time it is launched This means that every user who starts the PALM Ro boSoftware will have access to your data base To prevent unauthorized access to the data base it is therefore not advisable to enter the User ID and password here in the launch settings These will then be requested each time the PALM RoboSoftware is launched e If required enter the relevant User ID and password Note It is not possible to switch between File and Database Mode or to change database from within the PALM RoboSoftware You should exit from the PALM RoboSoftware and restart it if you wish to change to a different mode or database If necessary you must change the launch settings as described above If more than one user is working on the sys tem and using various different databases or modes you are advised to choose the start up setting Databas
9. Info The selected color only applies to the type of el ements that you selected prior to choosing the color If for example you have selected a color for Line type elements and then change to the Dot graphic tool new elements of this type will be displayed in the color that is select ed in the Colors window for LPC Dot PALM RoboSoftware 3 1 0106 EN Changing Colors and Line Thickness To change the color settings in the Colors window for new elements you want to draw wy Colors Click on Colors in the Graphic Toolbar The Colors window will open Colors Cancel Elements O Cub rea LPC Dat AutoCircle AutoLPC Dot RoboLPC Line Robol FC O ot Ruler Line Test Field MumberField mM E E E o x z x z z x a x x In the left pane of the window you will see a list of element properties whose color can be changed From the Elements list in the left window pane select the element the color of which you want to change Cut Line Color of the lines along which the laser is to cut Cut Area Color of the element areas if you activate the function for showing or calculat ing areas refer also to chapter 7 10 Mea suring LPC Dot Color of the dots the laser beam touches with the automatic laser function LPC in order to catapult the area AutoCircle Color of the circle that the laser cuts out and color of the point at w
10. o o e ts 73 PALM RoboSoftware 3 1 0106 EN 7 8 7 8 1 7 8 2 7 8 3 7 9 7 10 8 1 8 2 8 3 8 4 8 5 9 1 9 2 9 3 9 31 9 3 2 9 4 9 5 10 10 1 10 2 10 3 10 4 10 5 10 5 1 10 5 2 10 5 3 10 5 4 10 5 5 10 6 10 7 10 8 10 9 10 10 11 11 1 11 2 11 3 11 4 11 5 11 6 11 7 11 8 11 8 1 11 8 2 11 8 3 Contents Changing Colors and Line Thickness o ooo 74 Selecting Colors for Elements to be Drawn New o e 74 Selecting Line and Dot Thickness for Elements to be Drawn New 76 Changing Colors of Elements that have already been Drawn o o 77 Working with Element Groups a a a 78 MESSUNG uri dr Aad tl ea SRS ON ee we ee ee ee a A 80 Saving and Printing Elements 00000 eee e eee a 81 Settings for Saving Elements in File Mode 1 naaa a 81 Saving and Loading Elements in File Mode o a 82 Saving and Loading Elements in Database Mode o e ee 2 2 84 Exporting and Importing Elements o es 85 PINUNO ElEIMNEN S eames amp at ne ere ark ik Ra eee oe rr se ld 86 Saving MAGES a durara a ane yee See ee 87 Inserting a Copyright Note is ar aki a a r on Gh iea aak aah aie A a aao i ata a Ia arta aiai ade a Sa 87 Specifying Folder to Store Images ID a a 88 Saving Images Manually a csr ieat a a a dae ede a ea a a eee wh R 89 Savi
11. with one fixed and one 3D movable laser beam with two 3D movable laser beams If you have purchased a system with 3D Laser Beam Positioning you can move a trapped object with the laser beam parallel to the plane of the slide The systems with two laser beams enable you to trap two objects at the same time and move one or both parallel to the plane of the slide PALM RoboStage P A L M Microlaser Technologies GmbH offers var ious microscope stages PALM RoboStage Motorized microscope stage for laser micro manipulation High precision work within the Sub micrometer area is possible as is the employment of various holders for slides Petri culture dishes and capillary tubes PALM RoboSoftware 3 1 0106 EN PALM RoboStage II As a further development of PALM RoboStage it is now possible with PALM RoboStage II to process not only Petri culture dishes for live cells but also to process up to three slides simultaneously which opens up entirely new possibilities especially for work with serial sec tions Devices for positioning caps or vessels P A L M Microlaser Technologies GmbH offers var ious possibilities for positioning different caps PALM CapMover The PALM CapMover has been developed for the rapid positioning of caps The cap for which various inserts are available is positioned with the highest precision over the sample to be col lected PALM RoboMover The PALM RoboMover is our special
12. D Stop e Click on the Stop icon button in the main window of the PALM RoboSoftware or Press the Esc key on the keyboard Scanning progress is cancelled The partially fin ished scan will then be lost To change between the proof scan and the scan of the section Section Navigation Section Navigation al Section 1 P Slide Section e Click on the arrow button at the upper right of the window Whether you now see the section 1 or the proof scan Slide Section is indicated below the button To display your elements in the scan Draw Elements o Draw Marker Elements e Check the Draw Elements box in order to dis play all elements with the exception of text ele ments e Check the Draw Marker Elements box in order to display text elements The elements appear both in the overview scan and the section scan Navigator Navigation Scan Cellenger Analyze Axiovision Analyze Section Navigation 4 Section 1 Scanned with 5x lens Save Thumbnail Load Thumbnail Use autofocus suspend scan for focus Draw Elements Draw Marker Elements 51 Positioning the Microscope Stage Info Text elements serve for marking and informa tion purposes they do not describe a section of your sample in the way for instance that freehand elements do They therefore always appear the same size on the screen irrespec tive of the magnification setting If you have chosen a low l
13. Freeze Mode Move gt Freeze Mode Delete last element gt Edit gt Delete last element Edit gt Undelete Delete selected elements Edit gt Delete selected elements 147 Appendix B Keyboard Shortcuts ds Icon Button Menu Command End Laser gt Focus down Esc D Stop Move gt Stop Home Laser gt Focus up Page up Laser gt Energy Power more Page Down Laser gt Energy Power less 148 PALM RoboSoftware 3 1 0106 EN Index Numerics 3D Laser Beam positioning function 145 3D Mode exit 127 keyboard 127 select 127 set speed 125 toggle between 3D 1 and 3D 2 127 3D Offset 125 3D Mode 127 define Reference Position 122 keyboard 127 set speed 125 target Reference Position 123 A ApoTome status display 144 Autofocus activate 104 139 automatic focusing 141 deactivate 104 139 AxioVision 11 54 C Calibration 3D Offset 35 movement of stage 34 reset 36 Catapult 95 Cellenger 11 53 Center 145 Centering tool 41 Centric Attribute 60 Change color 77 Check Point 145 define 47 return to original position 47 target 47 PALM RoboSoftware 3 1 0106 EN Index Check Position define 141 meaning 141 set 139 141 Color Palette 75 Combination IR laser focus 118 Comment add 73 change 73 Context Menu 17 Control microscope 139 Copyright Note specify 87 Copyright note switch display on off 16 Crosshairs show hide 15 Cursor Mode select 40 Cust
14. 63x and 100x e If itis hidden enable the screen center display click on item Screen Center in the View menu e For the 3D Laser Beam Positioning choose a low to medium speed setting 3D for instruc tions on how to proceed please refer to chapter 11 8 1 Setting the Speed of the 3D Laser Beam Positioning e Choose low power and focus settings for laser beam IR 1 as described in chapter 11 2 Set ting IR Laser Power and Focus e Activate the IR laser click on the IR button on the right border of the monitor PALM RoboSoftware 3 1 0106 EN e Adjust the power and focus so that you can see an effect of the laser beam if you are using a slide colored with a black felt pen a hole in the colored coating will be visible at the appropriate settings e Switch to 3D 1 Mode click on item 3D 1 Mode in the Move menu The cursor will disappear every movement of the mouse will be reflected in a movement of the laser beam e Move the laser beam by dragging the mouse into a corner of the image in your image win dow Note The farther the dot is from the image center both vertically and horizontally the more ac curate your calibration will be e Double click there to exit 3D 1 Mode again Setup Calibration j New Calibration e Click on New Calibration in the Setup gt Cali bration menu A submenu will open e Click on menu item 3D 1 The following message will appear S
15. AutoLPC Automatic catapulting of larger areas of samples mounted on glass With glass samples only a li mited amount of cell material per laser pulse can be catapulted out with one laser shot Larger areas have to be catapulted with multiple laser pulses The distance between individual laser pulses de pends on tissue properties and is preselected in the LPC Parameter window Center The center point of the microscope image displayed on the monitor It may be marked with crosshairs The crosshairs can be displayed or hidden with the Screen Center command in the View menu Check Point A position of the stage If you target the Check Point the circular opening of the stage is positio ned within the beam path of the microscope only Stage II After catapulting the sample into the cap it can be observed with the microscope through this opening Check Position Freely definable focus setting of the microscope to set by amenu command motorized microscope only The setting should be defined so that the ca tapulted sample in the cap is displayed sharply on the monitor or in the ocular Close amp Cut Closing and Cutting An incompletely closed ele ment is automatically closed and cut completely according to the given conditions PALM RoboSoftware 3 1 0106 EN Close amp Cut AutoLPC Closing the marking line and cutting before cata pulting With critical samples the marked area must first be detached from its
16. Calibrating the 3D Offset 4 5 Calibrating the 3D Offset Note Calibrating the 3D Laser Beam Positioning is only necessary or indeed possible if you have purchased a system that includes this function In Double Beam Systems with two 3D movable laser beams both offsets 3D 1 and 3D 2 must be calibrated Recalibration is necessary if the position of the La ser Marker and the active laser position do not be have together in a proportionally consistent manner when in motion If they do move together in a proportionally con sistent manner but do not coincide only the Laser Marker must be readjusted refer to chapter 4 3 Setting the Laser Marker Calibration is done in several steps PALM Ro boSoftware will guide you through this process Note Before you carry out the calibration you must have set the Laser Markers as described in chapter 4 3 Setting the Laser Marker To perform the calibration e Insert a sample and focus the image Info The 3D Laser Beam Positioning can only be cal ibrated with the IR laser switched on Accord ingly you should use a sample that you no longer need for your work A slide that has been colored with a black felt pen is particularly suitable e Follow the instructions in chapter 4 2 Setting the Magnification to set the magnification cho sen for the microscope in the Lens menu Note Note that you can only use the IR laser in con junction with oil objectives 60x
17. EE 1 Freehand 63173 sil w 2 Freehand 73528 5l a 3 Freehand 78559 Sil 4 Slide1 Group The next two steps are omitted if you have drawn elements of the Reference Point type In this case continue with Step 3 Drawing and Process ing Elements as described on the following page Edit Define Group Reference Figure e Select the three drawn elements and click on the menu item Define Group Reference Fig ure in the Edit menu Groups ES cal Select a Group From List Name nr Grp Comment Slide 1 4 This opens the Groups window e Choose the group to which the elements belong and click on the OK button to close the win dow PALM RoboSoftware 3 1 0106 EN The Serial Sections Function Step 3 Drawing and processing elements Element List automatic and manual adaptation File Edit Move Laser e Draw th lements which are to be processed DES sm Proa qe el j on all samples e Add these elements to the reference group Select the elements and click on the menu item Add to Group in the Edit menu Slidel Slidez Slides Summary Show Types El 1 Freehand Ref 1 63173 Sli EM 2 Freehand Ref 2 73528 5l Note E 2 Freehand Refa rsak pal If you transfer elements to another sample 4 Slidel Group E Reference Ref 1 m as described below only those elements 6 Reference Ref 2 Si within the group will be considered 7 Reference Ref 3
18. Note In the Basic configuration with the manual microscope the Fluorescence Tool is not ac tive The selected settings record is activated i e all the settings defined in this record are implement ed to change the settings see chapter 12 9 Note When using a manual microscope only the set tings of the filter wheel and the shutter are ac tivated The position of the reflector revolver and the microscope illumination has to be Switched on manually on the microscope 130 12 5 Freeze Mode With the Freeze Mode of the PALM RoboSoftware you can display the actual fluorescence image on the monitor and edit it for example by drawing a freehand line or marking objects while the fluo rescence light is switched off again In this way sensible fluorescence samples are protected from fading To switch into and out of Freeze Mode Move ae Freeze mode Alt F e Click on menu item Freeze Mode in the Move menu With this menu command you can toggle between Freeze Mode and Cursor Mode Note In Freeze Mode all functions for moving the stage or functions that cause a movement of the stage e g all positioning functions as well as all laser functions are blocked In this way an unintentional movement of the stage is pre vented After editing the freezed image you can exit the Freeze Mode to perform a laser function or to observe other areas with fluorescence Note Before switching on a laser fu
19. Now please insert the slideholder and select the correct type Typ Slideholder E Return to the working area e Change the slideholder and or the slide and choose the corresponding entry from the Sli deholder field Note To avoid damage to the objective or to the Sample Move the stage back again to the Original position after changing the slide and the slideholder For this proceed solely as described below 48 To return the stage to its original position Return to the working area e If you do not have a motorized microscope Move the objective down as far as possible e Click on Return to the working area in the PALM Robo Select Slideholder window The stage will be returned to the position it was in before it was automatically moved to target the Load Position PALM RoboSoftware 3 1 0106 EN Start and Quit PALM Navigator 6 PALM Navigator The PALM Navigator will provide you with an over view of your sample or a section of your choice You scan your sample and get an image in the PALM Navigator window With one mouse click on any spot within the scanned image you can posi tion the stage to this spot and display particular ar eas of your sample on the monitor very rapidly From within the proof scan you can select an area and scan this at an enlarged size You can save and reload the scan The scan is saved with its coordinates This means that you can work with the reloaded image in
20. Serial Sections function refer to chapter 10 9 The Serial Sections Function e Select the Reference Point Tool in the Graphic Toolbar e Click on that point in your image at which you want to set a reference point 7 2 3 The Freehand Tool Ab Freehand Tool Use the Freehand Tool to draw irregularly shaped elements e Select the Freehand Tool in the Graphic Toolbar e Move the cursor to the dot where you want to begin your drawing e Hold the mouse button down while you draw the required shape and release it at the end dot 59 Drawing New Elements Info If you are in Cursor Mode you can only use the Freehand Tool inside the Scroll Rectangle When you reach the edge of this rectangle the line stops automatically The program switches to Scroll Mode the stage is moved As soon as you release the mouse button and the stage stops moving you can continue the line as explained in chapter 7 7 Processing El ements In Freeze Mode however you can continue to draw with the Freehand Tool outside the Scroll Rectangle for a detailed explanation refer to chapter 7 2 1 Drawing in the Various Program Modes 7 2 4 The Line Tool y Line Tool l Quadratic Attribute Use the Line Tool to draw straight lines e Select the Line Tool in the Graphic Toolbar If you select Quadratic Attribute as well the drawing function is limited to horizontal and vertical lines You now have two options for drawing y
21. Via the Microscope Window you can make the following settings Selecting an objective Switching the lamp on and off Setting the focus to any position Setting the speed with which the focusing is performed Set the focus to one of three defined positions Load Position Work Position Check Position Position of the reflector Position of the condenser Aperture diaphragm and speed with which the aperture diaphragm Is set Filter selection Opening and Closing the Shutter Settings for the beam path sideport or base port Settings for the Optovar Info The setting options available to you in detail depend on the configuration of your system To open the Microscope Window View T Microscope Window FE e Click on menu item Microscope Window in the View menu Microscope Axiovert 200 M Focus Lamp Shutter LightPath Setup Close so Pos Name J gt Objective 1 E Fluar 570 25 4 gt Light Bo 1 10 gt Focus 1355 AF Speed J A gt Reflector 1 Laser w A H Cocc 1 MET Aperture 0 14 Speed E apo e gt The window Microscope opens the designation of the window depends on the type of the microscope your system is equipped with 140 13 2 1 Setting Objective Reflector Condenser and Filter You select the magnification objective filter and condenser as well as the desired position of the re flector in th
22. be calibrated by the operator with the help for example of an ocular micrometer Saving and loading of the geometry and posi tion of defined elements Export of the element properties sequential number type number of laser operations per formed size of area position etc Storage of the portion of the microscope image viewable on the monitor either with or without the elements included in that portion Loading and saving of settings movement speed of the stage laser settings pre selected folders for storing images and elements Processing of the defined elements with the laser automatic cutting along defined lines fol lowed by catapulting Processing of a series of samples with the UV laser i e the areas to be cut and catapulted are defined on one sample and can be trans ferred to other samples If you have purchased a system with a motor ized microscope you can also create settings for the microscope via PALM RoboSoftware The software can be upgraded in line with your specific requirements e g with automatic image recognition software or special functions e g for Time Lapse experiments 1 3 Information Center The function of the Information Center program is to display and post process the sample data generated using the PALM RoboSoftware The In formation Center recognizes two operating modes File Mode and Database Mode refer also to chap ter 1 6 File Mode and Database Mode
23. continue 110 stop 110 Laser energy UV link Cut and LPC 102 set 102 Laser focus IR set 118 set with the mouse 120 Laser focus UV link Cut and LPC 102 set 102 Laser functions UV AutoCircle 96 AutoLPC 95 Close amp Cut 95 Close amp Cut AutoLPC 95 Cut 95 J ointCut 95 LPC 95 preview 98 RoboLPC 95 select 97 set number of operations 98 start 108 Laser Marker change appearance 16 meaning 146 Laser power IR set 118 Laser settings IR find suitable values 122 footswitch 123 power and focus 118 Laser settings UV 100 energy and focus 102 find suitable values 106 footswitch 116 general 104 Laser Tools IR laser settings 119 settings for UV laser 102 Lens Offset adjust 36 reset 37 Licensing 27 Line tool 60 151 Index Linking Cut and LPC energy and focus 102 Load elements 83 Load Position return to original position 48 Load Position objective meaning 141 set 139 141 Load Position stage define 48 targeting 48 M Measuring 80 area 80 length of lines 80 Microscope motorized Activate autofocus 141 activate autofocus 139 adjust light intensity 139 142 Check Position meaning 141 default settings 143 define Check Position 141 define Work Position 139 141 Load Position meaning 141 set aperture diaphragm 142 set beam path 142 set Check Position 139 141 set focus 139 140 set Load Position 139 141 set Optovar 143 set Work Position 139 141 speed f
24. ferent laser functions for example elements that are to be cut and catapulted completely Robo LPC beside those that are only to be cut Cut or those that are only to be catapulted LPC You can specify which laser function is to be applied prior to releasing the laser For instructions on how to do this refer to chapter 10 7 Releasing the Auto matic Laser Functions Elements for which the se lected laser function cannot be applied are ignored The display of the elements on the monitor de pends on which laser function is currently select ed the selection of the laser function also serves as a preview Elements that can be processed in the selected laser function are represented by thi cker lines or filled dots elements that cannot be processed are represented by thin lines and dots that are not filled circles In the preview dots and lines that are calculated by the program are also displayed lines that enclose open elements as well as AutoLPC and RoboLPC dots In chapter 10 4 Preview of the Automatic Laser Functions you will find further explanations and examples of the preview Previously drawn elements can be changed by their shape and size they can be numbered anno tated and deleted again You can calculate their area or length and you can select colors and line thicknesses in which your elements are to be dis played Note The measuring functions of the PALM Ro boSoftware and the scale bar are not
25. line of the element the arrangement of the dot configuration diag onal or parallel the selection if a laser pulse should be set to the center of the element AutoLPC Center Shot Note The distance settings always apply to the mag nification that you have set in the program As soon as you have selected another magnifica tion the values that were last used or the fac tory defaults are automatically applied Therefore before setting the speed first select the magnification that you want to work with PALM RoboSoftware 3 1 0106 EN Specifying Laser Settings To enter these settings proceed as follows Laser LPC Parameter e Click on the LPC Parameter entry in the Laser menu The LPC Parameters window opens In the aver age part of the window you will see the section LPC Distances LPC Parameter Ea Cancel Values for Lens Ox T LPC Distances ym 14 Distance of AutoLPC shots 2 Distance of 4utoLPC shots from line W AutoLPC shots diagonal W AutoLPC Center shot 12 Distance of RobolPC joint Autolincle pun 30 Radius of 4utoCircle e Enter the required value for the dot distance in um in the Distance of AutoLPC shots field e Enter the required um value for the distance of the points from the border of the elements in the field Distance of AutoLPC shots from line e Activate the AutoLPC shots diagonal check box to change the arrangement
26. quired also manually To begin with an overview of the necessary process steps follows Subse quently the procedure will be explained in detail PALM RoboSoftware 3 1 0106 EN To carry out automatic adaptation proceed as fol lows 1 Prepare the samples create three markings in your block before cutting the tissue sam ples e g three pinpricks These markings must be clearly visible under the microscope Then cut the tissue samples and mount them on the slides If required treat one sample the source sample in order to make the structures visible 2 Creating reference elements on the source sample Insert the first sample your source sample into the microscope and define three reference points on the markings made before cutting 3 Drawing and processing elements Now draw the elements for all the samples which are to be processed with the laser and join them to gether with the reference points into a group 4a Preparing the target sample position the tar get sample under the microscope and likewise define three reference points on its markings 5a Transferring elements to the target sample The PALM RoboSoftware moves the reference points on the source sample automatically to the locations of the corresponding reference points on the target sample and recalculates all the transferred elements so that they match up with respective structures on the Slide Similarly for the manual adaptation
27. sli Element List File Edit Move Laser lide ide ide3 Summar The PALM RoboSoftware creates three new ele A EE ments of the Reference type Show Types Info ry i Freehand Ref 1 63173 Si With the command Undefine Group Refer 7 Freehand Ref 23 Sada la ence Figure you can undo the definition of an ce 3 Freehand Ref 3 78559 Si element as a reference element The original a Group i p 5 Reference Ref 1 Sli element along with the Reference type el 6 Reference Ref 2 si ement created by the PALM RoboSoftware are Reference Ref 3 si preserved just the connection between the ra rae two elements will be deleted E 10 Freehand 55959 Si Within each of the three drawn elements the re spective reference element created by the PALM RoboSoftware will be shown Step 4a Preparing the target sample automatic adaptation Likewise you will define three reference points or reference figures on the target samples Proceed in exactly the same way as described for the source sample e Insert the slide onto which you want to trans fer the elements into the slideholder If you use a multiple slideholder it is advisable to leave the source sample in place and insert the target sample in a vacant position e Position the stage successively on the three markings on the target sample and draw an element on each in the same way as you did for the source sample You have now created your reference f
28. 1 3 as with automatic adaptation 4b Preparing the target sample position the tar get sample under the microscope You do not need to define any reference points in this in stance 5b Transferring elements to the target sample To do this move the reference elements of the source group manually to the position of the corresponding structures on the target sample The PALM RoboSoftware recalculates all the other elements to bring them into line with the corresponding structures on the slide PALM RoboSoftware 3 1 0106 EN The Serial Sections Function Step 1 Preparing the samples automatic and manual adaptation e Before cutting create three markings in your Sample block that are easily recognizable under the microscope e g three pinpricks The markings should form a triangle i e not a Straight line the further away they are from each other the more accurate the adaptation will be Make sure that you place the markings exactly vertical to the cutting plane e Produce the tissue samples from the sample e Select a sample for the source sample and treat it if necessary with a coloring substance or similar Step 2 Creating reference elements on the source sample automatic and manual adapt ation e Insert the source sample into the slideholder of the stage Info The images shown below are examples asso ciated with a 3 slide holder The source sam ple has been inserted in position 1
29. 89 Time Lapse 91 Scale bar switch display on off 15 Scanning detail 51 interrupt scan 51 load scanned image 52 overview 50 show elements 51 store scanned image 52 Screen Center show hide 15 Scroll bar position stage 41 Scroll Rectangle show hide 15 Select elements element list all 68 single or multiple 68 within a group 79 Select elements monitor 67 all 67 deselect selection 67 multiple 67 single 67 within a group 79 Serial Sections edit elements 113 function 110 prepare samples 111 PALM RoboSoftware 3 1 0106 EN Index reference elements of original sample 111 reference elements of target sample 113 transfer elements automatically 114 transfer elements manually 115 Set color 74 color window 74 current elements 77 for new elements 75 Set dot thickness 76 Set Laser Marker 32 Set line thickness 76 Set magnification 32 Set RoboLPC joint 105 Set speed 3D Offset 125 increment 127 keyboard commands 126 menu commands 126 PALM Robo speed window 125 Speed Tools 126 units 125 Set speed stage 43 Increment 45 keyboard commands 45 menu commands 45 PALM Robo speed window 44 Speed Tools 44 units 43 Settings 23 adjust Reflector Offset 136 automatic saving 26 fluorescence 129 Initial settings 23 Laser Marker 32 Lens Offset 36 magnification 32 reset calibration 36 reset Lens Offset 37 reset Reflector Offset 137 restore factory defaults 27 save elements File Mode
30. As with function Close amp Cut the laser cuts the sample along defined lines Start and end dot of an open path figure are connected by a straight cut line It remains a joint between start and end dot With this function geometrical figures can be cut 145 Appendix A Glossary The cut area remains fixed within the tissue The entire area can be catapulted later by a single laser pulse Laser Marker There is a Laser Marker for each laser beam Its tip should display the point where the laser beam meets the sample when released For a fixed laser beam such as the UV laser and the IR laser beam without 3D Laser Beam Positioning this is roughly the middle of the monitor for a beam that is 3D movable this point may be located elsewhere PALM RoboSoftware calculates the cutting func tions of the UV laser from the position of the Laser Marker The position of the Laser Marker should therefore always match that of actual laser focus Only then the correct results can be achieved with the automatic laser functions In the same way you will only be able to trap objects reliably with the IR laser and if your system is equipped with such move them precisely with the 3D Laser Beam Positioning if the position of its Laser Marker is correctly set You should therefore check the po sition of the Laser Marker before you start work The position of the Laser Marker may vary with dif ferent objectives Laser Microdissection and Lase
31. If you want to change the line thickness of the border enter a numerical value in the field for line or change the value set there by clicking on the arrow buttons to the left of the field Info The line thickness setting only has an effect if you have selected line as the style e To change the color of the Laser Marker click on the Color Button This opens the Color window Basic colors Custom colors 1E U ip ip BEE Eee ee Define Custom Colors gt gt Conca e Choose a color or define a new color e Close the Color window by clicking on OK e Close the Laser Marker window by clicking on OK 2 3 Operation All program functions are accessible via the menu bar or the Graphic Toolbar Many frequently used functions can also be activated via buttons in the icon bars or by means of special tools or keyboard commands 2 3 1 Mouse You will almost always be working with the prima ry mouse button that is the left mouse button in the default configuration If it is necessary to use the secondary mouse button the right button in the default configuration this will be indicated ex plicitly PALM RoboSoftware 3 1 0106 EN 2 3 2 Keyboard Shortcuts There are additional keyboard shortcuts for sever al functions These are Summarized in Appendix B Keyboard Shortcuts 2 3 3 Context Menu Several functions can also be accessed via the Context Menu STOP Stop ESC m Conti
32. LPC Parameter PALM Robo Laser Settings and PALM Robo IR Laser Settings windows Note To obtain a correct display of the drawn ele ments and to achieve a correct laser function the setting in the Lens Menu must match the magnification that is selected on your micro scope If the settings do not correspond to each other the actual cut lines and catapult points of the laser will not be correctly displayed on the monitor 32 4 3 Setting the Laser Marker In the default configuration of the program the Laser Markers are represented as a solid green equilateral triangle UV Laser Marker in the center of the monitor and a red IR 1 Laser Marker or green IR 2 Laser Marker Double Beam System only square The apex of the triangle or the cen ter of the squares should indicate the point at which the respective laser beam will strike the sample upon release Note PALM RoboSoftware calculates the cutting functions of the UV laser from the position of the Laser Marker The position of the Laser Marker should therefore always match that of actual laser focus Only then the correct results can be achieved with the automatic laser functions You should therefore check the posi tion of the Laser Marker before you start work For the IR laser also you can only achieve ac curate results if the IR Laser Marker is exactly aligned with the laser focus Remember that the position of the Laser Mark ers may vary for use with dif
33. Laser Position or Click Cursor at new IR Laser Position e Click in the center of the laser focus for that laser beam The message UV Laser Position is set or IR La ser Position is set will appear This message will disappear automatically after a few seconds Note Be sure to assign the Laser Markers to the cor rect laser beams 33 Calibrating the Movement of the Stage 4 4 Calibrating the Movement of the Stage Calibration is done in several steps PALM Ro boSoftware will guide you through this process To perform the calibration Setup Calibration j New Calibration Note The menu entries 3D 1 and 3D 2 appear only if you have installed a system with one or two 3D movable IR laser beams e Insert a sample and focus the image e Set the chosen magnification for the micro scope as described in chapter 4 2 Setting the Magnification e In Stage Mode choose a low to medium speed setting for the stage refer to chapter 5 3 Set ting Speed Note Do not use the Invert stage motion function when calibrating refer also to chapter 5 1 1 Positioning the Stage with the Mouse Stage Mode e Click on New Calibration in the Setup gt Cali bration menu A submenu will open e Click on menu item Stage A message appears Step 1 of 2 Click any point for moving to screen center e Choose any dot preferably as small but as sig nificant as possible in your image Note t
34. Microlaser Technologies P A L M is a registered trademark License License Key A679 3416 66B5 3745 Enter New License Support P A L M Microlaser Technologies Am Neuland 9 12 82347 Bermried Germany Phone 49 0 8158 9971 0 Fax 49 0 8158 9971 249 EMail support palm microlaser com Internet www palm microlaser com Info You can click on eMail and Internet to ac cess the manufacturer s Website or send an e mail via your e mail program as long as you are connected to the network e Close the window by clicking on OK 19 Help and Information about PALM RoboSoftware 2 4 4 System Information To display a range of information about the system Help PALM Robo Systemlnto e Click on PALM Robo Systeminfo in the Help menu e A window containing information about your system will open PALM Robo 5ysteminto Frame grabber MATROS Meteor ME Resolution 0d s 544 Color RGE 32 bit Frame rate 37 sec Screen rate 3 9 sec Stage type Stage Ilb Memory Usage O Bytes 20 2 4 5 PALM Robo Information The PALM Robo Information window shows all the messages output by the program both when it is launched and whilst it is running To open the PALM Robo Information window For Help press F1 e Double click in the field at the leftmost end of the Status Bar PALM Robo Information Show the message history History 13 27 56 Wrong
35. Selecting Line and Dot Thickness for Elements to be Drawn New Lines e Click on the Lines icon button This opens the Thickness window Thickness Cancel Select values for _ Cancel Line thickness E 1 to 20 Dot thickness 8 1 to 50 Ruler thickness 4 1 to 50 all values are in piel In the three fields in this window you can set val ues for the parameters Line thickness 1 20 Thickness of Figure elements Dot thickness 1 50 Diameter of Dot ele ments Ruler thickness 1 50 Thickness of measur ing lines IN pixels e Enter the desired values in the corresponding fields and confirm your entries by clicking OK Info The set thickness of the lines or dots is not re lated to the width of the laser cut line or the di ameter of the laser beam These are dependent on the laser parameters setting laser energy laser focus and movement speed of the stage refer also to chapter 10 5 Specifying Laser Settings Info Under certain conditions you may not see the effect of a changed line thickness on the mon itor If a laser function that is not used to cut is currently set LPC or AutoLPC the cut lines are displayed as hairlines regardless of the specified line thickness PALM RoboSoftware 3 1 0106 EN Changing Colors and Line Thickness 7 8 3 Changing Colors of Elements that have already been Drawn the colors of elements that have already been drawn ca
36. The function can be turned on or off by clicking the Auto Focus box A tick in the checkbox indicates that the function is enabled 104 10 5 3 Other General Laser Settings You can determine whether the elements should be displayed on the monitor while you are performing the laser function and whether the stage Is to be returned to start po sition on completion of the laser function To enter these settings proceed as follows e Inthe Setup menu click on menu item PALM Robo The Preferences and Configuration window opens W Return to start position after Laser function e Select tab Laser e Inthe Laser function section click in the checkbox in front of the item Display Elements during Laser function and the checkbox in front of item Return to start position after Laser function to enable or disable these func tions A tick in the checkbox indicates that the function is enabled 10 5 4 Special Laser Settings for the AutoLPC and Close amp Cut AutoLPC Functions Note These laser functions only apply for glass mounted samples When you release the AutoLPC or the Close amp Cut AutoLPC function the contents of an area ele ment is catapulted into the cap with single laser shots Four particular settings have to be carried out for this laser function the distance of the laser pulses to each other LPC distance the distance of the laser pulses from the border
37. and finish dots is shortened by a straight line to the set value of the RoboLPC joint parameter If you want to deselect this function enter the value 0 Note Should the value of the RoboLPC joint param eter be too small or at zero then it can occur that automatic catapulting does not take place in the RoboLPC function as the sample pos sibly will not remain fixed within the tissue when cut PALM RoboSoftware 3 1 0106 EN Specifying Laser Settings Elements as they were drawn and would be cut with the Cut function The same elements with laser function J ointCut selected for element 1 the distance from start to finish dot is increased for element 2 it is re duced Close element 3 will be opened The same elements with laser function Robo LPC selected for element 1 the distance from start to finish dot is increased for element 2 it is reduced Close element 3 will be opened In the center of the joint the catapult point is displayed AutoLPC Center shot With this parameter you define if a laser shot Should be set into the center of an element This makes sense when working with very Small or narrow elements for example If the distance of the AutoLPC shots to each other and or their distance from the border line of the element is approximately as big as the element or bigger possibly no LPC shot Is set into the element without this function i e the element would not be catapu
38. be used if an appropriate license has been purchased for the PALM RoboSoftware In File Mode you can save microscope images and elements in folders on a data carrier e g your lo cal hard disk Elements are geometric figures that you draw You may for instance identify areas for processing with the laser In Database Mode the PALM RoboSoftware is linked with a database If you save images then they are stored in the database Furthermore any elements that you draw are automatically stored in the database in exactly the same way as informa tion on samples experiments and microdissection operations The Information Center allows you to create databases and to display and edit their contents read the section in the manual about the Information Center for more details 1 7 Launching and Exiting the Program To launch PALM RoboSoftware Edi PALMRobo e Click on the PALM Robo icon button on your desktop if you have a desktop shortcut or click on Programs gt Palm gt PALMRobo in the Windows Start menu If your system does not include a database license then the PALM RoboSoftware will be launched If your system does include a database license then you can choose whether to work in Database or File Mode refer to chapter 1 6 File Mode and Database Mode Depending on the initial set tings refer to chapter 3 1 Launching Settings the PALM RoboSoftware will be launched either in File Mode
39. button pressed down until the focus has reached the desired position The focus will be adjusted In the field on the left the current position is displayed PALM RoboSoftware 3 1 0106 EN The Microscope Window To focus automatically Info This function is only available if your micro scope has an autofocus facility Focus 6922 9 AF Speed al e Click on the AF button in the Focus row 13 2 3 Defining and Setting Fixed Posi tions for the Focus You can target three defined focus positions two of it are freely definable the third position is pre scribed Work Position freely definable The objective is set so that the focus is in the same plane as the slide In this position you can view your sample on the monitor or in the ocular of the microscope Info The exact setting of the focus is variable for various objectives However you only have to define the setting of the Work Position for one objective The differences with the other ob jectives are automatically compensated To achieve this the parfocality must be adjusted correctly in the microscope see microscope manual However if you are working with slides or samples of different thicknesses you may have to readjust the focus Check Position freely definable The objective is set such that the focus is on the same plane as the cap into which your samples are cata pulted using the LPC functions In this position
40. can change the settings during the cutting process as long as the cut line remains incomplete PALM RoboSoftware 3 1 0106 EN If the cutting process has been completed before you have found your optimal settings you will have to draw a new element and restart the laser If possible try with multiple cutting operations e Change the laser focus and try to aim for a thin ner cut line e If you have achieved that then reduce the energy also with the intention of getting a thin ner cut line e Now try to continue optimizing your results by repeatedly changing both focus and energy e Once you have attained the best possible result you should either save or note the appropriate settings Refer to chapter 3 4 Sav ing and Loading Settings for instructions on how to do this b Without using the Automatic Laser Func tions e As described in chapter 10 10 Cutting and Cat apulting without Automatic Laser Functions adjust the footswitch in such a way that it can be used to activate the cutting function e Set the value for the microscope stage move ment speed so that you can easily follow the movement with the naked eye when in Stage Mode Enter Stage Mode and try moving the microscope stage with the mouse Change the speed during movement in order to find an appropriate value Remain in Stage Mode e Release the laser with the footswitch and at the same time move the stage The laser remains in operation as long
41. clicking on OK Whenever you save images subsequently the im age name you entered will be supplemented with a sequential number 01 02 PALM RoboSoftware 3 1 0106 EN To save your image Info Please note that the image is stored with the el ements unless the option to display them has been switched off The elements are stored in the same way as they are displayed on the screen i e perhaps with their area content specified with or without the element number and if relevant with changed image parameters refer to chapter 3 9 Set Image Parameters The Copyright Note and scale bar are only in cluded with the saved image if they are dis played on the screen However other features such as Laser Mark ers crosshairs etc are not saved fel Save Image File ET Save mage e Click on the Save Image icon button in the Archive Toolbar or click on menu item Save image in the File menu The Save Image As window opens the pre defined folder and the predefined file name with the additional sequential number 01 02 03 are preselected and offered the se quential number is incremented by one every time you save an image refer also to chapter To enter a file name for saving images manually e Select a different folder if you do not want to save your elements in this folder e Enter a name for your image file if you do not want to use the preset na
42. commands the first or last element drawn the element drawn immediately before or after the element currently centered You can center any element using the Element List To center specific elements using icon but tons Lil Last Element i4 First Element 4 Previous p Next Element Element e Click on one of these buttons in the Tool Bar First Element Last Element Previous Element or Next Element The required element is centered on the monitor To center defined elements using menu com mands Move ld Goto First Element Goto Prey Element P Goto Next Element P Goto Last Element e Click on one of these commands in the Move menu Goto First Element Goto Prev Element Goto Next Element or Goto Last Element The required element is centered on the monitor 66 To center a defined element using the Ele ment List e Open the Element List in one of the modes described in chapter 7 3 The Element List Window e Select the element you want to center click in the corresponding row with the element you want to select but do not click in the checkbox on the left border gt Goto Move Go to Element e Click on the Goto icon button or e click on menu command Goto in the Ele ment menu The desired element will be centered on the monitor Info You can change the movement speed of the stage when targeting the desired element Re fer to cha
43. database credentials The User ID password combination does n match A Switch to File Mode now A 13 27 21 Start controller 13 27 21 Start controller 13 27 21 Start controller 13 27 21 Start controller 13 27 20 Start controller 13 27 20 Start trace 13 27 20 Start fluorescence 13 27 18 Startup Microscope 13 27 10 Initializing Zeiss Axiovert 200M 13 27 08 Start axiovision l engine 13 27 06 Start cellenger engine The PALM Robo Information with its message history is opened PALM RoboSoftware 3 1 0106 EN Status Display 2 5 Status Display The PALM RoboSoftware States window provides you with details of the current status of your sys tem components To open the States window Stage 101175 6 47235 4 um e Double click in the field at the rightmost end of the Status Bar System and controller states System stIdle x Y z Stage side o oza fazo ooo UV Laser side fas fis fo Robomover stilotavallable o fo fo IR1 Laser ETT O CS IR2 Laser O la lo D31 side fa 000 foo fo pe stnotavatable fo fo fe y y y x y y y xX x Not available The system components are shown in the left hand column and the current status in the right hand column Columns X Y and Z provide information on the current coordinates of each component PALM RoboSoftware 3 1 0106 EN 21 22 PALM RoboSoftware 3 1 0106 EN Launching Settings 3 Settings When working w
44. dealt with earlier in this chapter If you have installed a Single Beam System the window contains only one tab IR 1 for setting power and focus Both values are set in analog form e Drag the slider or click on the arrow buttons or click and hold the mouse button down until the required value is displayed in the field between the slider and the arrow button or click in the text box enter the required value and press Enter e Close the window by clicking on OK PALM RoboSoftware 3 1 0106 EN To set IR laser power and IR laser focus with menu commands or with keyboard shortcuts Laser Energy Power more Fage Up Energy Power less Fage Down Focus up Home Focus down End Note With menu commands or keyboard shortcuts you change the settings for the laser functions that are set in Laser Tools You also change the settings when the Laser Tools are not dis played however you then have no control over the changes that have been carried out There fore before you use these commands switch to the Laser Tools display and select the laser function for which you want to change the en ergy and focus as described earlier in this chap ter Note If you have installed a Double Beam System then menu commands and shortcut keys are used to set the focus of the IR beam selected using the Wheel buttons left IR 1 right IR 2 see Page 120 Setting with menu commands e Click on items Energy Power mo
45. displayed If you wish to draw elements for more than one slide you must enter data for each slide to be processed 44 Please select or enter data Slide1 Slide2 Collection Device Data Source Data Label Liver Type Description Specimen Data Label Description Holder Label Holder Type Experiment Data Label Description Blue labeled Fields are mandatory You must enter all blue fields to enable a position For working Abbrechen e Enter the details for your sample Start with the uppermost field and move down one field at a time Info All fields shown in blue must be filled in before you can start working Other fields may be left blank Info If you would like to resume work on an existing experiment or with an existing sample you can specify this using the selection lists for the La bel fields PALM RoboSoftware 3 1 0106 EN The more buttons bring up further fields for you to enter additional details of your source your sample and or your experiment e Click the more button in the area for which you have supplementary information to enter One of the windows Source Data Details Spec imen Data Details or Experiment Data Details will open Source Data Details Label of source Description of source Type of source u Archived flag nu Archive name a Archive location re Additional Information FO cancel _ Specimen D
46. e Inthe Element List window select the group you want to ungroup do not select the ele ments Edit E Delete selected elements Del e Click on menu item Delete selected in the Edit menu The selected group is ungrouped the elements are retained e click on the Renumber icon button to update the numbering of the remaining elements PALM RoboSoftware 3 1 0106 EN To select single elements within a group on the monitor K Ctrl key Pointer Tool e Press and hold the Ctrl key e Click on the element within the group that you want to select The element will be selected in the Element List window e Click on the element you want to select 79 Measurin 7 10 Measuring PALM RoboSoftware provides functions for calcu lating the length of lines or the size of areas Area sizes are only calculated for elements of type Fig ure Line Freehand Rectangle or Circle and Au toCircle There is no point in showing area sizes for any of the other element types Note The measuring functions of the PALM Ro boSoftware are not calibrated at the factory they are an estimated value only P A L M does not guarantee the accuracy of the per formed measurements The measuring functions have to be checked and if necessary calibrated by the user for example with a ocular micrometer Correct determination of lengths and areas re quires that your system Is correctly calibrated If in doubt re
47. e Move the slider in the Light row to the desired position or click on one of the arrow keys to the left reducing intensity to the right increasing intensity 142 13 2 7 Opening and Closing the Shutter Shutter Open Reflected Light Shutter Shutter Close Reflected Light Shutter e Choose Open Close Reflected Light Shutter or Open Close Transmitted Light Shutter from the Shutter menu Info The Open Close Reflected Light Shutter command is only included on the menu if your system is equipped with a fluorescence unit The shutter will opened or closed respectively Note If you want to work with the laser the shutter must be open 13 2 8 Setting the Beam Path With the computer you can set the beam paths that are to be cleared on the sideport and the baseport The settings that you must make de pend on the configuration of your system refer to the manual for the microscope LightPath Sideport LightPath Bazeport gt e Click on the desired menu item in the Light Path menu Sideport or Baseport and select the desired entry in the submenu The selected setting will be made PALM RoboSoftware 3 1 0106 EN The Microscope Window 13 2 9 Setting the Optovar With the Optovar you can set an additional magni fication for the microscope image if your micro scope is equipped with an Optovar Note If you want to work with the laser the Opto var must be in pos
48. energy and focus for the cut and catapult functions e Click on the Energy Focus UV Laser entry in the Laser menu or press F9 on the keyboard or open the context menu with a right mouse click on an arbitrary location within your image Click here on the Energy Focus UV Laser entry The PALM Robo Laser Settings window opens It contains two tabs for setting energy or focus In the center of each tab you will find settings facili ties for combining the values of the cut and cata pult functions Cut lt gt LPC Auto i Change Cut Delta LPC Delta w 102 e Select the tab for the parameter for which you want to combine or separate settings Click the checkbox beside Auto Change in order to select or deselect a combination of these settings A tick in the checkbox indicates that the function is enabled e Inthe Delta field enter the value for the dif ference between the energy or focus values of the LPC functions and the Cut functions e Where appropriate select the other tab and carry out the same process e Close this window by clicking on OK To set the laser energy and laser focus with the Laser Tools Lazer le Uy CIR Cut LPC Energy Energy 100 100 le Footer C e Open Laser Tools if it is not displayed click on the entry Laser Tools in the View gt Menu or press the keyboard combina tion CtrI L e If you have a sy
49. he 28 3 9 Set lmage Parametros a a EA a oa 29 4 Calibrating the SYSTeM cs iia a aie Wels ee em es ww Pe ee a ea ee ee 31 4 1 Preliminary RCMalKS 0 intel a Eke ners arado et Bak Bee 31 4 2 Setting the MagniniCationy 2 22 2 4 286 a A A EE ee DR RE li ee 32 4 3 seting ther Laser Marker i ea hope aoe ea ah Press ae Pet ee Od cae hah ed ee Bees Es 32 4 4 Calibrating the Movement of the Stage aa a a 34 4 5 Calibrating tne 3D Otis mue gate oh teed SO doe das oak BR ee a ie a a ts a 35 4 6 Resetting CallDratlOn cana ae ae oh oe ERA a Bee Ge ee AAA A 36 PALM RoboSoftware 3 1 0106 EN 3 Contents 4 7 4 8 5 1 5 1 1 Deez 5 1 3 5 1 4 5 1 5 5 1 6 5 1 7 5 2 5 3 5 4 5 5 5 6 6 1 6 2 6 3 6 4 6 5 Ton 7 1 1 7 1 2 7 2 7 2 1 Tr 2e2 7 2 3 7 2 4 7 2 5 7 2 6 7 2 7 7 2 8 7 2 9 7 3 7 3 1 EZ 133 7 3 4 7 4 7 5 7 6 7 6 1 7 6 2 EA Tekal Pz 7 7 3 7 1 4 EVO Adjusting the Lens OSet 2200 aa edad ds me Ohne oe A eg ARS See ey ee 36 Resetting the Lens ONSel vdd ap dd Seed ees Se Vee tee eee eek See ee 37 POSITIONING ENG Stage ii a Sie ee ee A a da Se Ae a eee 39 M vement TYD Snr ot bite dar da A Stee ds eae Shee Bip eee eres Us ak ete 39 Positioning the Stage with the Mouse Stage Mode 0000 ee 39 Positioning the Stage with the Mouse Scroll anaana aaa es 40 Positioning the Stage Using the Scroll Bars 0 es 41 Positioning the Stage with the Keyboard o o e es
50. immediate surroun dings by a clear cut line Only in this way can pure Sample preparation be assured when catapulting This function can only be used with glass mounted samples Cut Cutting along a selected line The laser cuts pre cisely along the pre drawn line so that a clear line of detachment is created between required and unrequired material In the cut area all biological material is destroyed by ablation Thus a pure sample preparation can be carried out without fear of contamination Double Beam System The PALM MicroTweezers is available as a single beam or a double beam system In the double beam system the IR laser beam is split into two beams Depending on the configuration one or both of the beams is equipped with a 3D Laser Beam Positioning This means that you can trap two objects and move them relatively towards each other You can select different power and fo cus values for each of the two split beams Element An element is a drawing on your monitor Ele ments serve to define selected areas for the appli cation of laser functions to annotate selected points of the microscope image to measure length and areas and to define reference points Accor dingly the program distinguishes six types of ele ments Figure Dot AutoCircle Ruler Text and Reference Point Figure The element types Line Freehand Rectangle and Circle are known collectively as element type Figure JointCut
51. in a folder The date and time are used to create a name for this folder which is created automatically in the specified folder refer to chapter 9 2 Specify ing Folder to Store Images In If you are working in Database Mode the images will be stored in the database Show Log File button every time the Time Lapse function is activated the PALM RoboSoft ware creates a file showing the sequence of events for the function This file is saved in the same folder as the images Click on the button to open this file the button remains disabled until the sequence has been completed Set the sequence of events for your recordings in the Time Lapse Definition Data section If you tick the check box all time data inter vals and durations in minutes then all times will be expressed in minutes rather than the default of seconds The Video Time Lapse 1 and Time Lapse 2 check boxes allow you to choose whether you wish to save a video sequence or a series of images You can also tick more than one check box if for instance you wish to record a video and a Series of images or indeed two series of images with different settings Enter an interval between images to be stored in the Interval field applies only to series of images Enter the length for a video sequence or how long images should be stored in the Duration field For series of images the number of images can thus be calculated from the tim
52. is indicated by a white line Info If you have drawn elements that are currently positioned outside of the monitor section some or all of the borderlines of the selection rectan gle will also be outside the monitor section In this case the border lines are not visible PALM RoboSoftware 3 1 0106 EN Resetting the Lens Offset You will receive the following prompt Step 1 of 2 Click Selection Rectangle e Click on any dot within the selection rectangle and hold the mouse button down Note If you click inside the rectangle and inadvert ently do not hold the mouse button down the selected elements will not be moved to the cor rect position In this case reset the Lens Offset as explained in the following chapter 4 8 Re setting the Lens Offset and start the operation again You will receive the following message Step 2 of 2 Now move the Figure to the new position e Move the element or elements holding down the mouse button to the correct position and release the mouse button Then even elements that are outside the current monitor section will be moved The following message will appear New Lens Off Set is set This message will disappear automati cally after a few seconds PALM RoboSoftware 3 1 0106 EN 4 8 Resetting the Lens Offset You can restore the default factory settings for the Lens Offset at any time To reset the Lens Offset Setup Calibration j Factory Lens Uff
53. is the standard mode which opens by default when the program is launched In Cursor Mode you can open menus select functions click on icon buttons or draw elements Stage Mode In Stage Mode you can synchro nize the movement of the stage with that of the mouse Chapter 5 1 1 Positioning the Stage with the Mouse Stage Mode includes instruc tions on how to proceed and how to switch between Cursor Mode and Stage Mode In Stage Mode you can only access commands and functions that can be operated with key board commands Freeze Mode In Freeze Mode the movement of the stage and the microscope image on the screen is frozen In Freeze Mode all functions that cause or require movement of the stage such as scroll ing positioning of elements targeting specific points positioning a Laser Marker laser func tions are blocked This function is particularly useful when work ing with fluorescence refer to chapter 12 5 Freeze Mode 3D 1 Mode and 3D 2 Mode only if you have purchased a system with one or two 3D mov able IR laser beams In 3D 1 3D 2 Mode you move the IR laser beam synchronously with the mouse Chapter 11 8 The 3D Laser Beam Posi tioning includes instructions on how to pro ceed and how to switch between Cursor Mode and 3D Mode 18 2 4 Help and Information about PALM RoboSoftware PALM RoboSoftware includes a general Help Func tion PALM Robo Help as well as a tutorial for sev eral ve
54. laser then you should click the UV button The Laser Tools for the UV laser will be displayed e Under Footer click the button on the left below Cut if you want to perform cutting with the laser in Stage Mode or the button on the right below LPC if you want to perform catapulting with the laser in Stage Mode 116 PALM RoboSoftware 3 1 0106 EN Essential Settings 11 Tweezers Option The PALM MicroTweezers System is an optical tweezers with which can you trap and move ob jects under the microscope It works in conjunc tion with an IR laser and may be purchased as a Single Beam or Double Beam with or without 3D Laser Beam Positioning in each case Double Beam with two 3D movable IR laser beams With this system you can trap and move two objects independently of one another Double Beam with one fixed and one 3D mov able IR laser beam With this system you can also trap and move two objects independently of each another with the limitation that one IR laser beam is fixed and therefore one object can only be trapped and moved by moving the stage in the x and y axes Single Beam with 3D Laser Beam Positioning With this system you can trap and move one object by moving the IR laser beam or by moving the stage Single Beam with one fixed IR laser beam With this system you can trap an object and move it in the x and y axes by moving the stage In all four configurations you can lift trappe
55. of each rectangle side a white square is displayed Info The white selection rectangle partly covers the lines of the elements You cannot see the se lected element completely if you have select ed a rectangular element for example you see the selection rectangle only To select all elements Edit Select All Ctrl e Click on menu item Select All in the Edit menu main window or press key combination Ctrl A The keyboard command works as a toggle With this command you can select all elements only if no element was selected before ie their effect de pends on the previously applied settings If all elements were deselected they will now be selected If single or all elements were previously select ed they will now be deselected PALM RoboSoftware 3 1 0106 EN To select a single element ts Pointer Tool e Select the Pointer Tool in the Graphic Toolbar e Click once with the mouse on the element or move the cursor with the mouse button depressed to pull up a rectangle that surrounds the element you require entirely or partly ts Ctrl key Pointer Tool If your elements are overlapping you might not be able to select a single element with the described methods without selecting adjacent elements also e In this case keep the Ctrl key pressed and click on the desired element until it is selected with each click the next of the stacked elements will be selected To select m
56. of the dots A tick in this box indicates that the dots will be ar ranged diagonally If there is no tick in the box the dots will be arranged vertically or horizontally e Click in the box AutoLPC Center shot if a laser pulse should be set to the center of the element e Close this window by clicking on OK PALM RoboSoftware 3 1 0106 EN 10 5 5 Special Laser Settings for the ROboLPC Function Note These laser functions only make sense with membrane mounted samples If you release the RoboLPC function an area el ement will be cut out but a small RoboLPC joint will be established between the start and end dots of the cut line A laser pulse on this RoboLPC joint will catapult the area contents into the cap This function requires a special setting establish the length of the RoboLPC joint remaining after cutting To establish the length of the remaining joint for RoboLPC e Click on the LPC Parameter entry in the Laser menu The LPC Parameters window opens In the lower part of the window you will see the section LPC Distances Laser LPC Parameter 12 Distance of RobolPC joint e Enter the desired value in um for the width of the remaining RoboLPC joint in the Distance of RoboLPC joint field e Close this window by clicking on OK Note Should the value of the RoboLPC joint param eter be too small or at zero then it can occur that automatic catapulting does no
57. of type Dot as unfilled circles elements of type AutoCircle are indicated by a thin line with an un filled circle in the middle Close amp Cut AutoLPC a a As with Close amp Cut the positions of the laser pulses are displayed as filled circles with the diam eter that you have set RoboLPC Cut lines are displayed with the line thickness specified by you In addition the dot where the la ser pulse will focus to catapult is displayed in the center between the start and end point of Figure elements If the distance between start point and end point of an element should be greater than the parame ter Distance of RoboLPC joint as shown in the picture above the distance is automatically re duced by a straight line to the set value 99 Specifying Laser Settings AutoCircle The circular cut line of elements of type AutoCir cle appear with the line thickness specified by you the laser focus of the laser pulse as a filled cir cle with the diameter specified by you 100 10 5 Specifying Laser Settings In order to achieve correct results with automatic laser functions you must set a series of parame ters Several settings are required for all laser functions others only for some When relaunching PALM RoboSoftware the set tings apply that were in use when the program was last exited Laser energy and laser focus You can make independent settings for the Cut and L
58. on the Tool Bar The Alarm Bell window opens Alarm bell Alarm message Coptional cancel e Select the required time and enter a message if you want to e Start the alarm timer by clicking the OK but ton PALM RoboSoftware 3 1 0106 EN Set Image Parameters 3 9 Set Image Parameters You can modify the way in which your microscope image Is displayed in order to make any areas that are of particular interest for your work stand out more clearly You may for instance want to run an analysis under Cellenger or AxioVision see preceding chapter because of the clearer results they produce Facilities for adjustment are available in the Life Image window You can also avail yourself there of the facility to read colors and brightness values from your image and display them Info Any changes made here will only affect the image on the screen and not the original camera image in other words you are not re configuring any camera settings Info You should bear in mind that one conse quence of changing the image parameters is that there may be a delay before the image appears on the screen because each time a change is made e g the stage is moved the original camera image must be recalculated So it is worth trying to obtain a optimum result from the camera in the first place The settings can be seen immediately in the live image but cannot be seen in the Navigator refer to chapter 6
59. or in Database Mode with the database pre selected under Settings or there will be a dialog for you to specify which mode you wish to start in Operating Mode Selector Please select database Database CAPALMDatalPalhs1 RoboDataBbig4dd mdb TE User ID bj025705 Password sekolokdetekstet DB Mode File Mode 10 e Inthis case you should select the database you wish to work with and click the DB Mode but ton or click on the File Mode button if you wish to work in File Mode Note When the PALM RoboSoftware is launched you can choose between database and File Modes Once the program Is running this is no longer possible Once the program is running it is also not possible to change the database You should exit the PALM RoboSoftware and restart it if you wish to change to a different mode or database If necessary you must first modify the initial settings as described in chapter 3 1 Launching Settings To exit PALM RoboSoftware File Exit PALM Robo Alt F4 e Click on Exit Palm Robo in the File menu or click on the Close button in the top right corner of the window or press ALT F4 on the keyboard Info When you exit the program a dialog box may appear prompting you to confirm whether you want to save your settings or your elements only in File Mode for future use Refer to chapter 3 4 Saving and Loading Settings or 8 2 Saving and Loading Elements in File Mode for
60. path to an I maging Software Setup PALM Robo e Inthe Setup menu click on menu item PALM Robo The Preferences and Configuration window opens e Click on the Images tab Imaging Software Configuration Path T ml e Click on the button with three dots in section Imaging Software The Select the imaging software window opens e Select the desired program and close the win dow by clicking on Open In the Configuration Path field the program path IS now entered e Close the Preferences and Configuration win dow by clicking on OK Chapter 1 8 2 Imaging Software explains how the imaging software can be launched 28 3 8 Short Term Alarm The PALM RoboSoftware features a short term alarm which can be set for a pre defined time 1 2 3 5 or 10 minutes or for any time of your choice in seconds and minutes There is also an option to enter a message text At the end of the specified period a brief audible tone is sounded and a message window is displayed showing any text that you may have entered You may wish to use this short term alarm as a timekeeper in time critical operations or as a re minder to switch off the IR laser with temperature sensitive samples or to switch off the fluorescent illumination with samples sensitive to fluorescence before the sample begins to suffer any damage To set the short term alarm ib Alarm Bell e Click the Alarm Bell icon button
61. performing them Each chapter begins with a brief summary of the functions that it covers followed by instructions for using and executing the functions The instructions are organized so that they can be followed step by step However you should al ways read the summary at the start of each chap ter first and familiarize yourself with all the operational tasks of a section before you put them into practice A laser function carried out by mis take or with incorrect settings could destroy your sample Until you gain experience with the pro gram functions work on samples that are no long er required for testing This User Manual is prepared on the assumption that you have basic computer skills and that you can use a computer mouse Info This manual describes all the functions which are available to control all possible options refer to chapter 1 1 Functionality of PALM MicroLaser Systems Like PALM MicroLaser Systems the PALM Ro boSoftware has been designed on a modular basis During system installation by P A L M Microlaser Technologies GmbH it will be adapted exactly to your system configuration i e the program will provide those tools and commands which you require to control your particular system Tools and commands which relate to components which have not been installed will not appear PALM RoboSoftware 3 1 0106 EN About this User Manual 1 4 2 Conventions To help you find your way around the
62. position and shape by hand It is therefore recommended when transferring elements from one slide to another that this is done using the Serial Sections function refer to chapter 10 9 The Serial Sections Func tion This function adjusts the position and Shape of elements automatically You can also reload the most recently saved ele ments using the list of most recently used element files on the File menu if you haven t saved any elements this list will be empty File 1 testslide palm on Position 2 e testelide palm on Position 1 3 3324yElements palm 4 193440E lements palm e Inthe File menu click on the name of the ele ment file listed there which you wish to reload Info If you have used a slideholder that can ac commodate more than one slide then the po sition occupied by the slide when the elements were saved is shown to the right of the file name However you can also load the elements file for a different position The loaded elements will appear on your monitor and the name of the elements file will be displayed in the title bar of the program window Any ele ments that were already on the monitor will be de leted Note Take particular care to insert your sample care fully and correctly If the slide is canted or twisted the loaded elements will not match to the corresponding structures of your sample 84 8 3 Saving and Loading Elements in Database Mode In Database Mode you d
63. section of your Sample on the monitor However you can also draw elements that extend beyond the section dis played on the monitor Chapter 7 2 1 Drawing in the Various Program Modes includes instructions on the factors you must consider for this When processing elements you will often be work ing with the Element List window This window provides a summary of your elements and their properties and allows you to make changes to them and to perform various program commands Refer to chapter 7 3 The Element List Window for a description of the options offered by this win dow 55 Creating a New Set of Elements 7 1 Creating a New Set of Elements The procedure varies according to which mode you are working in Database Mode or File Mode Info Database Mode Is only an option if your system is configured accordingly Please contact P A L M Microlaser Technologies GmbH to up grade your system if required Info When the program is launched you decide whether you want to work in File Mode or Da tabase Mode refer to chapter 1 7 Launching and Exiting the Program It is not possible to switch between these two modes whilst the program is running If you want to change mode you must first shut down the PALM Ro boSoftware and then relaunch it If necessary you must first modify the initial settings as de scribed in chapter 3 1 Launching Settings 7 1 1 Working in File Mode You can start drawing elements a
64. text it has been organized into distinctive sections Indented paragraphs with a dash provide a swift overview of various functions or proper ties of PALM RoboSoftware e indented and bulleted paragraphs provide instructions for executing certain functions Menu names menu commands and the names of icon buttons and windows are set in inverted com mas In addition you will always be referred to the menu or bar where the command or button is lo cated Special markers in the left margin indicate text that you must not fail to read Caution This indicates a potentially dangerous situa tion You must observe the precautions marked like this to avoid injury or damage Note This indicates important information Read this information to maintain your system s high level of performance and safety and to prevent malfunction or damage to your sam ple Info This indicates tips on correct use and offers useful notes for getting the best use of PALM RoboSoftware 1 5 Installation The PALM RoboSoftware is already installed on the computer upon delivery or initial operation of your system If you experience problems or if you want to work with another computer please contact P A L M Microlaser Technologies GmbH File Mode and Database Mode 1 6 File Mode and Database Mode The PALM RoboSoftware recognizes two operating modes File Mode and Database Mode File Mode is always available Database Mode can only
65. the movement of the stage be fore switching to Stage Mode You cannot ac cess this command when you are in Stage Mode Note Do not use the function Invert stage motion if you want to calibrate the system 39 Movement Types To switch to Stage Mode Stage Move am Stage mode Fy e Click on the Stage icon button in the Tool Bar or click on menu item Stage Mode in the Move menu or open the context menu and click on menu item Stage Mode or press F7 on the keyboard Info The cursor is not displayed in Stage Mode In Stage Mode you can only change the speed and energy or power of the stage and the focus of the laser by means of keyboard commands note that you can only change the settings for the laser beam that is selected in Laser Tools You cannot access any other functions To switch from Stage Mode back to Cursor Mode e Click once with the left mouse button or Press ESC on the keyboard or press F7 on the keyboard 40 5 1 2 Positioning the Stage with the Mouse Scroll In Cursor Mode you can use the scroll function to move the stage e Move the cursor towards an edge of the image outside the Scroll Rectangle but still within the image itself if you want to move the stage horizontally or vertically or Move the cursor into a corner of the image out side the Scroll Rectangle if you want to move the stage diagonally Info If the Scroll Rectangle is not displayed yo
66. time you enter an appropriate com mand If you are working in File Mode the images are stored in a folder and when in Database Mode they are stored in the linked database refer to chapter 1 6 File Mode and Database Mode 9 3 1 Saving Images in File Mode You can specify a folder and a name for the image being saved refer to chapter 9 2 Specifying Fold er to Store Images In and text below When you Save images later the specified image name will automatically be supplemented with a sequential number 01 02 If you want to save pictures subsequently these assignments are selected automatically However you can decide whether you want to accept them You can also save your images later in a different folder You can also create a new folder when sav ing and assign different names to your images Note If you select another folder or another image name than the one proposed for saving these settings are adopted as the default settings and used for all subsequent saving operations To enter a file name for saving images manu ally Setup PALM Robo e Inthe Setup menu click on menu item PALM Robo The Preferences and Configuration window opens e Click on the Images tab Saving Images Folder stadminiMy Documents My PalmDatalImages Filename e Enter a name for images to be saved in the Filename field of the Saving Images sec tion e Close the window by
67. to use the freehand tool to draw el ements The subsequently necessary allocation of the other samples to the respective markings will then as a rule be more exact This procedure is described in the following e Position the stage so that one of the three markings in your sample is displayed on the monitor e g with the help of the PALM Navi gator e Select the desired drawing tool the freehand tool is recommended and draw an element to the shape of your marking e Position the stage on the second marking and draw an element here also to the shape of this marking e Proceed likewise with the third marking e Open the Element List window Your three elements are now listed there e Select the three elements Edit Create Group e Inthe Edit menu click on menu item Create Group The three elements are joined together to form a group The group appears as a fourth element in the Element List window Allocate a name to the group e g Slide 1 e Double click on the element group The Element Properties window will open e Type a name and click the OK button Info It is not essential to allocate a name to the group As you will no doubt later draw further elements and create further groups you will thus be easily able to retain an overview 112 Element List File Edit Move Laser Oth amp Pm k e 1 cu Slidel Slidez Slides Summary Show Types Area pm Gr
68. tool in use is alSo selected with the exception of the text tool and ruler For tools with which you draw cut lines this is the Cut function for the Dot tool it is the LPC function and for the AutoCircle tool it is the AutoCircle function The starting point of the element to be drawn is the point of the line tools and the center of the crosshairs in the case of the circle and rectangle tools LPC dots are set at the tip of the cursor this is also where the center point of AutoCircle ele ments are set The tool selected remains active until you select another drawing tool or another function You can see which graphic tool is currently in use by the appearance of the cursor and of the associated icon button the button in question appears de pressed 7 2 1 Drawing in the Various Program Modes Info This section is concerned with the software modes in the PALM RoboSoftware These are totally unrelated to the Database and File oper ating modes As is described in chapter 2 3 4 Software Modes PALM RoboSoftware works in a number of modes You can draw elements in Cursor Mode and in Freeze Mode Cursor Mode is the default mode for PALM RoboSoftware it is set automatically when the program is launched Chapter 12 5 Freeze Mode includes instructions for switching into and out of Freeze Mode PALM RoboSoftware 3 1 0106 EN In both modes you can draw elements inside the Scroll Rectangle without limi
69. where the laser beams strike the sample To check the cor rect setting of the Laser Markers you must release the laser Hold a sample that you no longer need for examination or one containing areas that are no longer needed for examination ready A slide that has been colored with a black felt pen is particularly helpful for setting and checking the IR Laser Marker You must check the positions of the Laser Markers after every magnification change and correct them as necessary 31 Setting the Magnification 4 2 Setting the Magnification Before you start work you must set the micro scope magnification in PALM RoboSoftware To select the required magnification Lene Ex T En e Click on the triangle at the right edge of the field in the Lens menu A list will open displaying the available magnifica tions settings e Click on the option corresponding to the mag nification that has been set on the microscope e Adjust the microscope to the desired magnifi cation only necessary for a manual micro scope on motorized microscopes adjustment is automatic Info Depending on the lenses provided on your mi croscope the selection list in your program may include entries other than those shown in the list above Note Note that the IR laser can only be used in con junction with 63x and 100x lenses Info You will also find a drop down box for selecting the magnification in the PALM Robo Speed
70. which the 3D Laser Beam Position ing is performed as well as the definition of a Ref erence Position may have to be carried out individually for each sample or sample type The following section describes how to proceed You only need to calibrate the 3D Laser Beam Po sitioning and adjust the IR Laser Marker setting if the results from using the laser are incorrect For instructions on how to do this refer to chapter 4 3 Setting the Laser Marker and chapter 4 5 Cali brating the 3D Offset 117 Setting IR Laser Power and Focus 11 2 Setting IR Laser Power and Focus There are several options available for setting the IR laser power and IR laser focus with the Laser Tools on the left side of the program window in default configuration with the PALM Robo R Laser Settings window which you can open via a menu or keyboard Shortcut using menu commands in the Laser menu using keyboard shortcuts There is another option for setting the IR laser fo Cus with the scroll wheel on the mouse If you have installed a Double Beam System you can set a different power value for each beam You can also set the focus differently for each beam or you can couple the focus settings together If you change the focus for one IR laser beam the focus of the other beam will automatically be adjusted to the same value Note The optimal settings for power and focus de pend on the objective in use on the m
71. you can check the results of the catapulting Caution Set the focus to Check Position only if you have previously moved the stage to the Check Point as described in chapter 5 5 The Check Point Otherwise the objective will col lide with the slide or the stage and may be damaged Load Position prescribed The objective is moved into the lowest possible position In this position you can for example move the stage to the check point and back or change the slide without any fear of hitting the objective with the slide PALM RoboSoftware 3 1 0106 EN Caution Using a non motorized microscope If you want to return the stage from the Check Point you must set the Load Position manu ally so that the objective does not collide with the slide refer to chapter 5 5 The Check Point for instructions on moving the stage to the Check Point and back To define the Check Position Focus Define gt CheckPos e Set the focus to Load Position refer to chapter 13 2 3 Defining and Setting Fixed Positions for the Focus e Move the stage to the Check Point refer to chapter 5 5 The Check Point e Set the focus so that a catapulted sample in the cap Is displayed sharply e Click on menu item CheckPos in the Focus gt Define menu e Set the focus to Load Position again e Move the stage back from Check Point To define the Work Position Focus Define d WiorkPos e Set the focus so that yo
72. 41 Positioning the Stage with the Centering TO0l o o es 41 Moving the Stage during an Automatic UV Laser Function oo o 41 Further Possibilities for Positioning the Stage o oo a 41 Stopping the Stage in an EMergenCy a 42 SeLUING SPCCGs id A ES IA AA A A A ee 43 The Reference Position of the Stage o a a es 46 TRE Check POMC amp vs ee wa me me eM Bah ee a ca a ee ete A ae ad 47 Tie LOGO POSO tri dc Oh eck SAM A Roe ar Sea ewe te Becks Se daees 48 PAEM INavigator siria aaa 49 Start and Quit PALM Navigator 49 Scanning a Sample with PALM Navigator o e e na 50 Positioning the Microscope Stage a a a 52 Analyzing Images Using the Cellenger SoflWare o o a e e 4 53 Analyzing Images Using the AxioVision Software o a a es 54 Drawing and Measuring lt a ia A AAA A a 55 Creating a New Set of Elements aa 56 Working in File MOde sg 200 iv ra a bd eee he eh bed Be a gai aE aia a he oe o de ee ee 56 Working in Database Mode a 56 Drawing New Elements sa 4 4000 e ii a Pe ee IEA A wee da AAA 58 Drawing in the Various Program Modes aa a a es 59 The Reference Point TOO s 1 44 4 6 8 adm ewe Se he ee Gaai E A he OUR et ee ee 59 The Freehand TOO sriti dng Goh tee Se ae eee kee ae ee eS amp Oe ee See 59 Tieline TOO W ard k
73. 6 2 Scan ning a Sample with PALM Navigator e Select the AxioVision Analyze tab Analyze scripts test Slide Section Adjust parameters Analyze ROI Use autofocus suspend scan for focus You see the same scanned image as on the Scan tab If you have switched on the display of ele ments as described on Page 51 then your ele ments will also be displayed here The AxioVision program allows you to create and Save scripts that you can then use to analyze your sample The AxioVision manual explains how to use the program e If you wish to call up AxioVision click the Adjust parameters button Scripts created in AxioVision are shown in the Nav igator s Analyze scripts selection list e From the Analyze scripts selection list choose the script you wish to use for analysis e Click on Analyze ROI button ROI Region of interest Your sample is analyzed 54 PALM RoboSoftware 3 1 0106 EN 7 Drawing and Measuring The automatic laser functions allow you to cut along defined lines and catapult individual points or areas out of your sample For a summary of the automatic laser functions refer to chapter 10 Cut ting and Catapulting Before you use these laser functions you must de fine these cut lines or areas they are called ele ments PALM RoboSoftware offers you a range of tools in the Graphic Toolbar for drawing elements You can draw elements one after another for dif
74. 81 Save images automatic 90 slideholder 24 UV laser 100 Short term alarm 28 Single Beam system 146 Slideholder settings 24 Software modes 18 3D Mode 127 Cursor Mode 40 Stage Mode 40 Speed Tools 44 126 153 Index Stage reference 146 Stage Mode exit 40 invert movement 39 select 40 set speed 43 Status Bar 14 Status display 21 T Time Lapse function 91 cancel 93 settings 91 Toolbars reposition 15 Show hide 15 Tools Fluorescence Tool 130 Laser Tools 119 Microscope Tools 139 Show hide 15 Speed Tools 44 126 U User Interface customize 15 V Video sequence Time Lapse 91 W Window colors 17 74 Element List 62 Laser Marker layout 16 LPC parameters 105 microscope window 140 PALM Robo information 20 PALM Robo IR laser settings 120 PALM Robo laser settings 103 PALM Robo speed 44 125 states 21 System Info 20 154 Work Position define 139 141 meaning 141 set 139 141 PALM RoboSoftware 3 1 0106 EN
75. A PALM Microlaser Technologies PALM RoboSoftware Software Manual 55 PALM Robo fot Wew Hove aser Lgser Marker Setup Devices Help SE y Ln sil Fitu 4ROPARtY SOOC O BO 24a 24 et ee IE A a Ty For Help press Fl MicroLaser Systems Version 3 1 0106 EN We make it visible This document is delivered only to those persons who are trained and authorized by P A L M Microlaser Technologies GmbH No part of this document may be reproduced or distributed in any form or by any means without the prior written consent of P A L M Microlaser Technologies GmbH The information in this document is subject to change without notice P A L M Microlaser Technologies GmbH assumes no responsibility for any errors or unclear formulations that may appear 6 edition January 2006 Regulatory Notice PALM MicroLaser Systems are intended for research use only and are not approved for medical applications in the United States and Canada LMPC is a trademark of P A L M Microlaser Technologies GmbH PALM is a registered trademark of P A L M Microlaser Technologies GmbH Adobe and Photoshop are trademarks of Adobe Systems Incorporated ACDSee is a registered trademark of ACD Systems Ltd The following instruments components or methods have been patented by P A L M Microlaser Technolo gies GmbH Laser catapult technology Laser Pressure Catapulting LPC Patents US 5 998 129 EP 879408 B1 and others Three dimens
76. ALM RoboSoftware via icon buttons in the main window of the PALM RoboSoftware viaa menu command in the Element List win dow Via an icon button in the Element List window via a keyboard shortcut via an icon button in the PALM RoboMover win dow if your system is equipped with a PALM RoboMover Note If you want to use the PALM RoboMover to po sition the caps automatically you have to se lect the desired laser function in the PALM RoboMovers window there you must start these functions too refer to the Manual of the PALM RoboMover rd Start Laser Laser gt Start Lazer function F1 In the Element List Window e Click on the Start Laser icon button or click on the Start Laser function entry in the Laser menu In the PALM RoboSoftware main window e Click on the Start Laser icon button in Cut Tools or click on Laser below Speed Tools or click on the Start Laser function entry in the Laser menu With the keyboard command e Press F11 on the keyboard Inthe PALM RoboMover window refer to the Manual for the PALM RoboMover After the laser function has been released the stage moves automatically along the defined cut lines or from LPC dot to LPC dot the laser is acti vated and deactivated as necessary When all ele ments for processing have been processed the la ser is switched off The stage is returned to the PALM RoboSoftware 3 1 0106 EN starting
77. C Icon Button Menu Command Help gt PALM Robo Help Open Imaging File gt Open Imaging T Information Center File gt Open Information Center File gt Exit PALM Robo Navigator Window ch g View gt Navigator Window E Element List View gt Element List Microscope Window View gt Microscope Window a Stage Move gt Stage Mode Move gt Setup Speed Laser gt Energy amp Focus UV Laser Laser gt Energy amp Focus IR Laser Start Laser function Laser gt Start Laser function Laser selected elements context menu Edit gt Select All DeSelect All Edit gt Copy PALM RoboSoftware 3 1 0106 EN Keyboard Shortcuts Ctrl E Ctrl F Ctrl G Ctri K Ctri L Ctri N Ctrl P Ctri S Ctri U Ctrl V Ctrl Y Alt P Alt Enter Alt F Backspace Alt Back space Del Appendix B Keyboard Shortcuts Icon Button Menu Command View gt Menus gt Speed Tools Move gt Speed faster View gt Menus gt Graphic Tools Move gt Goto Check Point Return View gt Menus gt Laser Tools File gt New Elements m ColorPalette On Off View gt Menus gt Color Palette Move gt Speed slower View gt Menus gt Cut Tools Edit gt Paste i Delete all unprocessed EK elements Edit gt Delete all unprocessed elements Toggle switch between standard pointer and reference pointer Edit gt Change
78. P A L M solution for working at high throughput rate Time saving automatic sample capture and additional sorting functions of the captured Samples enable above all deployment in the routine laboratory The PALM RoboMover makes automatic positioning of caps over your Sample possible There are various inserts col lectors available each of which contain one or more vessels All of the PALM RoboMover func tions can be controlled by the PALM RoboSoft ware Fluorescence Unit With the addition of a fluorescence unit the PALM MicroBeam System can be used as a fluorescence microscope According to the equipment basic or advanced this module can be used for non simul taneous application of fluorescence and laser func tion or even for simultaneous laser function under fluorescence illumination PALM RoboSoftware 1 2 PALM RoboSoftware The PALM RoboSoftware is designed to facilitate operation and control of PALM MicroLaser Sys tems The PALM RoboSoftware places at your disposal all the functions you require for precise control of the following functions Display of the microscope image recorded by a video camera on the monitor Software controlled movement of the stage Definition and processing of lines and areas elements for marking and further processing with the laser Measurement of dimensions of the sample parts length and area The measuring func tion offers an approximate value only and must
79. PALM Navigator until the image has been scanned To set the image parameters G Life image tools e Click on the Life image tools icon button in the Tool Bar This opens the Lifelmage window In the View settings area you will find sliders for setting vari ous parameters The Measurement tools area provides information about the image e Alter the parameters in the View Settings area to produce the desired result move the sliders click on the arrow icon buttons or enter the values in the relevant fields Contrast When contrast is increased light parts of the image become lighter and dark parts become darker i e differences in brightness are accentuated Reducing the contrast has the opposite effect Brightness the entire image becomes lighter or darker You should be aware that if the brightness setting is altered too much the light parts of the image may become completely white or the dark parts com pletely black PALM RoboSoftware 3 1 0106 EN Saturation changes color saturation Reducing this setting results in a weakening of the colors leading ultimately to a black white image increasing it results in extremely intensive colors Hue changes the coloration of your image Imagine every selectable color arranged in a circle from blue through violet to red orange yellow green and cyan then back to blue again When you change this setting the colors are shifted in this circle e g b
80. PC functions However you can also link the energy and focus values for cutting and catapulting to each other Set a Delta value and determine in this way the difference between the two values As soon as you change the energy or focus for Cut or LPC the value for the other laser function is automati cally adjusted to reflect the Delta difference The Delta value has to be set separately for energy and focus The set values apply to all laser functions with which you cut or catapult Speed at which the microscope stage moves during an automatic laser function You can set various values for the cutting pro cess and for the speed at which catapulted dots are sequentially targeted LPC RoboLPC joint This parameter is important for the functions RoboLPC and JointCut only It is used to define the length of the joint that remains between the start and finish dots of an element when the element is cut out refer also to the Description of the RoboLPC and JointCut functions in chapter 10 1 Laser Functions If you select the RoboLPC or J ointCut func tion all elements of type Figure whose start and finish dots are closer together than the value of this parameter are opened to this value including closed elements such as rect angles or squares and circles or ovals For ele ments whose start and finish dots lie farther apart e g elements drawn with the Freehand Tool the distance between the start
81. Robo Instructor e Click on PALM Robo Instructor in the Help menu The PALM Robo Instructor window will open PALM Robo Instructor What do you wanttoa do 7 Calibrate the system Detine the Lens Ottset Save elements on file Load elements trom file e Click on the function about which you want more detailed information The PALM Robo Help window opens the required help topic is displayed The PALM Robo Instructor window stays open If you arrange both windows side by side on your monitor you can select several or all topics in the Instructor one after the other and have them dis played in the Help window To continue working with PALM RoboSoftware you must close the PALM Robo Instructor window again You can leave the PALM Robo Help win dow open e Click on OK in the PALM Robo Instructor window or click on the Close button in the top right corner PALM RoboSoftware 3 1 0106 EN 2 4 3 Program and Manufacturer To view information about the program and the manufacturer 2 About Help fF About PALM Robo e Click on About in the Tool Bar or click on About PALM Robo in the Help menu A window opens with information about the ver sion number and the manufacturer About PALM Robo X PALM Microlaser Technologies mamm Wersions PALM Robo software Y31 0 0 PALM Robo DLL Y 3 0 0 1 PALM Robo Mic Y 3 1 0 PALM Controller 1 136 109M Copyright C 2005 P A L M
82. Settings window opens The folder that you have specified for your settings files opens automatically e Select a different folder to store your settings files as necessary e Enter a name for your file and click on Save The window will close The new file name will ap pear in the Status Bar 26 To load a settings file Setup Load Settings e Click on Load Settings in the Setup menu The Load Settings window opens e Select the settings file you want to load and click on the Open button The window will close The name of the settings file will appear in the Status Bar PALM RoboSoftware 3 1 0106 EN Restoring Settings 3 5 Restoring Settings You can restore the factory default settings at any time To restore settings Setup Load Factory Defaults e Click on Load Factory Defaults in the Setup menu A prompt will appear Do you really want to take factory settings e Confirm the prompt by clicking on Yes The Status Bar no longer displays the name of a settings file All the settings you have made are now replaced by the factory default settings Note If you restore the settings all those settings which you created will be deleted including any change in the position of the Laser Marker PALM RoboSoftware 3 1 0106 EN 3 6 Unlocking Add On Software Compo nents PALM RoboSoftware contains a number of compo nents that need to be unlocked befo
83. To show or hide the numbering of the ele ments mg Number on off View Show Numbers e Click on the Number on off icon button or click on menu item Show Numbers in the View menu PALM RoboSoftware 3 1 0106 EN To update the numbering of your elements Renumber Edit Rernumber all e Click on menu item Renumber all in the Edit menu in the main window or in the Element List window or click on the Renumber button in the Ele ment List window or open the context menu and click on menu item Renumber all or All elements will be numbered sequentially 7 7 5 Adding a Name or a Comment to Elements You can add a name and or a comment to all ele ments not only text elements The name of an el ement is displayed at its start dot for the number as well as in the Element List window in the Name column A comment is displayed in the El ement List window in the Comment column You can edit a name or a comment at any time Proceed as you would for writing a name or com ment To add a name or comment to an element Name Comment If you do not have the Element List window open Edit rma Change Alt Enter e Select the element to which you want to add a name or a comment e Click on menu item Change in the Edit menu The Element Properties window will open e Enter your name in the Name field or your comment in the Comment field e T
84. Using one of the methods described in chapter 11 8 The 3D Laser Beam Positioning move the IR laser beam to the location where you want to mark the new Reference Position and switch the IR laser off Laser Marker Position Laser Marker P UYY Laser IRA 1 Lazer IR 2 Lazer e Drag the cursor to item Position Laser Marker in the Laser Marker menu A submenu opens containing a list of all available Laser Markers PALM RoboSoftware 3 1 0106 EN Settings for the Footswitch e Click on the name of the Laser Markers the positions of which you want to redefine IR 1 or depending on your system R 2 The message Click Cursor at new IR Laser Posi tion will appear e Click in the center of the laser focus for that IR laser beam The message I R Laser Position is set will appear This message will disappear automatically after a few seconds Note It is essential to ensure that you assign the cor rect Laser Marker to the laser beam To move the 3D movable IR laser beam to its Reference Position Move Goto Reference j e Click on menu item Goto Reference in the Move menu A submenu will open e Click on item IR 1 or IR 2 The selected 3D movable beam will be moved to its Reference Position Info Note that the Reference Position for the IR la ser beam has an entirely different significance than the Reference Position for the stage PALM RoboSoftware 3 1 0106 EN 11 5 Set
85. all Laser Markers Click on the name of the Laser Markers whose ap pearance you want to change This opens the La ser Marker window 16 Laser Marker Cancel Cut marker Triangle LaserMlarker filled hi L 15 Height air 15 width 4 O Line Color The top section of the window contains three se lection fields In the Laser Marker field you can select the Laser Marker whose appearance you want to change The Laser Marker you chose in the menu for opening the window is selected by de fault Laserklarker Cut marker Cut marker Trap 1 marker Trap 2 marker If you want to change the appearance of more than one Laser Marker you can change from one to the other here Type Triangle Box Rectangle e If you want to change the shape of the Laser Marker open the Type selection list by click ing on the arrow in the left border of the field and select the shape you want Style filled filled transparent line e If you want to change the style of the Laser Marker open the Style selection list by click ing on the arrow in the right border of the field and select the style you want PALM RoboSoftware 3 1 0106 EN Operation e To change the height and width of the Laser Marker enter the appropriate figures in the Height and Width fields or change the existing settings by clicking the arrow buttons on the left side of the fields e
86. amp Cut AutoLPC This function combines the functions Close amp Cut with AutoLPC refer to appropriate sections Line elements are closed and cut completely along the border lines the cut area contents are then catapulted to the cap using multiple laser pulses RoboLPC With this function defined structures are cut out and catapulted intact into the cap When cutting automatically a straight line between the start and end dots of the border line is left as a joint You can determine the length of this with the parameter Distance of RoboLPC joint A laser pulse is then set exactly into the center of the RoboLPC joint which catapults the entire cut area into the cap This function is only possible when there is an LPC membrane between your sample and the slide 96 As soon as you release one of these functions the stage is moved automatically along the defined cut lines or to the defined dots and the laser is activat ed and deactivated as necessary AutoCircle With the AutoCircle function circular areas are cut out of your sample and catapulted intact into your cap by a laser pulse This function is only possible with membrane coat ed samples Apart from these automatic laser functions you also have the option of releasing the laser with the footswitch refer also to chapter 10 10 Cutting and Catapulting without Automatic Laser Func tions This function is only available in Stage Mode In this cas
87. and the target sample in position 2 Slide 1 and Slide 2 tabs in the Element List window Info Before further processing it is advisable to create a proof scan of the sample with the help of the PALM Navigator refer to chapter 6 PALM Navigator This makes the target ing of individual areas of your sample far eas ier tal Reference Tool Reference Point Ad Freehand Tool Now you will create the reference elements In addition you can Insert an element of the type Reference Point into the center of each marking or draw three elements which correspond exactly to your markings For this it is advisable to use the freehand tool With which you can repro duce the shape of the markings exactly The PALM RoboSoftware then calculates the center of each element in succession and at each of these points automatically inserts an element of the type Reference Point Info It is advisable not to mix both methods Ei ther set or draw three reference elements 111 The Serial Sections Function The procedure is essentially the same whether you draw elements with the freehand tool or set reference elements with the reference point tool If you have drawn elements you will create a ref erence element in the center of the elements using the Define Group Reference Figure command This point is omitted if you work with the reference point tool refer to the following description It is advisable
88. as the foot switch is depressed e f necessary increase the energy until the laser achieves a cutting effect You will get a cut line in the sample This line will initially be unclear and wide or the sample will be cut incompletely as a result of an inadequate set ting of the laser focus and energy e Continue cutting and change the laser focus Aim for a thinner cut line e If you have achieved that then reduce the energy also with the intention of getting a thin ner cut line e Now try to continue optimizing your results by repeatedly changing both focus and energy e Once you have attained the best possible result you should either save or note the appropriate settings Refer to chapter 3 4 Sav ing and Loading Settings for instructions on how to do this 107 Releasing the Automatic Laser Functions 10 7 Releasing the Automatic Laser Functions Prior to releasing one of the laser functions you must have drawn elements refer also to chapter 7 Draw ing and Measuring made laser settings energy focus and possibly auto change and LPC distances refer also to chapter 10 5 Specifying Laser Settings set the stage movement speed refer also to chapter 5 3 Setting Speed selected the required laser function refer also to chapter 10 3 Selection of Automatic Laser Functions selected the elements to be processed with the laser positioned the collection device cap or well
89. ata Details Label of specimen Liver 1 none Specimen staining Description of specimen firsteut Label of holder slide 5647 Description of holder FE Cancel Experiment Data Details Label of experiment FO Name of experimenter FE Description of experiment FO Video file name of Time lapse video u Log of experiment FO Cancel The first field in each of these windows is pre pop ulated with the Label entries from the window Please select or enter data If you have added your own additional fields to the database these will also appear in the Data De tails windows the Information Center manual explains how to create additional fields e Enter the details you wish to record then close the window by clicking the OK button e Proceed in the same way for each slide position if you wish to work with more than one slide e If you want to catapult samples select the Collection Device Data tab 57 Drawing New Elements 54 Please select or enter data Slide1 Slide2 Collection Device Data Collection Device Data Label Collector Type Collector config Blue labeled fields are mandatory You must enter all blue fields to enable RoboMoyver For collecting Abbrechen e Enter details of your collection device Cap Mover RoboMover Info If you want to catapult the fields shown in blue must be filled in Otherwise these fields may be left blank If you wis
90. ate value However focusing is also temperature dependent This means that if the temperature is different when an element is approached than it was when the element was drawn there is a possibility that the focus setting will be incorrect Correct focusing is only possible if the work area has a stable tem perature and if your system has been moderated for a sufficient length of time see Zeiss micro scope manual for further information The fluctu ation in room temperature must not exceed 2 degrees and your system should be moderated for a minimum of four hours In order to avoid focusing problems you can turn the automatic mode on and off the system will then automatically use a stored value To turn automatic stored value focusing on and off Setup PALM Robo e Inthe Setup menu click on menu item PALM Robo The Preferences and Configuration window opens e Click on the Elements tab Positioning Elements W Go to the element z Focus e Tick the check box Go to the element z focus if focusing is to be carried out automatically PALM RoboSoftware 3 1 0106 EN 3 4 Saving and Loading Settings You can save your settings in a file and reload them for future work This also means that a back up copy is created of all the settings you have made To save settings you can specify a folder When you save your settings later this folder will be selected automatically However y
91. bars and tools as well as the Status Bar The icon bars the tools and the Status Bar can be toggled on and off as shown in chapter 2 2 Cus tomizing the User Interface Info The possible options refer to chapter 1 1 Func tionality of PALM MicroLaser Systems which are avallable are dependent upon the configu ration of your system This can be adapted to your personal requirements Please contact P A L M Microlaser Technologies GmbH if you wish to upgrade your system PALM RoboSoftware 3 1 0106 EN Device Tool Bar Image Process ing Tool Fluorescence Archive Tools JOR Speed IP G0 ERS m FL Laser y Lens J40xLD y Tools Focus AF active Scroll y Autofocus Q Pd pm sec Set Workpos 3200K 2000 10000 um Light Speed Time Lapse Tool 100 2 Microscope Tools IR Laser Status Slide2 gt 3 Elements 9 Stage 112 3 37499 9 um Idle Status Bar Info If the camera image does not appear when the program is launched check that all connections to the microscope and also the settings on the microscope have been made correctly In the area of the camera image you will see vari ous markings and information Red crosshairs these mark the exact center of the image A green solid triangle the UV Laser Marker a red square and if you purchased the Double Beam System a green square the IR Las
92. beam 1 or IR laser beam 2 Double Beam Sys tem with two 3D movable IR lasers only you will need to use different key combinations To move the IR laser beam 1 with the key board e Press the Shift key and one of the four arrow keys together The IR laser beam 1 will con tinue to move until you release the key This allows you to move the IR laser beam 1 up down and to the left and right If you press the shift key and two arrow keys simultaneously e g gt you can also move the IR laser beam diagonally To move the IR laser beam 2 with the key board e Press the Ctrl key and one of the four arrow keys together The IR laser beam 2 will con tinue to move until you release the key This allows you to move the IR laser beam 2 up down and to the left and right If you press the shift key and two arrow keys simultaneously e 9 1 gt you can also move the IR laser beam diagonally 127 128 PALM RoboSoftware 3 1 0106 EN General Introductory Remarks 12 Working with Fluorescence Option A supplementary fluorescence unit is available as an option According to the configuration this is suitable for the following applications Configuration Basic non simultaneous appli cation of fluorescence and laser functions Configuration Advanced a simultaneous application of fluorescence and laser functions IS possible For more information about the application and the basic settings re
93. boSoftware 3 1 0106 EN Note Close the shutter chapter 12 6 before switching on the fluorescence lamp and before finishing or interrupting your work This will prevent unintentional exposure of your sample to fluorescence light 12 3 Settings for Working with Fluo rescence Motorized Microscope In order to use the fluorescence illumination you must switch on the fluorescence illumination refer to User Manual select the desired fluorescence filter unit chap ter 12 4 Activating a Fluorescence Settings Record With this the functions Excita tion filter Reflector position Microscope light Timer and Shutter are performed via a single command The basic settings of these functions have been made during installation of your PALM system The PALM RoboSoftware provides you with the capability of saving various combinations of these parameters and retrieving them when they are needed chapter 12 9 Note Close the shutter chapter 12 6 before switch ing on the fluorescence lamp and before finish ing or interrupting your work This will prevent unintentional exposure of your sample to fluo rescence light 129 Activating a Fluorescence Settings Record 12 4 Activating a Fluorescence Set tings Record You can activate a fluorescence settings record as follows FL Laser Texas Red e Open the drop down list FL in Fluorescence Tool and select the desired settings record
94. calibrated at the factory they are an estimated value only P A L M does not guarantee the accuracy of the performed measurements The measuring functions have to be checked and if necessary calibrated by the user for example with a ocular micrometer You can save your elements as a Set or load a pre viously saved set of elements In addition you can save the properties of your elements in a file re load them in another program and process them further For instructions on how to do this refer to chapter 8 Saving and Printing Elements PALM RoboSoftware 3 1 0106 EN If your system includes database functionality and if you are working in Database Mode then your el ements will be saved automatically in a database and can be reloaded from there refer to chapter 1 7 Launching and Exiting the Program and the Information Center manual You can also save extracts of the microscope im age with the superimposed elements in a file for documentation purposes Elements stored in this way cannot be reloaded into the PALM RoboSoft ware For instructions on how to do this refer to chapter 9 3 Saving Images Manually and chapter 9 4 Saving Images Automatically during a Laser Function In order to draw elements you must display the section of your sample on the monitor that you want to process Refer to chapter 5 Positioning the Stage for instructions on how to move the st age in order to display the required
95. con button or click on menu item Export Elements in the File menu The Export Elements window will open The pro posed saving location is the folder selected the last time you used this function e Select a folder to store your elements as neces sary PALM RoboSoftware 3 1 0106 EN e If you have saved your elements previously the name of the file in which they are saved is offered by default If you want to overwrite this file do not change the name in the input field or enter a new name for your element file e Click on the Save button You can now open the file with a text editor and read it print it or edit and save it again To import your elements File fat Import Elements e Click on menu item Import Elements in the File menu in the PALM RoboSoftware main window or in the Element List window The Import Elements window will open e lfnecessary open the folder in which you have saved the file e From the displayed list select the file you want to load and click on the Open button The imported elements are displayed on your monitor Any elements that were already on the monitor will be deleted 85 Printing Elements 8 5 Printing Elements The Print Elements function can be used to ei ther print your elements on a printer or to gener ate an Excel import file To print your elements File e Print Elements e Click on menu item Print Elements
96. correctly if you want to catapult Info If your system is equipped with the PALM RoboMover all settings can be controlled via the software comfortably refer to the Manual for the PALM RoboSoftware If your system Is equipped with the PALM CapMover the positioning of the cap Is car ried out with a joystick refer to the Manual for the PALM CapMover Note Take care that you have fully completed all set tings prior to carrying out any of the laser func tions Incorrectly set parameters can cause your sample to be destroyed Note Note that when a laser function is released only elements that are compatible with the function can be processed For example if you have se lected the Cut laser function only elements of type Figure will be processed if you have selected elements of other types e g Dot these are ignored If you want to process dif ferent element types with the laser you must do this in several separate operations You will need to proceed differently depending on whether all the elements you have drawn provid ed they are compatible with the laser function or only certain of them are to be processed with the laser 108 To select elements for processing with the laser proceed as follows in the Element List window e Open the element list press F5 or use one of the methods explained in chapter 7 3 The Element List Window e Select the required laser function and the num ber o
97. crement in the Speed Delta for faster slower functions field Close this window by clicking on OK 11 8 2 Moving the IR Laser Beam with the Mouse 3D 1 3D 2 Mode In 3D Mode you control the movement of the IR laser beam with the mouse Each mouse action is translated into a movement of the IR laser beam You can perform movements in any direction To switch to 3D Mode 3D 1 3D 2 Move Lal 3D 1 mode iL 30 2 mode e Click on the 3D 1 3D 2 icon button or click on menu item 3D 1 Mode 3D 2 Mode in the Move menu or open the context menu and click on the item 3D 1 Mode 3D 2 Mode Info The cursor is not displayed in 3D 1 3D 2 Mode In 3D 1 3D 2 Mode you can alter the speed of the 3D Laser Beam Positioning and the power of the laser beam using keyboard com mands and also change the focus with the scroll wheel on the mouse Note that you can only change the settings for the laser beam that is selected in Laser Tools Several func tions can also be accessed via the context menu PALM RoboSoftware 3 1 0106 EN To switch between 3D 1 and 3D 2 Mode during a movement e Open the context menu by right clicking the mouse e in context menu click on that 3D Mode to which you want to change To switch from 3D Mode back to Cursor Mode e Click once with the left mouse button 11 8 3 Moving the IR Laser beam via the Keyboard Depending on whether you want to move IR laser
98. ctor Shutter and Light from selection lists PALM RoboSoftware 3 1 0106 EN Editing a Fluorescence Settings Record Fluorescence Entry Ea Cancel Specity the Fluorescence Entry Pr 4 Mame PO Filter dontcae Shutter dontcae e Reflector dontcae e Laser Elo B Light don t care When you create a new Settings record every field is populated by default with don t care This set ting means that no change will be made to the cur rent setting for the respective part when you activate the settings record as described in chapter 12 4 e Enter any name for your settings record in the Name field e Open the drop down lists Filter Shutter Reflector and Light one after another and select the desired menu items Filter g don t care don t care mon E oo Pa mon E Dd Pi You can define the entries of the drop down list Filter on your own refer to chapter 12 8 2 Specifying Names for the Fluorescence Excitation Filters Reflector dontcae e don t care 4 Rhodamine F Texas Red The entries of the drop down list Reflector are prescribed in the control program for the microscope available with the motorized micro scope only shutter p don t care z don t care CLOSE OPEN CLOSE OPEN PALM RoboSoftware 3 1 0106 EN The three setting options for the shutter have the following effect if you ac
99. d Therefore prior to setting the speed first select the magnification that you want to work with Before you initiate a movement of the stage all three setting options are available for all move ment types While the stage is moving you can only change the speed of the Cut movement type using the Speed Tools tool In the case of the other movement types the speed can be changed with keyboard commands Info If you have selected too fast a speed for Cut Mode it may happen that your sample is not cut through completely In this case reduce the cut speed To select the unit for speed setting For the speed setting you can select between two different units percent and um sec Setup PALM Robo e Inthe Setup menu click on menu item PALM Robo The Preferences and Configuration window with the Configuration tab opens e Click in Radio Button Logic units percent or Micrometer sec units um sec in section Metric e Close the window 43 Setting Speed To change the speed using the PALM RoboSpeed window Move Setup Speed Fa e Click on Setup Speed inthe Move menu or press F8 on the keyboard PALM Robo Speed ES e cos Values for Lens 5x kaj Stage Arrow Scroll Position Cut LPC 30 30 Arrow Speed setting for Stage with Lens 5s Ln Sc Speed Delta 1 for faster slower functions The PALM Robo Speed window will op
100. d ob jects in the z axis away from the plane of the slide Thus in combination with PALM MicroBeam you can for example trap objects transport them to the UV laser and process them with it The following chapters will explain how to operate your PALM MicroTweezers together with PALM Ro boSoftware A few of the functions described are not available if you have purchased a Single Beam System and or a system without 3D Laser Beam Positioning Please contact P A L M Microlaser Technologies GmbH to upgrade your system if re quired PALM RoboSoftware 3 1 0106 EN 11 1 Essential Settings Before you can work with your PALM MicroTwee zers System you must make a number of prelim inary settings Optimum results can only be achieved if the initial settings are correctly carried out The settings you must make depend on the configuration of PALM MicroTweezers System that you have purchased You must align the IR Laser Marker in the Dou ble Beam System both IR Laser Markers with the IR laser focus You must set the IR laser power and the IR laser focus If you have a system with 3D Laser Beam Position ing you must also calibrate the 3D Laser Beam Positioning set the speed with which the 3D Laser Beam Positioning is performed In addition for systems with 3D Laser Beam Posi tioning you can define a Reference Position for the IR laser beam The settings for the IR laser power and focus and the speed with
101. d the currently set folder appears underneath in the Folder field e Select the desired folder or create a new folder 88 e Close the Image folder window by clicking on OK You are back in the Images register in the Pref erences and Configuration window e Close the window by clicking on OK To select a folder other than the preset folder or to create a new folder Folder File J New Image Folder e Click on the Folder icon button in the Archive Toolbar or click on menu item New Image Folder in the File menu The Image Folder window will open Image folder Select the image Folder Desktop fy My Documents ES 4a My Computer i 8 My Network Places Folder My Documents New Folder Cancel e If you want to save the image in an existing folder select this in the directory tree and con firm your selection with OK e If you want to create a new folder select the folder in the directory where you want to save this folder click on New Folder and enter a name e Click on OK The window will close The next time you select a save command the folder selected is preset as the save location Info You can also decide not to select or create a new folder until later when you are ready to Save an image PALM RoboSoftware 3 1 0106 EN Saving Images Manually 9 3 Saving Images Manually When images are stored manually one image is stored every
102. e ment List window If you want to select multiple elements keep the Ctrl or Shift keys pressed and click on the corresponding entries successively e Ifyou want to deselect the elements click in an empty line in the table Info With this method you can select groups of ele ments of the same type and color by clicking on the corresponding entry in the table on the Summary tab Depending on the choice made in the Show summary for field refer to chapter 7 3 2 Items Shown on the Summary Tab either all elements or the elements on one slide are shown 68 7 7 Processing Elements You can process and change drawn elements in to tal or in part in various ways you can delete elements or restore deleted elements refer to chapter 7 7 1 edit elements refer to chapter 7 7 2 copy elements refer to chapter 7 7 3 update the numbering of the elements if you have deleted some elements refer to chapter 7 7 4 add comments to the elements or edit existing comments refer to chapter 7 7 5 change the color in which the elements are dis played on the monitor refer to chapter 7 8 3 7 7 1 Deleting Elements and Restoring Deleted Elements You can delete all elements at once or only the last drawn element or one or more selected elements Info Elements that have already been processed by the laser cannot be deleted To delete all current elements ek Delete all unprocessed elemen
103. e interval between individual images and the overall duration The Endless Mode check box only for series of images can be used to specify that images should be captured at a defined time interval until such point as the sequence is broken off manually The Approx Size in MByte fields indicate the amount of storage required to save the video or image sequence with the configured time settings Images are saved in tif format with LZW compression and videos in avi format PALM RoboSoftware 3 1 0106 EN Savin Images and Videos Using the Time Lapse Function The Use auto focus check box can be used to activate a mode in which the focus is read justed for each new image Above all this can be important if the stage is moved whilst the image is being captured and images of different parts of your sample are being stored You should be aware that it takes around 30 seconds to adjust the focus This means that if this function is chosen then it is not an option to have an interval of less than 30 seconds If you check the Switch off transmitted light close transmitted light shutter between picture recording box then in the time between differ ent images being saved the microscope light is switched off and the shutter on the microscope is closed to prevent any light from reaching the sample Please note that it takes around 10 seconds to switch the light off and on again This means that if this fu
104. e whose color matches Color column and type cific types are to be listed Lines Freehands matches Type of Elements column Dots AutoCircles Rulers Texts References etc Area um Area of the elements only ele If you are using a multiple slideholder e g 3 slide ments of type Figure and AutoCircle for el holder you can use the field Show summary for ements of other types an area value has no to specify whether total values are to be shown for meaning only one slide or for all the slides combined Remarks specifies how many elements exist The elements are grouped in the table by color and and of what type in the relevant color element type PALM RoboSoftware 3 1 0106 EN The following summary information for all ele Color color in which the elements are dis ments can be found below the table played on the monitor Type of elements element type figure con tains all elements formed with a line total number of elements the total area of all elements of type Figure in the wider sense and AutoCircle 63 The Element List Window To specify which slide the total values should be shown for only for multiple slideholder Show summary For e Click on the arrow on the right by the Show summary for field on the Summary tab A list containing all the slides will open example shows the 3 slide holder e Click on the name of the slide for which you would like total
105. e Click on the sample in the table at the right onto which the elements are to be transferred Info In the example above only one group of ele ments exists on each sample If you have cre ated several groups they will also appear in the tables Make sure you always choose the correct group e Close this window by clicking on OK PALM RoboSoftware 3 1 0106 EN Element List File Edit Move Laser D HG B g apa e 1 cu Slide Slide2 Slides Summary Show Types Area um Gr ire 11 Freehand Ref 1 138521 sli 12 Freehand Ref 2 128639 sli a 13 Freehand Ref 3 135713 sli 14 Slidez Group 15 Reference Ref 1 sli 16 Reference Ref 2 sli 17 Reference Ref 3 sli 18 Dot sli 19 AutocCircle sli 20 Freehand sli The elements are copied to the target sample At the same time an adaptation occurs The refer ence elements are moved to the respective posi tion on the target sample Any differences in the position and shape of the elements between the two samples will be balanced out Note Check the position of the matched elements if your tissue block contains diagonal struc tures Match the position if required the Shape also of such elements by hand if necessary refer to7 7 2 Editing Elements Now you can process your elements on the target Sample with the laser Info Elements which have changed shape are identified by an asterisk following the ele ment type
106. e Mode with selectable DB e Close this window by clicking on OK 24 3 2 Setting Slideholder PALM RoboStage supports a variety of different in serts slides Petri dishes or capillaries Several windows in the PALM RoboSoftware contain tabs which depict the insert or which provide a separate tab for each slide refer to chapter 6 PALM Navi gator or chapter 7 3 The Element List Win dow The software must be notified about which insert you are using To set the type of slideholder that is being used Setup PALM Robo e Inthe Setup menu click on menu item PALM Robo The Preferences and Configuration window opens The Configuration tab will be displayed Stage Typ Slideholder 3 5lide Holder e Inthe Stage section choose the slideholder you are using Close this window by clicking on OK PALM RoboSoftware 3 1 0106 EN Turning on Automatic Focusing for Elements 3 3 Turning on Automatic Focusing for Elements In addition to the coordinates describing its posi tion on the drawing plane x and y coordinates each element also has a z coordinate This z coor dinate is represented by the focus value of the mo torized microscope When you save elements this Z coordinate is also stored with each element re fer to chapter 8 Saving and Printing Elements for details of how to store elements As you approach elements the focus is automati cally set to the appropri
107. e Re flector Offset With that you move all existing ele ments Note Please note that on adjusting the Reflector Offset all existing elements are really moved not only the elements drawn with fluores cence illumination If you wish to draw ele ments under normal as well as fluorescent lighting you should first check whether the Reflector Offset is set correctly It may be possible that you do not see the struc tures of the elements with normal illumination In this case you have to draw a reference element on a point of your sample that is to be seen with both types of illumination To adjust the Reflector Offset Setup Calibration j New Aeflector Offset e Draw the required elements with fluorescence illumination Draw at least one element on a point that is to be seen with normal illumina tion also Suitable for this is any type of ele ment for example Freehand or Dot refer to chapter 7 2 3 The Freehand Tool or chapter 7 2 7 The Dot Tool This element serves as a reference for the Reflector Offset e Switch off the reflector and the fluorescence illumination e Move the stage so that the reference element and the corresponding point on your sample is displayed on the monitor l Pointer Tool e If itis not yet active select the Pointer Tool in the Graphic Toolbar e Click on menu item New Reflector Offset in the Setup gt Calibration menu PALM RoboSoftware 3 1 0106 EN Adjus
108. e element The num ber and position of the individual laser pulses are then automatically calculated by the PALM RoboSoftware Position of the AutoLPC shots You determine whether the positions of the laser shots are to be arranged horizontally or diagonally PALM RoboSoftware 3 1 0106 EN tions on how to do this 101 Specifying Laser Settings 10 5 1 Energy and Focus There are several options available for setting the laser energy and laser focus with the Laser Tools on the left side of the program window in default configuration with the PALM Robo Laser Settings window which you can open via a menu or keyboard Shortcut using menu commands in the Laser menu using keyboard shortcuts You can combine the settings for Cut and LPC If you change the setting for one laser function then the other laser function is automatically changed to compensate Note The optimal setting for energy and focus de pend on the objective in use on the micro scope The settings that you enter always apply to the magnification that you have set in the program As soon as you have selected another magnification the values that were last used or the factory defaults are automatically applied This also applies to combining the settings for Cut and LPC Therefore prior to setting laser energy and la ser focus first select the magnification that you want to work with To combine and separate laser
109. e image Slide Navigation aa Selected slide 2 e Select in the PALM Navigator window the required slide if necessary Click on the slide or click on one of the arrow keys at the top right side to change from one slide to another e Click on the New Slide button to delete an image which may be present e With the mouse pull up a rectangle that sur rounds the area which you want to scan e Select the Scan tab Displayed on the tab is a monotone area with the same proportions as the rectangle you dragged out 50 Navigation Scan Cellenger Analyze Axiovision Analyze Section Navigation gt Slide Section Save Thumbnail Load Thumbnail Use autofocus suspend scan for focus Draw Elements Draw Marker Elements As soon as you execute the scan command see below the sample will be scanned section by section For this the focus setting will be applied which was valid before the command was sent You can choose whether the focus setting should be corrected for each scanned section automati cally only if you have a motorized microscope with automatic focusing or manual Use autofocus suspend scan for focus e Activate the Use Autofocus checkbox if you have a motorized microscope with automatic focusing that you want to use or activate the suspend scan for focus checkbox if you want to manually readjust the focus for each section to be scanned Info If you readjust the
110. e inserted in the stage the picture shows for example the 3 Slide Holder which can accept three slides Slide 2 is currently selected Note that the type of slideholder must also be set in the PALM RoboSoftware Refer to chap ter 3 2 Setting Slideholder for instructions on how to do this Info If you are working in Database Mode the slides for which you have not yet entered any data refer to chapter 7 1 2 Working in Da tabase Mode are shown shaded To quit PALM Navigator Close e Click on menu command Close in the PALM Navigator window 49 Scanning a Sample with PALM Navigator 6 2 Scanning a Sample with PALM Navigator To scan your sample proof scan e Choose the desired magnification Info The duration of the scan is dependent upon the magnification set on the microscope Set on a high magnification e g 100x the scan operation would take several hours There fore choose a low magnification at first e g 2 5x or 5x In this way you can produce a proof scan within a few minutes Magnification and required area are to be synchronized Note If your system is equipped with a manual mi croscope make sure that you have set the same magnification on the microscope asis in the PALM RoboSoftware refer to chapter 4 2 Setting the Magnification with a motorized microscope the magnification will be auto matically set to that selected in the software e Insert a slide and focus th
111. e same way e Open in the Objective Reflector Con denser or Filter row depending on which setting you want to change the drop down list and select the desired entry Click on one of the arrow buttons on the right side to change to the next or previous setting Info The entries in the lists are made in the control program for the microscope and can only be changed there If only the entry lt none gt is available in a list this means that the corresponding part Is not motorized in your system and therefore cannot be set via PALM RoboSoftware 13 2 2 Setting the Focus You can either adjust the focus almost constantly in small steps of about 1um each or you can de liberately target defined positions Load Position Work Position Check Position If your microscope is equipped with autofocus you can perform the fo cusing automatically These setting options are also available in the MI croscope Tools Additionally in the Microscope Window you can set the speed with which the focusing Is per formed To set the speed of focus setting Focus 6922 9 AF Speed HL al gt e Drag the slider Speed to the desired value To set the focus Focus 6922 9 AF Speed H al e If you want to make small changes click in Focus row on the left or right arrow key to adjust the focus up or down If you want to make extensive changes click on the left or right arrow key and keep the mouse
112. e whi a hak ae he er ie A eRe ee Oe Aol a ee ea aa Pad e ee 60 The Rectangle TOO 2 2343443 da e ba eee Oe hee Ree Bo ee ee eee 60 Te CIRCE TOOL vi a tm we Oe eR a he ee ale eh OS we Eh a e 60 TRe DOE TOOL te apta dar ira Ss ee eRe he ie Uke Bice a Rak ay it aia et dota 60 The AUlOCINGcle TOO as a Re ee eee a ee hee ae we ee dk eee ee 4 61 The Text TOO veias Ae E een bee eed eee ead e eee ee eee ees 61 The Element List WindOWs aa dea 4 06 4 ade aa be hak eae aie he eke oe A ee bee ek ee ee 62 Items Shown on the Slide Tab o es 62 Items Shown on the Summary Tab 0 0 es 63 Change Items SNOWN isai 2a 2s oh ed ORGS a e EO aR Ree ew Be aoe ae 64 Functions in the Element List Window 0 00 aa a es 65 Displaying and Hiding Elements 0 0 0 ee aaa 65 Centering Elements on the Monitor 0 aaa a o e a 66 Selecting and Deselecting Elements 0 a a a es 67 Selecting and Deselecting Elements on the Monitor 0 00 cee ee ee 67 Selecting and Deselecting Elements in the Element List Window 68 Processing EIEMCNIS se saw eee ee eee a ee a Ee eae ead ate eA 68 Deleting Elements and Restoring Deleted ElementS o o 68 Editing ElIEMENtS isis 2284442 A a db A a ee 70 COPYING Elements sr acs a a as e add ara o oe oe 72 Updating the Element NUMberiNQ o e es 73 Adding a Name or a Comment to Elements
113. e window The window will close the changed entry will appear in the table of the Fluorescence window e Close this window as well by clicking on OK The window will be closed your changed entry will be saved 12 9 3 Deleting a Settings Record e Inthe Setup menu click on menu item Fluorescence This opens the Fluorescence window e Click on the row in the table containing the set tings record that you want to delete Delete e Click on Delete The selected settings record will be deleted e Close the Fluorescence window by clicking on OK 12 9 4 Changing the Sequence of Entries in the List e Inthe Setup menu click on menu item Fluorescence This opens the Fluorescence window t Click on the row in the table that contains the record you want to move up or down Click on the Up or Down button depending on the direction in which you want to move the entry The entry will be moved all entries will be renum bered 136 12 10 Adjusting the Reflector Offset If you have set or changed a reflector for fluores cence the microscope image on the monitor may appear to be shifted slightly although the stage has not been moved If you now draw elements and then change the reflector again your elements will not be positioned correctly The automatic laser functions will then not process exactly the areas you wish You must compensate for this by setting th
114. e you must move the stage with the mouse or the arrow keys PALM RoboSoftware 3 1 0106 EN Preparations 10 2 Preparations Before you can use automatic laser functions you must ascertain various parameters The laser settings energy focus dot density with AutoLPC etc Refer to chapter 10 5 Specifying Laser Settings for instructions on how to do this It is recommended that you experiment in order to ascertain which settings best suit your working requirements Chapter 10 6 Finding Suitable Laser Settings gives you instructions on finding the optimal values The moving speed of the microscope stage dur ing the operation of the laser function Refer to chapter 5 Positioning the Stage for instruc tions on how to set the speeds for moving the microscope stage Setting the required laser function Refer to chapter 10 3 Selection of Automatic Laser Functions for instructions on how to do this The areas to be cut and catapulted elements For instructions on how to do this refer to chapter 7 Drawing and Measuring and chap ter 10 7 Releasing the Automatic Laser Func tions If you release the laser with the footswitch you do not need to have drawn any elements Note Do not process your sample or at least the ar eas Important for your work until you have as certained your optimal setting by experiment Application of the laser with unfavorable set tings can cause irreparable destruction t
115. ear on the left hand side of the window Info The parameters that you can set here depend on which set of rules you have chosen As a rule one does not know at first which param eter settings will deliver the optimum analysis re sult e Configure initial values for the parameters e Click on the Test button PALM RoboSoftware 3 1 0106 EN Delete elements Analyze ROI 2 Elements found in 13 sec The parameter settings are tested with the current microscope image The result is displayed below the buttons on the right hand side of the window e Optimize the parameter values if necessary If you have found the optimum values e Click on Analyze ROI button ROI Region of interest Your sample is analyzed 4 Elements found in O sec Evaluate 5 of 63 53 Analyzing Images Using the AxioVision Software 6 5 Analyzing I mages Using the Axio Vision Software The AxioVision software enables images of sam ples to be analyzed using scripts that have been defined in advance and also allows elements to be generated This helps with routine operations to automate the selection of the areas of interest Info You can only avail yourself of this analytical facility if your system includes the AxioVi sion software Please contact P A L M Micro laser Technologies GmbH if you wish to upgrade your system e Scan the section of your sample that you wish to analyze as described in chapter
116. ected beam in the same way Drag the slider or click on the arrow buttons or click and hold the mouse button down until the required value is displayed in the field between the slider and the arrow button or click in the text box enter the required value and press Enter Close the window by clicking on OK PALM Robo IR Laser Settings Cancel Values for Lens Ex IR 1R 2 9 IR 1 2 Power R142 Focus A Power 108 IR Balance En 2 gt PALM RoboSoftware 3 1 0106 EN Setting IR Laser Power and Focus PALM Robo IR Laser Settings Cancel Values for Lens 5x IRA IR 2 IR 142 Power 1R 1 2 Focus IR 1 lt gt 1R 2 IR Focuz 1 IR Focus 2 Focus 100 rj Simultaneous 10C z BJ oT 32 32 Y kE If you want to work with both IR laser beams e Select tab IR 1 2 Power e Use the sliders or the arrow keys to set the power and balance or click in the text box and enter the desired value e Select tab IR 1 2 Focus e Use the sliders or the arrow keys to set the focus of both IR laser beams or click in the text box enter the desired value and press Enter e Close the window by clicking on OK Info You can only adjust the focus values for both beams independently if you have deactivated the coupled settings option If coupling is ac tive any change made to one beam will auto matically be applied to the other Enabling and disabling the coupled feature was
117. ection device under the microscope For this purpose PALM RoboStage has a notch through which the microscope objec tive can be moved upwards The corresponding position of the stage is referred to as the Check Point You can automatically target the Check Point with a menu command Caution Set the objective to the lowest possible posi tion before you move the stage to or from the Check Point If the objective is set too high the stage will collide with it This can cause serious damage to the objective and to your sample if you have purchased a system with motorized microscope the objective is moved automati cally To move the stage to the Check Point Check Point Move 2 Goto Check Point Ctrl k e If you do not have a motorized microscope Move the objective down as far as possible e Click on the Check Point icon button in the Tool Bar or press key combination Ctrl K or Click on menu item Goto Check Point in the Move menu The stage is moved so that the Check Point is po sitioned in the center of the monitor Note To avoid damage to the objective or to the Sample Move the stage back again to the Original position after observing the cap For this proceed solely as described below PALM RoboSoftware 3 1 0106 EN The Check Point To return the stage to its original position Check Point w Move a Goto Check Point Chrl k e If you do not have a motorized microscope Move the
118. ed eye Move the microscope stage to test with the Scroll func tion refer also to chapter 5 1 2 Positioning the Stage with the Mouse Scroll Change the speed during movement in order to find an appropriate value Take a note of the value established and enter it as speed for the move ment type Cut e Draw an arbitrary Line image for example a Spiral or a staggering line The longer the line is the longer you have the opportunity of con trolling and possibly changing the laser settings during the cutting operation e Set the laser function Cut and select the num ber of operations to 1 refer to chapter 10 3 Selection of Automatic Laser Functions for Instructions on how to proceed e Ifyou have drawn one or more elements open the Element List window press F5 for exam ple and select the element to be cut with a mouse click If you have only drawn one ele ment then that one is selected automatically e Start the automatic laser function press F11 or release the laser using one of the other meth ods explained in chapter 10 7 Releasing the Automatic Laser Functions The stage is moved below the laser the laser is re leased e f necessary increase the energy until the laser achieves a cutting effect You will get a cut line in the sample This line will Initially be unclear and wide or the sample will only be cut incompletely as a result of an inadequate setting of the laser focus and energy You
119. ed or loaded any settings which means that you are working with the factory defaults this field is empty P 4 Elements The fourth field shows either the number of the drawn elements or the number of the element presently positioned at the center of the image x of and the number of all existing ele ments of y If you double click on the field the Element List window will open Chapter 7 3 The Ele ment List Window describes how to use this window 14 Te Idle The fifth field shows the current program mode Cursor Mode Stage Mode Scrolling Calibra tion Laser Point Continuous Position Laser ON 3D 1 3D 2 If you are just in the process of drawing or editing an element its current size is indicated If no action is currently in progress then Idle is displayed Stage 101175 6 47235 4 um The field on the right shows the coordinates of the unit currently being moved or most recently moved Stage current coordinates relative to the point of ori gin of the drawing plane x y When the stage IS actually moving the green ball in this field changes to yellow The point of origin of the drawing plane is per manently preset refer to chapter 5 4 The Ref erence Position of the Stage 3D Offset Coordinates of the laser focus point relative to the point of origin of the 3D adjustment You can change the point of origin of the 3D adjustment RoboMover Coordinates of the RoboMo
120. elements with one magnifica tion and then select another magnification a dif ferent objective the microscope image on the monitor may appear to be shifted slightly al though the stage has not been moved Your elements are no longer in the correct position The automatic laser functions will then not process ex actly the areas you wish You must compensate for this by setting the Lens Offset Note These differences between the objectives have in the case of those supplied and those marked with the P A L M Logo been compensated by a factory default setting of the Lens Offset You must only compensate the Lens Offset if a shifting of the elements occurs when altering the magnification Info If you did not draw an element before changing the objective you do not need to define the Lens Offset However if you still select this function you will receive the following mes sage You must draw some Figures for defin ing the Lens Offset Click on OK as necessary to confirm this message To adjust the Lens Offset Setup Calibration j New Lens Offeet e Move the stage so that at least one element is displayed on the monitor l Pointer Tool e If itis not yet active select the Pointer Tool in the Graphic Toolbar e Click on menu item New Lens Offset in the Setup gt Calibration menu All available elements are automatically selected and surrounded by a selection rectangle the se lection rectangle
121. en There are tabs located in the bottom part of the window that you can use to set the speed for the various movement types the last two tabs are used for setting the speed of the 3D Laser Beam Positioning for the IR laser refer to chapter 11 8 1 Setting the Speed of the 3D Laser Beam Positioning Set speeds for all movement types in the same way e Select the tab of the movement type for which you want to change the speed e Enter the desired value with the slider or Enter the desired value in the Actual Speed field e Close this window by clicking on OK 44 To change the speed of the Speed Tools tool e Open the Speed Tools tool if it is not displayed click on the menu item Speed Tools in View gt Menu or press key combination Ctrl E e Click on the arrow beside the field under Selected Speed A list of the different movement types will open e Select the movement type for which you want to change the speed Movement types 3D and 3DArrow will only ap pear if you have purchased the PALM CombiSys tem with 3D Laser Beam Positioning They are used to set the speed of the 3D Laser Beam Posi tioning for the IR laser refer to chapter 11 8 1 Setting the Speed of the 3D Laser Beam Positioning Seral umrzec 3390 07 alo The Speed Tool for the selected movement type is shown in the example shown above Scroll The maximum selectable speed is dependent upon the chosen mo
122. er a program for automatic detection of structures on your sample is desig nated here as image processing software To launch the I mage Processing Software IE Image Processing File VE Open Image Processing e Click on menu item Open Image Processing in the File menu of PALM RoboSoftware or click on the Image Processing icon button If the Image Processing icon button is not dis played follow the instructions in chapter 2 2 Cus tomizing the User Interface to switch on this display Info If you have decided to purchase a system without Imaging Software the symbol de scribed above does not appear in your PALM RoboSoftware and the menu command Is in active The Image Processing Software will be launched Instructions for working with this program are the Subject of the program s user manual 12 PALM RoboSoftware 3 1 0106 EN The PALM RoboSoftware Layout 2 Program Layout and Operation Tool Bar sa PALM Robo File Edit View Move Laser Laser MaXer Setup Devices Help Laser Tool Mh PW E Me eSR G es oD WAS OO S Laser Sey Footer C 4 ZERO SARPAJSIOO oO BSE Ya 44 E IA IA A LI IA oe For Help press F1 Graphic Tools Color Palette 2 1 The PALM RoboSoftware Layout When the program has been launched the main window of the PALM RoboSoftware as depicted above will appear showing the camera image the Menu Bar various icon
123. er Markers The Laser Markers indicate the target for the laser You will see a dotted line rectangle on the bor der of the image this is the Scroll Rectangle 13 The PALM RoboSoftware Layout At the bottom on the left a scale bar and Copy right Note if specified are displayed chapter 9 1 Inserting a Copyright Note explains the procedure Info The crosshairs the scroll rectangle the scale bar and the Copyright Note and also the inclu sion and appearance of the Laser Markers can all be turned on off or modified as explained in chapter 2 2 Customizing the User Interface The Status Bar at the bottom of the program win dow provides you with information on a number of current settings Some of the fields feature a win dow that opens when the field is double clicked For Help press F1 If you double click on the field the PALM Robo Information window will open refer to chapter 2 4 5 PALM Robo Information Slide The second field indicates which slide is being Shown on the monitor this only applies when you are using a slide holder that accommodates more than one slide or a message Is displayed whilst elements are being calculated by the PALM RoboSoftware If you double click on the field the Navigator window will open refer to chapter 6 PALM Navigator EDok set The third field shows the name of the current active setup file file extension set If you have not sav
124. er 5 1 1 Positioning the Stage with the Mouse Stage Mode explains how to switch into Stage Mode Info If you switch from Stage Mode to 3D Mode or vice versa the IR laser will be switched off if you have switched the laser on using the PALM RoboSoftware On the other hand the laser remains on if you switched it on using the footswitch in the Hold position refer to chapter 11 5 Set tings for the Footswitch and are continuing to keep the footswitch depressed The laser likewise remains on if you switched it on with the footswitch in the Switch posi tion refer to chapter 11 5 Settings for the Footswitch To activate and deactivate the IR laser with PALM RoboSoftware Laser IR Lager onloff e Click on the IR Laser on off entry in the Laser menu or click on the IR button on the right border of the program window As soon as you activate the IR laser the gray dot beside the button will turn to orange Note Particularly living objects may suffer injury due to the radiation of the IR laser Therefore you should not activate the IR laser until you actually need to use it and keep it activated only as while you are working with it Info You can also switch off the IR laser by clicking the Stop icon button on the Tool Bar However this results not only in the IR laser being switched off all other processes cur rently running will also be stopped The Stop icon button should there
125. er function by an amount that you can set Note that the cutting effect of the laser is not only dependent on the energy and focus settings but also on the speed at which the stage is moved The slower you move the stage with a fixed energy set ting and defined focus the greater is the effect of the laser since it works with a fixed pulse frequen cy This means that there are three parameters to set that reciprocally influence each other Work up to the optimal settings successively Essentially there are two options for ascertaining the optimal laser settings for the cutting function without using the automatic laser functions Enter Stage Mode move the stage with the mouse release the laser with the footswitch refer also to chapter 10 10 Cutting and Cata pulting without Automatic Laser Functions and change energy and focus with keyboard com mands The advantage of this method is that you can cut and test various settings at will within cer tain limits You should try to move the stage more slowly if possible so that you can evalu ate the cutting effect during the movement 106 with the automatic laser functions You draw an element cut along this line with the automatic Cut function and change energy focus and speed during the cutting operation by using keyboard settings The advantage of this method is that you do not have to work with the footswitch mouse and keyboard at the same time but with t
126. es and Configuration window 25 Saving and Loading Settings To save your settings automatically when exiting the program Info The PALM RoboSoftware essentially saves the settings which are valid on exiting the pro gram These settings are automatically load ed at the next start and remain valid until you change them or load a previously saved set tings file The function described here saves your set tings in a file that can be reloaded any time even if you have changed the settings in the meantime see note below Saving Settings Folder E Documents and Settings adminiMy Docume W Save Settings on program exit e To toggle this function off or on tick the Save settings on program exit check box in the Saving settings section of the Configuration tab in the Preferences and Configuration win dow A tick in the checkbox indicates that the function is enabled Note The default setting Save Settings on pro gram exit is effective only if you have saved your settings once already during your work or have loaded a settings file If you subsequently change the settings they will be saved automatically to the current settings file when you exit the program The name of the current settings file appears in the Status Bar The current file will also be overwritten To save your settings Setup Save Settings e Click on Save Settings inthe Setup menu The Save
127. esig nations for the second beam are enclosed in square holders The 3D Laser Beam Positioning also works when the IR laser beam is deactivated This means that you do not have to switch the IR laser on to move the beam to a different site There are two ways to move the IR laser beam with the mouse with the arrow keys You can vary the speeds for both modes of move ment 11 8 1 Setting the Speed of the 3D Laser Beam Positioning You can set the speed separately for every type of IR laser beam movement 3D Speed of movement in 3D Mode 3DArrow Speed when moving with arrow keys If you have installed a system with two 3D mov able IR laser beams the speed values set apply to both beams For the speed setting you can select between the units um sec and Percent You can save the speed settings together with oth er parameters in a settings file Refer to chapter 3 4 Saving and Loading Settings for instructions on how to do this You have the following options for setting the movement speed of the IR laser beam via the PALM Robo Speed window which you open with a menu command or a keyboard Shortcut with the Speed Tools tool with keyboard or menu commands Speed slower Speed faster For speed changes entered with keyboard or menu commands you can define an increment Every time the corresponding keyboard or menu command is entered the speed is increased or decreased by this value
128. evel of magnification it is there fore possible that text elements may cover areas that are significant for you In this case you can turn off the text elements other ele ments will continue to be displayed Info You may for instance use text elements to mark the area on the sample that you wish to scan In this way the section of interest can be found more easily on the overview scan To save the scanned image Save Thumbnail Click on Save Thumbnail button The Save scanned picture window opens Choose the file format in which you want to save the image the formats JPEG TIFF and Windows Bitmap are available Choose the folder in which you want to save your image Enter a name for the image Click on the Save button To load the scanned image Load Thumbnail Click on Load Thumbnail button The Open scanned picture window opens Choose the file format you used to save the scanned image Select the image you want Click on the Open button The image is loaded and displayed 52 6 3 Positioning the Microscope Stage If not already done scan your sample as described in chapter 6 2 Scanning a Sample with PALM Navigator Navigation Scan Cellenger Analyze Axiovision Analyze Slide Navigation aR Selected slide 2 M Draw Elements Draw Marker Elements With a mouse click or with the arrow button select the slide which you want to observe Click on the sp
129. exactly the Same way as with a new scan The Navigator window can be resized If the win dow is resized the scanned image is likewise in creased or reduced in size You can set a parameter to specify that your ele ments will appear on the scans If your system includes PALM RoboStage Typ II and you employ the corresponding slideholder you can produce scans of several samples and change between them in the display If your system includes the Cellenger or AxioVi sion software you can transfer the scanned sec tion directly to these programs for analysis To facilitate analysis the Life Image window en ables you to modify various parameters for the im age contrast brightness saturation color gamma value These facilities are described in chapters 6 4 Analyzing Images Using the Cellenger Software 6 5 Analyzing Images Us ing the AxioVision Software and 3 9 Set Image Parameters PALM RoboSoftware 3 1 0106 EN 6 1 Start and Quit PALM Navigator To start PALM Navigator View f Navigator window F4 e Click on menu item Navigator Window in the View menu Navigation Scan Cellenger Analyze Axiovision Analyze Slide Navigation E M Draw Elements M Draw Marker Elements The PALM Navigator window opens e Select the Navigation tab if not already selected An image of your slideholder appears Info The exact appearance is dependent upon the type of slideholder you hav
130. exas Red Select an Entry and DoubleClick to Change 12 9 1 Creating a New Settings Record Setup Fluorescence e Inthe Setup menu click on menu item Fluorescence This opens the Fluorescence window This con tains a table and a number of buttons The table lists your saved settings including the sequential number and name as well as the settings made for the Filter Reflector Shutter and Light parameters The sequential number is increment ed by one automatically for each new entry that you create The buttons are used for entering and editing the settings records Change Insert e Inthe table double click in the row with the entry new or click once in the row with the new entry and then click on the Change button or Click on the Insert button 134 A Texas A Yes default es es Info A new entry is always added at the end of the list over the new row If you have already created entries you can select one of the ex isting entries with a click of the mouse If you then create a new record using the Insert button a new record will be inserted above the marked record You can change the order of the records as will be explained later This opens the Fluorescence Entry window This contains several fields in which you can enter a name for your new settings record or in which you can select settings for the parameters Filter Refle
131. f operations as explained in chapter 10 3 Selection of Automatic Laser Functions If you wish to process individual elements with the laser e Select the element or elements you want to process click on the desired element To select multiple elements hold the Ctrl key down as you click on your selection If you want to process all ele ments of one type or one color with the laser click on the corresponding line in the table on the Summary tab If you wish to process all elements that have not yet been processed with the laser e Cancel the element selection if elements have been selected If you wish to process all elements with the laser including those that have already been processed with the laser e Select all elements To select elements for processing with the laser proceed as follows in the PALM RoboSoftware main window Laser selected elements Ctl F11 e Select the required laser function and the num ber of operations as explained in chapter 10 3 Selection of Automatic Laser Functions e Select the elements for processing with the laser in the main window e Open the context menu by right clicking the mouse e Click on Laser selected elements in the con text menu PALM RoboSoftware 3 1 0106 EN Releasing the Automatic Laser Functions To release an automatic laser function There are several options available for releasing the laser via a menu command in the main window in the P
132. f the elements refer to chapter 10 3 Selection of Automatic Laser Functions for instructions on how to proceed Otherwise you will not obtain the desired result P Ctrl key Freehand Tool e Select the Freehand Tool e Press and hold the Ctrl key e Move the cursor to the area you want to edit The nearest anchor point of the element is indicat ed by a rectangle with black lines e Keeping the mouse button depressed draw the changed line As soon as you have reached the element again the nearest anchor point is displayed as a rectan gle with black lines and the color of the element e First release the mouse button then the Ctrl key 71 Processing Elements The new drawn line is joined with the displayed an chor point see above The previous line between start and end dot of the correction is deleted Note If you first release the Ctrl key and then the mouse button the element between the start dot of your correction and the end dot of the el ement is removed see below To stretch elements lines rectangles ellipses AutoCircle la Pointer Tool e Select the element you want to stretch e Drag the Pointer on one of the squares at the corners or sides of the selection rectangle The cursor changes to a double headed arrow e Pull with depressed mouse button The selected element is stretched The square op posite to the selected square serves as a bench mark Info The original pr
133. fer to chapter 4 4 Calibrating the Movement of the Stage To measure the length of lines Ruler l Quadratic Attribute e Click on the Ruler icon button in the Graphic Toolbar or If you select Quadratic Attribute as well the drawing function is limited to horizontal and vertical lines The cursor changes to a white arrow e Click on the start dot of the line whose length you want to determine and keep the mouse button depressed e With the mouse button depressed move the cursor to the end dot of the line to be measured and release the mouse button The line will be displayed on the monitor as a line with a display of its length in um 80 To show and hide area sizes Element areas a e Click on the Areas icon button in the Graphic Toolbar Area sizes of all drawn Figure and AutoCircle type elements will be shown in um At the same time the areas measured will be displayed with a transparent color marking e To hide the area display click again on the Areas icon button in the Graphic Toolbar Note If elements are not drawn closed the start and end points of the elements are connected by a straight line for calculating the area Info Only the area of elements of type Figure and AutoCircle is indicated The displayed size of Dot type elements has no relation to the di ameter of the laser beam or the size of the cat apulted area the display of an area size for these ele
134. fer to the User Manual 12 1 General Introductory Remarks The procedure to use for working with fluores cence illumination depends on the way your sys tem is configured The differences concern the manipulation of the microscope motorized or manual version and the fluorescence shutter or filter wheel respectively If your system Is equipped with a motorized micro scope you can control all the elements that you need to adjust specifically in order to use the fluo rescence illumination via PALM RoboSoftware When using a manual microscope you have to make the settings on the microscope manually the functions of the filter wheel and the shutter are controlled by the PALM RoboSoftware In other words you can select the desired excitation filter or close and open the shutter via a program com mand 12 2 Settings for Working with Fluo rescence Manual Microscope In order to use the fluorescence illumination you must switch on the fluorescence illumination refer to User Manual select the desired fluorescence excitation filter necessary when using a filter wheel only refer to chapter 12 8 3 swivel in the desired reflector position in the microscope refer to User Manual switch the microscope light on or off as neces Sary activate the timer if you want to preset a time for which the excitation light should illuminate your sample refer to chapter 12 7 and open the shutter chapter 12 6 PALM Ro
135. ferent objectives To set a Laser Marker position correctly you must release the laser and determine its actual focus point Then you must set the associated Laser Marker to the focus point of the laser In order for you to be able to check the position of a Laser Marker and adjust it if necessary the Laser Marker must be displayed e Verify that the Laser Marker whose position you want to check is displayed and if necessary enable the display click on the View Laser Marker item in the Laser Marker menu a submenu will open Here you select the Laser Marker you want to check PALM RoboSoftware 3 1 0106 EN Setting the Laser Marker To determine the focus point of the UV laser e Using any of the methods described in chapter 5 Positioning the Stage move the stage so that an area of your sample which you do not require for your work appears in the middle of the monitor or insert a membrane coated slide a Stage e Activate Stage Mode if not already active click on the Stage Mode button in the Tool Bar or press F7 e Release the laser with a single short application of pressure to the footswitch e Check the result You should see a small almost circular area where material has been removed This is most easily visible on the membrane Info In some circumstances you will not see any ef fect from laser activation or there will be a large hole in your sample In this case check that there is
136. focus for each section to be scanned you will get a far sharper overall result However the duration of the scanning operation will increase considerably e Click on the Scan Slide button PALM RoboSoftware 3 1 0106 EN Scanning a Sample with PALM Navigator Navigator Navigation Scan Cellenger Analyze Axiovision Analyze Section Navigation gt Slide Section Scanned with 5x lens Save Thumbnail Load Thumbnail Use autofocus suspend scan for focus Draw Elements Draw Marker Elements The selected section will be scanned You can fol low the progress in the window Slide Section will be displayed at the upper right of the window To scan sections of the proof scan e With the mouse pull up a rectangle that sur rounds the area in the proof scan which you want to scan e Click on the Rescan button The selected section will be scanned At the upper right of the window Section 1 appears Navigation Scan Cellenger Analyze Axiovision Analyze Section Navigation gt Slide Section EP Scanned with 5x lens Save Thumbnail Load Thumbnail Use autofocus suspend scan for focus Draw Elements Draw Marker Elements Info You can pull up a frame in the scan of a sec tion in order to scan the area once more The previously scanned section will then be lost PALM RoboSoftware 3 1 0106 EN To interrupt scan progress
137. fore only be used to switch off the laser in the event of an emergency 124 11 7 Moving Objects in the Z Axis Regardless of the configuration of your PALM MicroTweezers you can lift trapped objects in the Z axis away from the plane of the slide by altering the focus of the IR laser beam The object will re main trapped in the plane of the IR laser focus It is simpler to alter the IR laser focus with the scroll wheel on the mouse Chapter 11 2 Setting IR Laser Power and Focus includes an explanation of your options for displacing the IR laser focus If you have purchase a PALM MicroTweezers with 3D Laser Beam Positioning you can also move trapped objects in the x and y axes i e parallel to the plane of the slide please contact P A L M Microlaser Technologies GmbH if you wish to up grade your system PALM RoboSoftware 3 1 0106 EN The 3D Laser Beam Positioning 11 8 The 3D Laser Beam Positioning Using the 3D Laser Beam Positioning function you can move trapped objects in both the x and y axes i e parallel to the plane of the slide The following section provides instructions for working with a 3D movable IR laser beam If you have purchased a system with two 3D movable IR laser beams you will use the same methods for working with both beams You will simply need to operate the corresponding icon buttons and menu commands for the 3D Laser Beam Positioning of the second beam as well In the following d
138. function Stopping a running laser function Info All the functions listed are available via menu commands or icon buttons in the PALM Ro boSoftware main window Info Even when the Element List window Is open you can still access all the functions that are of fered in the PALM RoboSoftware main window This means that you do not have to close the Element List to continue working PALM RoboSoftware 3 1 0106 EN 7 4 Displaying and Hiding Elements All elements are displayed in the default configu ration of PALM RoboSoftware You can hide the el ements To display or hide elements cA Graphic on off View E Show Elements e Click on the Graphic on off icon in the Graphic Toolbar or e Click on menu item Show Elements in the View menu Both commands work as a toggle Info If the display has been switched off then it is switched back on automatically when new ele ments are drawn 65 Centering Elements on the Monitor 7 5 Centering Elements on the Monitor The image on the monitor only represents a sec tion of your sample If you have drawn multiple el ements usually not all of these are visible on your monitor at the same time If you want to view a particular element you can either find it by mov ing the stage or you can have PALM RoboSoftware display it for you automatically at the center of the image Certain elements can be centered automatically using icon buttons or menu
139. ge as the point of origin for calculating the coordinates of your elements With each start of the controller the stage is mo ved to the end positions in x and y direction This position is defined as a reference point automati cally The Reference Position is retained for as long as the controller unit remains switched on and even if you shut down and restart the PALM Robo Software RoboLPC With RoboLPC an object is detached and catapulted into a cap in a single operation only possible with membrane mounted samples The drawn line is completely closed Then the laser first cuts along the line but leaves a narrow joint The width of this joint can be preselected in the LPC Parameters window The joint is displayed on the monitor with an LPC dot Finally the sample is catapulted from here Single Beam System The PALM MicroTweezers is available as a single beam or a double beam system The single beam system configuration is equipped with one IR laser beam and may optionally include 3D Laser Beam Positioning Work Position Freely definable focus setting of the microscope to set by amenu command motorized microscope only The setting should be defined so that the Sample is displayed sharply on the monitor or in the ocular PALM RoboSoftware 3 1 0106 EN Appendix B Keyboard Shortcuts Keyboard Shortcuts F1 F2 F3 Alt F4 F4 F5 F6 F7 F8 F9 F10 FLL Ctrl F11 Ctrl A Ctrl
140. h one mouse click you can freely center positions on the monitor Refer to chapter 6 3 Positioning the Microscope Stage for instructions on work ing with this program 41 Stopping the Stage in an Emergency 5 2 Stopping the Stage in an Emergency You can stop the movement of the stage at any time for example in an emergency or in the event of a malfunction An icon button and a menu com mand are provided so that you can stop the move ment in both the main window of PALM RoboSoftware refer to chapter 2 Program Layout and Operation and in the Element List window refer to chapter 7 3 The Element List Win dow To stop all current movements with one key board shortcut e Press Esc on the keyboard To stop all current movement in main window D Stop Move Em Stop ESC e Click on the Stop icon button in the Tool Bar or click on the Stop entry in the Move menu or open the context menu by right clicking the mouse and then click on menu item Stop To stop all current movement in the Element List window D Stop e Click on the Stop icon button in the Icon Bar Info You can still use the icon button and the menu command when the Element List window is open 42 All current operations are stopped immediately Repair the malfunction if necessary Note The Stop function stops all current opera tions i e any UV laser functions that happen to be running The Conti
141. h to resume work on an existing ex periment or with an existing sample you can do this using the selection list for the fields The more button provides access to further fields for entering supplementary details e Click on the more button The Collection Device Data Details window opens Collection Device Data Details Label of Collector Description of collector Label of collector type Description of collector type Cancel e Enter the details you wish to record then close the window by clicking the OK button The Please select or enter data window will be displayed again e Click on OK The window will close Now you can draw ele ments Info Slide positions for which you have entered in formation are represented in the Navigator as white areas Positions for which no data has been entered are shown shaded see illustra tion in chapter 6 1 Start and Quit PALM Navi gator 58 7 2 Drawing New Elements PALM RoboSoftware provides a range of tools for drawing elements Freehand Line Rectangle and Circle Tool With these tools you can draw shapes along which you can later cut with the Cut laser function or the area content of which you can catapult later with the AutoLPC or RoboLPC auto matic laser functions Elements drawn using these tools are collectively designated ele ments of type Figure Dot Tool Use the Dot Tool to mark individual cells for subse
142. he key board alone However you will probably have to draw and cut several elements until you have found your optimal settings With this method you can also set higher speeds but only evaluate the cut after conclud ing the automatic laser function Apart from that you can also find settings for multiple cutting processes with this method since the laser cuts along exactly the same line during each operation In order to find the optimal values for the catapult ing functions you must draw appropriate ele ments and release the required automatic catapult functions LPC AutoLPC RoboLPC or AutoCircle with varying settings of the LPC energy and LPC fo cus Disable the combination of Cut and LPC Energy as well as Cut and LPC Focus in order to be able to perform independent settings for these functions Note The optimal settings for energy and focus also depend on the objective in use on the micro scope The settings that you enter always apply to the magnification that you have set in the program As soon as you have selected another magnification the values that were last used or the factory defaults are automatically applied Therefore prior to setting laser energy and la ser focus first select the magnification that you want to work with To find the optimal laser settings for the cut ting functions Info To ascertain your optimal laser settings you have to know how you can set the laser focus t
143. he laser energy and the speed of the stage for the different types of movement If necessary refer to chapter 10 5 Specifying Laser Set tings or chapter 5 Positioning the Stage or to the User Manual for instructions on how to pro ceed e Insert a sample that has the same qualities as the sample that you want to examine select the required objective and adjust it into focus e Select the magnification set on the microscope in the Lens menu of PALM RoboSoftware e Switch on the display for Laser Tools and Speed Tools if these have been hidden if necessary PALM RoboSoftware 3 1 0106 EN Finding Suitable Laser Settings click on the entries Laser Tools and Speed Tools in the View gt Menu or press the key board combination Ctrl L or Ctrl E e Set the laser focus and laser energy at median values 50 50 or start with factory default set tings if necessary refer to chapter 3 5 Restor ing Settings for instructions on how to reset the Factory Defaults Note If you reset to factory defaults not only laser energy and laser focus are reset but also all other settings that you have made refer also to chapter 3 Settings The further procedure depends upon whether or not you want to use the automatic laser functions to find appropriate laser settings a Using the Automatic Laser Functions e Set the Scroll function for the microscope Stage speed so that you can easily follow the movement with the nak
144. he same positions at which you drew them To work with saved elements you need only insert the respec tive sample into the slideholder and reload your el ements Note Make sure that you reinsert the slide exactly as you had it inserted during previous pro cessing i e not tilted or even rotated 180 Info It may also happen that the drawn elements shift after you change objectives If the optical axes of both objectives do not match exactly the microscope image on the monitor will be displayed as slightly offset In this case you will have to redefine the Lens Offset Refer to chapter 4 7 Adjusting the Lens Offset for in structions on how to do this 46 To set the reference position of the stage Normally you will not have to determine the Ref erence Position yourself If however a disruption should occur e g you may have moved the stage unintentionally by hand you can reset the Refer ence Position of the stage Move Reference the Stage e Click on menu item Reference the Stage in the Move menu Note Note that the Reference Position for the stage has no relationship to the definition of a Ref erence Position for the 3D variable IR laser beam refer to chapter 11 4 Defining a Ref erence Position PALM RoboSoftware 3 1 0106 EN 5 5 The Check Point When you catapult samples into a collection device using the automatic laser functions you can then observe the contents of the coll
145. hich the laser strikes the sample to catapult the cut area in the automatic AutoCircle function AutoLPC Dot Color of the dots the laser beam touches in the automatic function AutoLPC 75 Changing Colors and Line Thickness 76 RoboLPC Line If an open element is envi sioned for the RoboLPC laser function but the distance between the two finish dots is greater than the value specified in Distance of RoboLPC joint refer also to chapter 10 5 Specifying Laser Settings the distance is automatically shortened to the preset value This automatically determined line is dis played in the color selected here Lines that close open elements are also dis played in this color if the functions Close amp Cut or Close amp Cut AutoLPC are selected RoboLPC Dot Color of the dot the laser beam touches with the automatic laser func tion RoboLPC in order to catapult the de tached area Ruler Line Color of the lines for measuring refer also to chapter 7 10 Measuring Text Field Background color of text ele ments the text itself is always black Number Field Background color of the field with the element number and where appli cable the comment number and comment are always black Select the desired color from the Color Palette in the right window pane Proceed using the same method for all ele ments whose color you want to change Close this window by clicking on OK 7 8 2
146. hich you begin your drawing becomes a corner of a perceived rectangle enclosing the circle or the ellipse e Select the Circle Tool in the Graphic Toolbar If you select Quadratic Attribute as well the drawing function is limited to circles If you select Centric Attribute as well the dot at which you begin your drawing becomes the center point of the ellipse or the circle e Move the cursor to the dot where you want to begin your drawing e With the mouse button pressed down drag the oval or circle out until it is the desired size and then release the mouse button 7 2 7 The Dot Tool Besa a ot Too Use the Dot Tool to mark for example individual cells that are to be catapulted with the AutoLPC automatic laser function e Select the Dot Tool in the Graphic Toolbar e Click roughly in the middle of the cell you later want to catapult The selected location is displayed as a dot PALM RoboSoftware 3 1 0106 EN Drawing New Elements 7 2 8 The AutoCircle Tool a AutoCircle Tool Use the AutoCircle Tool to quickly mark individual areas on your sample The tool draws a circle and the center of the circle with a click of the mouse When you release the AutoCircle laser function the marked area Is cut out along the circumference of the circle and is catapulted into your cap by a laser pulse to the center of the circle You can adjust the diameter of the circle To adjust the diameter of the AutoCirc
147. his dot and click on it with the cursor tip Note The farther the dot is from the image center both vertically and horizontally the more ac curate your calibration will be The cursor will disappear and the program will switch automatically to Stage Mode The following message will appear Step 2 of 2 Move this point to screen center and click again e Use the mouse to move your selected point to the crosshairs at the image center and click the left mouse button The cursor will reappear the program automati cally switches out of Stage Mode The following message will appear Calibration is finished 34 Check position of Laser Marker This message will disappear automatically after a few seconds Check the position of the Laser Marker as de scribed in chapter 4 3 Setting the Laser Marker if you did not already do this before the calibration You can check the calibration by drawing an ele ment and activating the automatic laser Cut function Calibration is correct when the cut line exactly follows the element you have drawn for instructions on working with these functions refer to chapter 7 Drawing and Measuring and chapter 10 Cutting and Catapulting Note The calibration always applies only to a magni fication that has been set before the calibra tion If you intend to work with various magnifications you must recalibrate for each change of magnification PALM RoboSoftware 3 1 0106 EN
148. how you can specify a different folder If you wish to save your elements in a folder other than the specified folder e Click on the button H The Please enter select a file name window opens e Select the desired folder enter a file name and then click the Save button The elements are saved and the folder closes To load an elements file File Open Elements e Click on menu item Open Elements in the File menu in the PALM RoboSoftware main window or in the Element List window The Load Elements from file window opens The exact appearance of this window depends on which slideholder you are using chapter 3 2 Setting Sli deholder explains how you should notify the soft ware of which insert you are using The following description applies to the 3 slide holder You can follow the same procedure for other slideholders PALM RoboSoftware 3 1 0106 EN Load elements from file lw Slide1 Es Pocuments and Settings adminiMy Documents E Documents and Settings adminMy Documents W Slides E Documents and Settings adminiMy Documents W all active E Documents and Settings admin My Documents If you are using a slideholder that can accommo date more than one slide this window can be used to load the elements for one or more slides at the same time e If you have saved the elements for several or all slides with the same name and now want to load them Check the a
149. icro scope The settings that you enter always apply to the magnification that you have set in the program As soon as you have selected another magnification the values that were last used or the factory defaults are automatically applied This also applies for coupled focus settings Therefore prior to setting IR laser power and IR laser focus first select the magnification that you want to work with 118 To couple and uncouple the focus settings for both IR laser beams Double Beam System only Window PALM Robo IR laser settings e Click on the Energy amp Focus IR Laser entry in the Laser menu or press F10 on the keyboard The PALM Robo IR Laser Settings window opens This contains four tabs for setting power and fo cus e Click on the IR 1 2 Focus tab In the middle portion of the tab is a checkbox for coupling the focus values of both IR laser beams IR 1 lt gt IA 2 Simultaneous ft es iv Focus e Click the checkbox beside Simultaneous Focus in order to select or deselect a combi nation of these settings A tick in the checkbox indicates that the combina tion is enabled e Close this window by clicking on OK In the Laser Tools e Open Laser Tools if it is not already displayed click on the entry Laser Tools in the View gt Menu or press the keyboard combina tion Ctrl L e Click on the IR button e Click on the Simultaneous Focus checkbox to enable or d
150. iew gt Menu or press the keyboard combina tion Ctri L e Under Wheel click on the button for the IR laser beam whose focus you wish to change using the mouse left for IR 1 right for IR 2 e Rotate the scroll wheel on the mouse until the desired value appears in the text box of the Focus Calibration Tool Info The Wheel buttons can also be used to spec ify the focus that menu or key commands will set see Page 121 To set the IR laser power and focus in the PALM Robo I R Laser Settings window Laser Energy amp Focus IR Lazer FIO e Click on the Energy Focus IR Laser entry in the Laser menu or press F10 on the keyboard PALM Robo IR Laser Settings window opens PALM Robo IR Laser Settings Values for Lens Ex IRA 1R 2 1 142 Power IR 142 Focus IR Focuz 1 TT alo 120 If you have installed a Double Beam System the window will include four tabs R 1 with tools for setting the power and focus of laser beam 1 R 2 with tools for setting the power and focus of laser beam 2 R 1 2 Power with tools for setting the power and balance for both beams IR 1 2 Focus with tools for setting the focus for both beams If you want to work with only one IR laser beam Select the tab for the desired beam IR 1 or IR 2 The power for the beam you did not select is set to zero automatically You set the power and focus of the sel
151. ime Lapse function optional this can be used to automatically store series of images or video sequences You can specify the time interval between images in a series and the duration of a recording How to work with the Time Lapse function is described in chapter 9 5 Saving Images and Videos Using the Time Lapse Function You can specify a Copyright Note If you have opt ed to display the Copyright Note then it will be in serted in every image that you store refer to chapter 9 1 Inserting a Copyright Note If you are working in File Mode refer to chapter 1 6 File Mode and Database Mode you can specify a folder or create a new folder for images to be stored in You can also specify a file name For instructions on how to make these specifications please refer to chapter 9 3 Saving Images Manually If you are working in Database Mode your images will be stored in the database The file name is generated automatically For automatic storage of images you can specify whether your images should be stored with or without the superimposed elements Please refer to chapter 9 4 Saving Images Automatically during a Laser Function for instructions on how to do this Note When you save the current image only the section of the microscope image that Is visible on the monitor will be saved optionally with or without the elements within this section Note If you want to save your elements for subse quent use you mus
152. in the table in the Element List window Step 4b Preparing the target sample manual adaptation e Insert the slide onto which you want to trans fer the elements into the slideholder Step 5b Transferring elements to the target sample manual adaptation Edit Match Reference Point manual e Select the group which you want to match in the example Slide 1 e Click on the menu item Match Reference Point manual in the Edit menu PALM RoboSoftware 3 1 0106 EN The Serial Sections Function Info This menu command is only available in the PALM RoboSoftware s main window e Inthe main window of the PALM RoboSoftware move the mouse cursor to the menu item Goto Reference gt Group in the Move menu and click the item Ref 1 Goto Reference j The reference element 1 will be displayed on the monitor e Drag the mouse cursor onto the microscope image The mouse cursor changes to the Group Refer ence Pointer recognizable by the attribute R With the Group Reference Pointer you can select reference elements and then move them e Select the reference element with a mouse click and then drag it to the respective marking in your sample Note Make sure that you do in fact move the ref erence element to the respective marking If you move it to another marking the match will be faulty e Proceed in the same way with the other two ref erence elements By moving the reference e
153. indow click OK 143 Status Display ApoTome 13 3 Status Display ApoTome If your microscope is equipped with an ApoTome and if the ApoTome is configured in the Micro scope Toolbox a status display will appear below Microscope Tools The displays are as follows pol ome a The ApoTome is set up in such a way that the light beam can pass through it unrestricted With this setting you can work with the laser without risk of damage to the ApoTome 4poT ome L The ApoTome is set up in such a way that the grid is positioned in the beam path Info The laser beam would destroy the grid If the grid is swiveled in the laser is automatically switched off and no laser function can be started 4poT ome al The ApoTome controller is switched off In this case the PALM RoboSoftware has no information on the status of the ApoTome Note Check the status of the ApoTome before switching the laser on The grid must not be positioned in the beam path as it would be de stroyed by the laser 144 PALM RoboSoftware 3 1 0106 EN Appendix A Glossary Appendix A Glossary 3D Laser Beam Positioning Using the 3D Laser Beam Positioning function on the IR laser you can move trapped objects in both the x and y axes ie parallel to the plane of the slide AutoCircle Cutting along a circular line and catapulting the entire area into the center of the circle with a pulse of the laser
154. instructions on how to do this PALM RoboSoftware 3 1 0106 EN Launching other Programs from PALM RoboSoftware 1 8 Launching other Programs from PALM RoboSoftware PALM RoboSoftware has interfaces with other pro grams which means that these programs can be launched directly from PALM RoboSoftware The programs concerned are programs for organizing and editing images and detecting structure Information Center a component of the PALM MicroLaser Systems Imaging Software any image editing program Image Processing Software Metafer can be accessed via P A L M AxioVision can be accessed via P A L M Cellenger can be accessed via P A L M 1 8 1 Information Center The PALM Information Center is a component of PALM MicroLaser Systems Its functionality is de scribed briefly in chapter 1 3 Information Cen ter Further details can be found in the Manual of the Information Center To launch the Information Center program se Information Center x File ft Open Information Center pa e Click on the Information Center icon button in Archive Tools or click on menu item Open Information Center in the File menu of the PALM RoboSoftware main window or press F3 on the keyboard The Information Center program is launched The Information Center manual explains how to operate this program PALM RoboSoftware 3 1 0106 EN 1 8 2 Imaging Software You can incorporate a
155. ional laser beam positioning system Patents US 5 689 109 EP 679325 B1 and others Element List Patent US 6 930 764 Additional patents pending P A L M Microlaser Technologies GmbH Am Neuland 9 12 82347 Bernried Germany Phone 49 0 8158 9971 0 Fax 49 0 8158 9971 249 E mail info palm microlaser com www palm microlaser com P A L M Microlaser Technologies GmbH 2006 All rights reserved 2 PALM RoboSoftware 3 1 0106 EN Contents Contents Chapter Page 1 N EFOdUCHON 1 2 rad daa aa e ata dra rA 7 1 1 Functionality of PALM MicroLaser SysteMS o 7 1 2 PALM RODO SOLOW ARC cue tasca due ota bal dd do ap ths a ri ata a eee 8 1 3 information Center coartada taria sd os idad cda Mba a 8 1 4 ADOUL this User Mandala ceca ds Oe is is dt epee ae ele ads Be od ee ek 9 Td CONOS acatar la a ee a ee ee ee a ee ee 9 A CONVENTOS ed ate atc ws ded a po di Mo di Mar coe Ge Sau ot is ay toe 9 1 5 Stadt Soe ete Oe ee Boe a ec Ale RD eee ee oe ae e et eS 9 1 6 File Mode and Database Mode a a a e 10 1 7 Launching and Exiting the PrograM oo aaa 10 1 8 Launching other Programs from PALM ROboSoftware o eee eee eee ees 11 1 8 1 AfoOrFmation Center out aa a doo BA 11 1 8 2 MAJN O OTWare ae oooh eset iB te bl sE A rd td os cd 11 18 3 AXIOVISION and Celle ger sisi da a A A dd TO a id 11 1 8 4 Image Processing Software 2 rai n a E act E A A ak ey we See A 12 2 Prog
156. isable the combination A tick in the checkbox indicates that the combina tion is enabled PALM RoboSoftware 3 1 0106 EN Setting IR Laser Power and Focus To set the IR laser power and IR laser focus with the Laser Tools e Open Laser Tools if it is not already displayed click on the entry Laser Tools in the View gt Menu or press the keyboard combina tion Ctrl L e Click on the IR button Laser e IR IR Power Balance 100 Ey JH ie L 64 liz 83 4 4 IR Focusl A Focus2 100 100 1 Y 1 J 48 5 4 4 le Wheel C Wo Simultaneous Focus The Laser Tools for the IR laser will be displayed If you have installed a Single Beam System the only settings you can choose between are IR Power and IR Focus 1 Both values are set in analog form e Drag the slider or click on the arrow buttons or click and hold the mouse button down until the required value is displayed in the field between the slider and the arrow button or click in the text box enter the required value and press Enter PALM RoboSoftware 3 1 0106 EN If you have installed a Double Beam System you can choose between the following settings R Power this is used for controlling the entire laser output for both IR beams Balance Tr1 Tr2 this is used to distribute the laser output between the two beams R Focus1 IR Focus2 this is used to control the focu
157. ite available Inthe Color Palette you can select a line or dot color for drawing new lines or dot elements Working with this palette is particularly advan tageous if you want to draw multiple elements of the same type which are to be in various col ors Inthe Colors window you can specify colors for all element types Line Dot Ruler and Text concurrently These settings are overwritten later by any subsequent changes to the Color Palette In addition you can select colors for the laser function preview However you cannot draw any further ele ments or carry out any other functions until you have closed this window The settings in the Colors window can be saved with your other settings in a file to be loaded later refer also to chapter 3 4 Saving and Loading Settings PALM RoboSoftware 3 1 0106 EN To change the color settings with the Color Palette for new elements you want to draw S Color Palette Show Hide View Menus d Color Palette Ctrl P In case the Color Palette is hidden Click on the Color Palette icon button on the Tool Bar or choose Color Palette from View gt Menus The Color Palette will be displayed LIL IL IL III HL IL INIA LIL AL Select the Graphic tool with which you want to draw the next element Left click on the desired color in the Color Pal ette to select it Draw the required element s The newly drawn elements are displayed in the de sired color
158. ith PALM RoboSoftware you will create various settings If you draw elements move the stage activate the laser save elements images and settings You will also make various general settings which enable you to adjust the PALM RoboSoftware to your specific requirements All settings can be changed at any time while you are working All of your settings will be retained until they are actively changed again even if you exit the pro gram and relaunch it For lasting reliability you can also save your settings in a file If you want to work on the same or a similar sample in future you can load these settings again and work with them All factory default settings can be restored at any time The following section provides instructions for configure settings for launching PALM RoboSoftware switch software over for different slideholders only PALM RoboStage Il turn automatic focusing on and off for saved and reloaded elements configure settings for the saving of settings saving and loading settings You can also find information on unlocking software components purchased later setting a short term alarm All other settings are explained in detail elsewhere Settings for moving the stage chapter 5 3 Setting Speed Settings for drawing elements chapter 7 8 Changing Colors and Line Thickness Settings for the laser functions chapter 4 Cal ibrating the System chapte
159. ith which it is supplied It is therefore not usually necessary to recalibrate only applies to the supplied objective or such objectives marked with the P A L M logo Recalibration is only necessary if despite exact positioning of the Laser Marker and correct choice of objective differences occur between the drawn contours and those processed by the laser Carry out a recalibration as described in chapters 4 1 to 4 5 and 4 7 You can reset the calibration to the factory default setting at any time refer to chapter 4 6 or 4 8 PALM RoboSoftware 3 1 0106 EN 4 1 Preliminary Remarks To achieve the desired results with the laser func tions the following points must be fulfilled the program must be set to the microscope s magnification setting andthe movement of the stage and the IR laser if you have installed a system with 3D Laser Beam Positioning must have been calibrated the Laser Markers in the default setting the green triangle or the red and green squares in the middle of the monitor must have been aligned with the laser foci and ifthe magnification is changed any deviations through the objectives must be compensated Lens Offset Calibrating with the software establishes a rela tionship between the dots in the image and the ac tual positions of the stage This ensures that automatic laser functions are performed at pre cisely the required locations The Laser Markers must mark the points
160. ition 1x otherwise the la ser will see a different image than you see in the ocular or on the monitor LightPath Optovar F s FirstPos e Click on menu item 1x First Pos in the LightPath gt Optovar menu The Optovar is set to position 1x Info Other settings for the Optovar can only be made manually on the microscope 13 2 10 Pre Settings for the Microscope You can specify a range of settings that will be made automatically when PALM RoboSoftware is launched and closed Reflector Condenser Filter Shutter Lamp Baseport and sideport Setup e In the menu bar of the Microscope Window click on Setup The Microscope Setup window opens Microscope Setup Ed Cancel OR Define the Microscope Settings for Program Startup Shutdown Reflector dontcare gt don t care Condenser don tcare gt don t care Filter don t care r don t care S Shutter Reflected dontcare gt don t care k Shutter Tranemitted dontcare gt Lamp don t care ka don t care r Baseport don tcare gt don t care Sideport dontcare dontcare Don t Care PALM RoboSoftware 3 1 0106 EN On the left there is a list of the microscope parts for which you can make prior settings There are two groups of fields in the middle and one on the right in which you can specify the se
161. k boxes to enable or disable the corresponding function A tick in the checkbox indicates that the respective function is enabled If the function is enabled images for each element processed with the laser will be saved every time the laser function is triggered Names have been assigned for the images as follows x BeforeCut x AfterCut x _beforeLPC x AfterLPC x stands for an element number that is assigned automatically by the PALM Robo Software This el ement number is incremented starting from 1 for each save operation PALM RoboSoftware 3 1 0106 EN Saving Images and Videos Using the Time Lapse Function If the images are to be saved together with the su perimposed elements W Save Elements in Image e Activate the checkbox in front of the Save Ele ments in Image option A tick in the checkbox indicates that this function is enabled every time you save an image it is saved together with the associated elements Info Please note that images you have saved by this method cannot be reloaded into PALM Ro boSoftware refer to chapter 8 Saving and Printing Elements PALM RoboSoftware 3 1 0106 EN 9 5 Saving I mages and Videos Using the Time Lapse Function Info Time Lapse is only available if you have pur chased an appropriate license Please contact P A L M Microlaser Technologies GmbH if you wish to upgrade your system The Time Lapse function enables yo
162. l Bars Graphic Tools Ctrl G Color Palette Ctrl P Cut Tools Cerl L Status Bar e In View gt Menus click on the name of the bar or tool that you want to show or hide A tick beside the name indicates that the bar or the tool is shown Some icon bars and tools can also be displayed or hidden using keyboard shortcuts PALM RoboSoftware 3 1 0106 EN To display or hide a bar or tool using key board shortcuts e Press one of the following keyboard combina tions Ctrl G to display hide the Graphic Tool bar Ctrl U to display hide Cut Tools Ctrl P to display hide the Color Palette Ctrl L to display hide Laser Tools Ctrl E to display hide Speed Tools To move an icon bar e click on the vertical white bar on the left edge of the icon bar that you want to move and e drag and drop the bar in the desired position Release the mouse button To switch the Scroll Rectangle or the screen center display on or off View Scroll Rectangle Screen Center e Inthe View menu click on Scroll Rectangle or Screen Center A tick in front of the menu entry indicates that the Scroll Rectangle or the screen center is being Shown These settings will be retained after you exit and relaunch the program To show or hide the Scale Bar View fardstick e Click on menu item Yardstick in the View menu A tick on the left of the menu entry indicates that the Scale Bar is displayed This se
163. le circle Laser LPC Parameter Autolincle pun 100 Badius of AutoCircle e Click on the LPC Parameter entry in the Laser menu The LPC Parameters window opens e Enter the desired radius in um in the AutoCir cle field e Close this window by clicking on OK Note Elements that have already been drawn are not affected when this setting is changed Note The radius of an element that has been drawn cannot be altered subsequently Therefore if your setting proves to be unsuitable you must delete the element change the radius as described above and draw a new element To draw an AutoCircle type element e Select the AutoCircle Tool in the Graphic Toolbar e Click close to the middle of the area that you want to cut out in a circle and catapult later The selected site is indicated by a dot which is surrounded by a circle Info If you have set a high magnification and or a large radius it may happen that the circle ex tends beyond the section of the microscope im age displayed on the monitor and therefore is not visible You will then see only the center point of the element PALM RoboSoftware 3 1 0106 EN 7 2 9 The Text Tool A Text Use the Text Tool to add markings with or without textual comments to your image The marked sites are displayed as flags the comments in a rectangle Comments are always displayed on one line You can select or subsequently change the co
164. lements the shape and position of the remaining elements will be auto matically matched e Click on the menu item Match Serial Section Point manual in the Edit menu The pointer reverts to the standard pointer Note Check the position of the matched elements if your tissue block contains diagonal struc tures Match the position if required the shape also of such elements by hand if necessary refer to7 7 2 Editing Elements 115 Cutting and Catapulting without Automatic Laser Functions 10 10 Cutting and Catapulting without Automatic Laser Functions Even without automatic laser functions cutting and catapulting are possible This is done by switching the laser on and off using the footswitch However to do so you must switch to Stage Mode and move the stage using the mouse refer to chapter 5 1 1 Positioning the Stage with the Mouse Stage Mode You can choose whether you wish to start the cut ting or catapulting function when you operate the footswitch To set the function of the footswitch Info You must perform this setting before switching to Stage Mode Lazer le Ly CIR Cut LPC Energy Energy 100 100 ll Focus Focus 100 100 1 7 le Footer e Open Laser Tools if it is not displayed click on the entry Laser Tools in the View gt Menu or press the keyboard combina tion Ctrl L e If you have a CombiSystem including an IR
165. les ellipses Note Select the laser function Cut before joining the elements refer to chapter 10 3 Selection of Automatic Laser Functions for instructions on how to proceed Otherwise you will not ob tain the desired result Ctrl key Freehand Tool e Select the Freehand Tool e Press and hold the Ctrl key e Move the cursor to the start or finish dot of the first element the start and finish dots of an ele ment are indicated by a small square with thick black lines and the same color as the element e Keeping the mouse button depressed draw the connecting line to the start or end dot of the second element As soon as you have reached the start or finish dot a small square with thick lines in the color of the element appears here Both elements are joined and are assigned a num ber as a new element Info If you have added a comment to each of the two elements you want to join the comment of the element with the higher sequential number is deleted by joining the comment on the ele ment with the lower sequential number is as signed to the new element If the elements you have joined were in two different colors the new element will be dis played in the color of the element that had the lower sequential number PALM RoboSoftware 3 1 0106 EN To change the shape of elements lines rectangles ellipses AutoCircle Note Select the laser function Cut before changing the shapes o
166. lick on items Focus up or Focus down to set the laser focus higher or lower Setting with keyboard shortcuts e Use the Page Up and Page Down keys to raise or lower the laser energy e Press Home or End to increase or reduce the value for the laser focus The values that are currently set are displayed in the Laser Tools 103 Specifying Laser Settings 10 5 2 Activating and Deactivating Autofocus Info This function is only available if your micro scope has the autofocus facility If the Autofocus is switched on then each time an element is targeted during a laser function and each time the lens is changed the image is fo cused automatically Autofocus can be turned on and off in Microscope Tools or on the Laser tab in the Preferences and Configuration window In the Preferences and Configuration win dow Autofocus can be turned on and off as follows e Inthe Setup menu click on menu item PALM Robo The Preferences and Configuration window opens e Select tab Laser Auto Focus i OF active e Inthe Laser function section click the AF active box to turn the function on or off A tick in the checkbox indicates that the function is enabled To turn autofocus on or off in Microscope Tools proceed as follows Ii AF active e If you want to hide the microscope tools they Should first be displayed choose Microscope Tools from View gt Menus e
167. ling your sample on the slide make sure that the areas to be processed do not come too close to the edge of the slide The display of the elements on the monitor also serves as a preview for the various laser functions The display of your elements thus also depends on the laser function that has been selected When the program is launched the laser function Cut PALM RoboSoftware 3 1 0106 EN Drawing New Elements is enabled by default When you switch among the tools for drawing Figure Dot and AutoCircle elements PALM RoboSoftware also switches auto matically between the laser functions Cut LPC and AutoCircle the display on the monitor is also altered accordingly Chapter 10 3 Selection of Au tomatic Laser Functions includes instructions on selecting a different laser function yourself and chapter 10 4 Preview of the Automatic Laser Functions describes the changes in the display of elements on the monitor The program automatically assigns a number to each drawn element If you have not changed the default setting this number is displayed Refer to chapter 7 7 4 Updating the Element Numbering for instructions on how to show or hide these num bers To activate one of the graphic tools e Click on the icon of the desired graphic tool in the Graphic Toolbar The icon button appears to be pressed the cur sor changes to a shape that symbolizes the tool in use An appropriate laser function for the
168. ll active box enter the common file name in the adjacent field to the right if necessary uncheck the check box for slides for which you do not wish to load elements and then click the OK button Your elements will be loaded for all the selected Slides e If you wish to load the element files for one or more slides and those files were saved with different names Check the relevant check boxes Slide1 Slide2 and or Slide3 in the example enter a file name for each you may be able to select the desired file using the button OD and then click the OK button Info If you have drawn and saved elements for a slide and now wish to continue working on the same slide you need not necessarily insert it at the same position it occupied when the ele ments were saved If for instance you had the slide inserted at position 1 you could now also insert it at posi tion 2 You must bear in mind however that the elements you had saved for position 1 must now be loaded for position 2 83 Saving and Loading Elements in Database Mode Info This method can also be used to transfer ele ments from one sample to another in that you draw and save elements for one slide and then load them for a different slide for two consec utive tissue samples for instance Remember though that the position and probably also the shape of the elements will not be exactly right for the second sample and you will have adjust the
169. lor of the com ment background and flag as described in chapter 7 8 Changing Colors and Line Thickness Text elements are ignored by the laser functions To place a marking e Select the Text Tool in the Graphic Toolbar e Click on the point at which you want to place your marking Text element Enter text pes element A marking will appear as a flag at the selected site The Text Element window opens e Ifyou want to add a comment to your marking enter the desired comment Close the window by clicking on OK If you do not want to add a comment close the window by clicking on Cancel Text element Your comment will appear at the foot of the flag You can also assign a name to the element this procedure is described in chapter 7 7 5 Adding a Name or a Comment to Elements In this case the name of the text element is shown in place of the element number 61 The Element List Window 7 3 The Element List Window Element List File Edit Move Laser Slidei 5lidez Slides Summary ci Rectangle Freehand Freehand AutoCircie AutoCircie Circle Reference Oo J om A w FR Circle E m DJ 11 Autocircie 12 orple Group gt A AutoCircle Show Types 4744 106064 30976 31224 31224 11409 5655 31224 The Element List window provides a summary in table format of all selected elements You can check various parameters change them and acti vate va
170. lted Monitor display during an automatic laser func tion You can determine whether the elements are to be displayed or hidden on the monitor while the laser function is in operation The default set ting is that the elements are not to be dis played Position of the stage after completing a laser function You can determine whether the stage Is to be returned to start position on completion of the laser function The default setting is that the position is maintained as it was during the last laser function Info When comparing microscope images in the case of serial images before and after cutting or catapulting it is advisable to return to the orig inal position The set values can be saved and reloaded You can reset all values to the factory default settings Re fer to chapter 3 4 Saving and Loading Settings and chapter 3 5 Restoring Settings for instruc tions on how to do this Info It is recommended that you experiment in or der to ascertain which values best suit your working requirements Refer to chapter 10 6 Finding Suitable Laser Settings for instruc Distance between AutoLPC shots and distance of the AutoLPC shots from the element border line only applicable in the case of AutoLPC These define the required distance between the dots which should apply when Laser pulses are released in the AutoLPC function and the min imum distance that these dots should have from the border line of th
171. lue becomes violet violet becomes red etc and cyan becomes blue The more you change this setting the greater the shift in colors Gamma corrects the linear nature of the brightness pattern between black and white Lifelmage view parameter view settings Measurement tools Contrast Pixeldata 0 20 a ae Brightness Red Green Blue Brightness Imagedata Minimum Maximum Brightness 176 220 Refresh 0 20 4 00 f 1 00 4 Reset to default To set the image parameters back to their default values e Click on the Reset to default button in the View settings section All parameters are reset to their default values 29 Set Image Parameters To display the color values and the bright ness of individual points in the image e Click on the Pickup button in the Pixeldata section e Click on the point in the image whose color val ues and brightness you wish to display The fields Red Green and Blue show the brightness values for the three primary colors and their ratio to one another while the Brightness field shows the brightness of the selected point in the image To display the lowest and highest brightness value for the entire image e Click on the Refresh button in the lmage data section 30 PALM RoboSoftware 3 1 0106 EN Preliminary Remarks 4 Calibrating the System Note The system is calibrated by the manufacturer for the objectives w
172. me Note The name you enter is taken as a default set ting for all subsequent image saving opera tions and thus overwrites the original default setting refer also to chapter To enter a file name for saving images manually e Choose the format in which you want to save your image Info You can choose between the formats bmp tiff and jpg The recommended formats are tiff of jpg since these formats allow addi tional information to be saved along with the Image stage position microscope settings e Click on Save The window will be closed your image will be saved 89 Saving Images Automatically during a Laser Function 9 3 2 Saving Images in Database Mode No settings need to be made in order to save imag es in Database Mode All images are stored in the database The file name is generated by the PALM RoboSoftware manual followed by the next num ber in sequence The sequential number is incre mented by one every time you save an image To save your image Info Please note that the image is stored with the el ements unless the option to display them has been switched off The elements are stored in the same way as they are displayed on the screen i e perhaps with their area content specified with or without the element number and if relevant with changed image parameters refer to chapter 3 9 Set Image Parameters The Copyright Note and scale bar are only in cluded with the
173. ment in the Edit menu Info You can repeat this function many times Each time the latest of the remaining elements will be deleted Info If the last element to be drawn has already been processed with the laser you can no long er delete it In this case the icon button and or menu command is are disabled PALM RoboSoftware 3 1 0106 EN To delete any elements There are several options available e Select the element you want to delete on the monitor with Pointer Tool or in the Element List window and press key Del or Edit E Delete selected elements Del e Select the element you want to delete on the monitor and click on menu item Delete selected elements in the Edit menu of the main window or e Select the element you want to delete in the Element List window and click on menu item Delete selected elements in the Edit menu The element will be deleted If you select multiple elements you can also de lete multiple elements simultaneously with this command To restore deleted elements Edit Undelete Alt BackSp e Click on menu item Undelete in the Edit menu in the main window of the PALM RoboSoftware or press key combination Alt Backspace The last performed deletion command is undone Info If you have deleted several elements you can call up the function repeatedly 69 Processing Elements 7 7 2 Editing Elements There are a number of way
174. ments You can copy any element and insert the copy in your image To copy elements Edit Copy Ctrl C Paste Ctrl e Select the element you want to copy either in the main window or in the Element List win dow refer to chapter 7 6 Selecting and Dese lecting Elements e Click on menu item Copy in the Edit menu in the main window or in the Element List window e Click on menu item Paste in the Edit menu in the main window or in the Element List window PALM RoboSoftware 3 1 0106 EN Processing Elements Info If you want to work with the keyboard short cuts Ctrl C and Ctri V you have to se lect the element you want to copy on the monitor not in the Element List window A copy of the element will be pasted the copy is selected e Move the copy to the required position Info You can also select and copy multiple ele ments at the same time 7 7 4 Updating the Element Numbering PALM RoboSoftware numbers all the elements that you draw As soon as you have drawn a new ele ment it is assigned a number automatically This number is increased by one for each additional el ement If you delete elements the remaining ele ments keep their respective numbers If you want to renumber your elements sequentially you can perform a renumbering operation You can enable and disable the numbering display In the default configuration of the software the numbering is displayed
175. ments therefore has no practical use PALM RoboSoftware 3 1 0106 EN Settings for Saving Elements in File Mode 8 Saving and Printing Elements PALM RoboSoftware has a number of options for saving exporting or printing the elements you have drawn or their properties to load them into the PALM RoboSoftware later on again This allows to reuse a sample in the stage and superimposing the associated elements on the monitor to observe and edit the element properties in another program to enable the element properties to be archived as a printout To help you with this three functions are available that generate the different output formats Save Elements generates a binary file That binary file can be reloaded or reimported into the PALM RoboSoftware refer to chapter 8 2 Saving and Loading Elements in File Mode Info In Database Mode refer to chapter 1 6 File Mode and Database Mode your elements are automatically saved in the database The menu commands Save Elements and Open Elements are therefore not avail able if you are working in Database Mode Chapter 8 3 Saving and Loading Elements in Database Mode explains how you can reload elements from the database Export Elements generates a text file That text file can be reloaded or reimported into the PALM RoboSoftware and opened with any text editor You can then choose to view print or edit the data refer to chapter 8 4 Ex
176. mporting Elements With the Export function you create a file that can be opened in any text editor Info The functions Export Elements and Import Elements are intended primarily for exchang ing data with other programs If you are working in File Mode the Save Ele ments function is recommended for saving el ements which you later want to reload into the PALM RoboSoftware refer to chapter 8 2 Sav ing and Loading Elements in File Mode In Database Mode you do not need to save the elements explicitly they are saved automati cally in the database and can be reloaded from there refer to chapter 8 3 Saving and Loading Elements in Database Mode Info In the Element List window you can specify that only elements of selected types are to be displayed in the window i e elements of other types are not included in the table in the Ele ment List refer to chapter 7 3 The Element List Window This has no effect on the Save operation all existing elements will be saved under all circumstances Info If you are using a slideholder that can accom modate more than one slide all the existing el ements are saved in one file irrespective of which sample you have drawn them for To export elements File ES Export Elements ES Export Elements In the PALM RoboSoftware main window e Click on menu item Export Elements the File menu The Element List window e Click on the Export i
177. n be changed using menu commands in the main window via commands in the Element List or using an icon button and the Color Palette To change the color using a menu command Info For text elements the color of the flag can be changed as described below Edit mA Change Alt Enter e Select the element whose color you want to change in the main window or Element List window e Click on menu item Change in the Edit menu in the main window or in the Element List window The Element Properties window will open e Click on the arrow at the top of the Color field A list opens to display all available colors Element properties gt E magenta T hr green 3 dark green Grp lightblue blue darkblue cut shot lightyellow yellow darkpellow H m w lightcyan Comment Span darkcyan lightmagenta J darkmagenta Type rea Cancel e Click on the desired color and close the window by clicking OK The selected element is displayed in the chosen color Info You can only change the color of one element at a time If you have selected multiple ele ments or parts of multiple elements only the color of the element with the lowest number of all the selected elements is changed PALM RoboSoftware 3 1 0106 EN To change the color using an icon button and the Color Palette Info For text elements the color of the flag can be changed as described below
178. n be used to shift any point on the microscope image into the center ka Centre e Click on the Centering Tool in the graphics tool bar e Click on the point in the image that is to be moved to the center The centering tool will continue as the active tool until you click on any other tool on the graphics toolbar 5 1 6 Moving the Stage during an Auto matic UV Laser Function As soon as you activate one of the automatic UV laser functions the stage automatically moves along the programmed cut lines or from dot to dot You do not have to select any particular type of movement in this case 5 1 7 Further Possibilities for Position ing the Stage Aside from the aforementioned possibilities you can move or precisely position the stage using the following methods Centering elements The stage is so positioned that a previously selected element will be dis played in the center of the monitor Refer to chapter 7 5 Centering Elements on the Moni tor for instructions on how to do this To move to the Load Position refer to chapter 5 6 The Load Position When the stage is in the Load Position you can easily change the slide and the slideholder PALM RoboStage Type II only To move to the Check Point refer to chapter 5 5 The Check Point When the microscope stage is at the check point you can observe catapulted samples in the collection device under the microscope Navigating with PALM Navigator Wit
179. n image processing program of your choice such as Adobe PhotoShop or ACD Systems ACDSee referred to simply as Imaging Software from now on into the PALM RoboSoft ware Refer to chapter 3 7 Incorporating an Im aging Editing Program for instructions on how to do this Info The image processing software is not a PALM product Neither does P A L M supply such software To launch the I maging Software Imaging Software File Open Imaging F2 e Click on the Imaging Software icon button in Archive Tools or click on menu item Open Imaging in the File menu of the PALM RoboSoftware main window or press F2 The Imaging Software you have incorporated will be launched Instructions for working with this program are the subject of the program s user manual 1 8 3 AxioVision and Cellenger To allow you to enhance your system P A L M of fers not only Metafer but also certain other ap proved image processing programs which for instance enable you to localize specific objects or structures within your microscope image with an automated search function these programs are AxioVision and Cellenger Both of these pro grams can be activated in Navigator refer to chapter 6 4 Analyzing Images Using the Cellenger Software and chapter 6 5 Analyzing Images Using the AxioVision Software 11 Launching other Programs from PALM RoboSoftware 1 8 4 Image Processing Software The program Metaf
180. nction a position with a beam splitter suitably coated for this wavelength must be swiveled into the reflector revolver of the microscope For systems with the fluorescence configuration Basic this is the first position only PALM RoboSoftware 3 1 0106 EN Opening and Closing the Shutter 12 6 Opening and Closing the Shutter Fluorescence Shutter You can open and close the shutter via a menu command or an icon button However you can use the timer function chapter 12 7 to preselect a time after which the shutter is to be closed again automatically after you have opened it Shutter Type Note Shutter Error In the Basic configuration with the manual Actual state CLOSED microscope the function Shutter is not ac tive In this case the shutter has to be oper ated manually at the microscope stative To open and close the shutter on the filter e Click on the Close button to close the shutter wheel via icon buttons or on the Open button to open the shutter e Close the window by clicking on OK Shutter S Closed Shutter is currently closed Shutter LA Open shutter is currently open Info There is only one button for opening and clos ing the shutter lts appearance changes ac cording to the current status e Click on the Shutter icon button in Fluores cence Tools The shutter will be opened or closed depending on its position before the command was sent To open and close the sh
181. nction is chosen then it is not an option to have an interval of less than 10 sec onds If you check the Close reflected light shutter between picture recording box which is only enabled if a fluorescence unit is installed then the fluorescence shutter is closed during the interval between images being saved This enables sensitive samples to be protected from too long an exposure to fluorescent light Info You can either check the check box for micro scope shutter control or the one for fluores cence shutter control In the Trigger Point Definition area you define which event will be used to start the Time Lapse function Manually the sequence is started manually by clicking on the button Start Time Lapse manu ally see below Foot switch Cut LPC the sequence is started as soon as you activate the laser using the foot switch chapter 10 10 Cutting and Catapulting without Automatic Laser Functions explains how you can set the footswitch for Cut or LPC LPC laser function the sequence is started as soon as you activate an automatic LPC laser function Scheduled You can set a time for the function to be started automatically PALM RoboSoftware 3 1 0106 EN In accordance with the start event that has set up one of the following symbols is displayed in the PALM RoboSoftware main window trie Manually Foot switch Cut LPC or th LPC Laser function ma Scheduled To star
182. nd Closing the Shutter oo e e aaa 142 Setting the Beam Pal ed A AE ae A A A 142 Satting he ODtOVal 24 so 2 eae team ia e a ORY Waianae eee Peewee es 143 Pre Settings for the Microscope es 143 Status Display ADO TOME i di 622 3 5400044 A Lae Stade bed ee hae ee tke oe bea 144 Appendix A Glossary irradia aa ed 145 Appendix B Keyboard Shortcuts 000 cee eee 147 MAS ri a Bi Ab ge a ook eG ene A For Edad EA 149 PALM RoboSoftware 3 1 0106 EN Functionality of PALM MicroLaser Systems 1 Introduction 1 1 Functionality of PALM MicroLaser Systems PALM MicroLaser Systems are modular in design and extremely flexible PALM MicroBeam The PALM MicroBeam offers the greatest degree of precision in the field of non contact laser microdissection and manipulation It can be used in virtually all areas of molecular research Your PALM MicroBeam is designed to enable you to cut sections from your sample and detach them from the surrounding material under the micro scope and then to catapult them into a collecting vessel PALM MicroTweezers With your PALM MicroTweezers you can trap ob jects under the microscope and lift them away from the plane of the slide It can be employed as stand alone technology or in combination with PALM MicroBeam as the PALM CombiSystem PALM MicroTweezers is available in four configura tions with a fixed laser beam with a 3D movable laser beam
183. nd off numbering 73 types 58 ungroup 79 update numbering 73 Z Focus 25 Enabling software components 27 Export of elements 85 F Factory defaults 27 File Mode change to Database Mode 10 Information Center 8 load elements 82 PALM RoboSoftware 10 save elements 82 save images 89 Fluorescence adjust Reflector Offset 136 filter wheel 132 name of filter 133 reset Reflector Offset 137 selecting a Fluorescence Excitation Filter 133 settings 129 shutter 131 timer 132 working with fluorescence 129 Fluorescence settings create new set 134 delete 136 edit 136 Fluorescence Tool 130 Footswitch IR laser 123 UV laser 116 Freeze Mode 130 Functionality 7 PALM RoboSoftware 3 1 0106 EN G Graphic Tool activate 59 AutoCircle 61 circle 60 dot 60 freehand 59 line 60 rectangle 60 Reference Point 59 ruler 80 text 61 types 58 Group of elements add elements 79 forming 78 remove elements 79 select single elements 79 ungroup 79 H Help general 18 PALM Robolnstructor 18 Imaging Software incorporating 28 start 11 Import of elements 85 Information Center functionality 8 start 11 Initial settings 23 mode 23 Installation 9 K Keyboard shortcuts summary 147 L Laser IR 3D Offset 125 PALM RoboSoftware 3 1 0106 EN Index combination focus 118 move objects in z axis 124 set focus using the mouse 120 settings 117 switch on and off 124 Laser UV
184. ndless approx fin sec fin sec Mode Size MByte Video gt Time Lapse 1 gt Time Lapse 2 10 Switch off transm lightfclose transm light shutter between picture recording Close reflected light shutter between picture recording Trigger Point Definition f Manually f Foot switch Cut LPC C LPE laser Function C Scheduled cora To enter the settings proceed as follows Activate the desired functions using the check boxes and or buttons and enter relevant infor mation in the fields All the settings are described in detail below Once you have configured your settings click on the OK button to close the window You should enter various items of information on your sample in the Experiment section 92 Enter a name for your series of images or video sequence in the Name field this field MUST be filled in You can enter a description in the Description field The File Prefix field contains the prefix for the name of your video or image files The content of the Name field is entered automatically as a default it can however be modified or added to although the field must not be left blank The PALM RoboSoftware automatically adds date and time to the prefix entered in the field Every video and image file thus receives a unique name If you are working in File Mode refer to chapter 1 6 File Mode and Database Mode all images and video sequences are saved
185. ng Folder to Store Images In 9 2 Specifying Folder to Store I mages In Note If you are working in Database Mode all your images will be stored in the database In this case the folder that has been specified for storing the images Is irrelevant You can specify a folder for storing all images that are stored in File Mode either manually automat ically or using the Time Lapse function If you are storing images manually then you can choose a different folder at the time you store them To specify a folder Setup PALM Robo e Inthe Setup menu click on menu item PALM Robo The Preferences and Configuration window opens e Click on the Images tab Saving Images Folder sjadminitly Documents My PalmDatalImages A Filename Image The name and full path of the currently set folder appears in the Folder field of the Saving Imag es section Now you can either specify a folder via the Folder field or via the button to the right of the field or create a new folder If you want to specify a folder in the Folder field e Enter the name and full path of a folder in the Folder field Info You can also enter the name of a folder that does not yet exist It will be created automat ically when you exit the window If you wish to select a folder using a button e Click on the button The Image folder window will open A directory tree structure is displayed in the window an
186. ng Images in File Mode 0 0 aaa a a 89 Saving Images in Database Mode a a a 90 Saving Images Automatically during a Laser Function o o a ee ee es 90 Saving Images and Videos Using the Time Lapse Function aaa aoaaa e o 91 Cutting and Catapulting ia A EES Slee eee eS 95 Laser FUNCOMS a sera a tele cae hed tar Bid ae tegen Pec ee A do Be ok Ee aa hoe Ae a de ee a OR 95 PrepahatlonS 4 4 a Bde ae Be Ri ba ee ee dle AD RA Bd ra Ad ae 97 Selection of Automatic Laser Functions o a e a a 97 Preview of the Automatic Laser Functions e a 98 Specifying Laser SettingsS lt 2 6 po sb awe AA dae AA a eee eae ee dd 100 Energy and FOCUS aaa ad a oe ee eee ES a ee ee Ee eS 102 Activating and Deactivating AUTO f0CUS a a a a a 104 Other General Laser SettingS ana saaa a a a a a 104 Special Laser Settings for the AutoLPC and Close amp Cut AutoLPC Functions 104 Special Laser Settings for the RoboLPC Function o o oo e ee 105 Finding Suitable Laser Setting9S oo es 106 Releasing the Automatic Laser FunctionS o o e 108 SLOP COMUNUG EIA 110 The Setlal Sections FUNCION sa ire A Ae Ae ae a a 110 Cutting and Catapulting without Automatic Laser FUNCtIONS o oo o 116 TWeezers Option curan a a amp E E E A AAA AA 117 Essential Settings ai se A Se a id Ee ie es 117 Setting IR La
187. nothing obstructing the laser beam in the microscope s laser light path or conduct preliminary tests to determine the op timum settings for laser energy and focus For instructions on how to do this refer to chapter 10 6 Finding Suitable Laser Settings To determine the focus point of the IR lasers e Insert a slide that has been colored with a black felt pen e Switch the IR laser on briefly toggle the IR button in the right border of the program win dow to switch the laser on and off e Check the result You should see a small almost circular area where color has been removed Info In some circumstances you will not see any ef fect from laser activation or there will be a large hole in your sample In this case check that there is nothing obstructing the laser beam in the microscope s laser light path or try again with different settings for laser power and focus instructions on making these setting are included in chapter 11 2 Setting IR Laser Power and Focus PALM RoboSoftware 3 1 0106 EN To set the position of a Laser Marker Laser Marker Position Laser Marker F UV Laser IRA 1 Lazer IR 2 Lazer e Inthe Laser Marker menu click on menu item Position Laser Marker A submenu opens containing a list of all available Laser Markers e Click on the name of the Laser Marker whose position you want to change Depending on your selection you will be prompted to Click Cursor at new UV
188. nt of the Save Ele ment files automatically option in the Saving Elements section to enable or disable this function A tick in the checkbox indicates that this function is enabled then the program is closed an ele ments file will be saved with the name you have assigned containing the changes you have made since it was last saved e g new elements drawn or previous elements deleted e Close the Preferences and Configuration win dow by clicking on OK 82 8 2 Saving and Loading Elements in File Mode In order to be able to use your elements later you must save them separately from your images Info In the Element List window you can set the display so that only selected types of element are displayed in the window i e elements of other types do not appear in the Element List table refer to chapter 7 3 The Element List Window This has no effect on the Save oper ation all existing elements will be saved under all circumstances To save your elements in a file Save elements File Al Save Elements In the PALM RoboSoftware main window or in the Element List window e Click on the Save Elements icon button in the Tool Bar or click on menu item Save Elements in the File menu The Save elements to file window opens The ex act appearance of this window depends on which slideholder you are using chapter 3 2 Setting Sli deholder explains how you should notify the
189. nue Laser selected elements Chrl F11 w Navigator Window F4 E Element List F5 3 Microscope Window FE am Stage mode FY Invert stage motion a Freeze mode i 30 1 mode iL 30 2 mode Undelete Alt BackSp Select All Ctrl DeSelect All Ctrl Change AIRE nter Renumber all Copy Ctrl C Ca Delete selected Del e Delete last Back5p E Delete all unprocessed elements Ctrl y Create Group Add to Group Remove from Group Define Group Reference Figure UnDetine Group Reference F igure Match Serial Section Group Match Reference Point manual To open the context menu e Right click on any dot in the image As soon as you have selected a command or when you left click anywhere outside the context menu the context menu will close 17 Help and Information about PALM RoboSoftware 2 3 4 Software Modes Info This section refers to the software modes of the PALM RoboSoftware These are totally unrelated to the Database and File operating modes PALM RoboSoftware works in a number of modes Cursor Mode Stage Mode Freeze Mode Scrolling Calibration Laser Point Continuous Position Laser ON 3D 1 3D 2 The active mode is displayed on the Status Bar You can and in fact must switch between Cursor Mode and Stage Mode or Freeze Mode and between Cursor Mode and 3D 1 or 3D 2 Mode manually All other modes are activated deactivated automatically by the program when the corresponding functions are se lected Cursor Mode This
190. nue function refer to chapter 10 8 Stop Continue starts the UV laser functions even if you only moved the stage without laser function before the stop page Therefore you should never use the Continue command to resume a movement of the stage in which the laser function was not activated because this will always switch the UV laser on as well instead you should use one of the op tions described in chapters 5 1 1 to 5 1 4 and SA7 The Stop function has no effect on the IR la ser PALM RoboSoftware 3 1 0106 EN Setting Speed 5 3 Setting Speed You can set the speed separately for every type of stage movement Stage Movement speed in Stage Mode move using mouse Arrow keys Movement speed using arrow keys For movement with the arrow keys as was described for Position you can set higher speeds than for the other movement types This movement type is therefore particularly suitable for rapid movements over larger dis tances Scroll Speed of movement when scrolling moving the stage by left clicking the mouse outside the Scroll Rectangle Position Speed for automatic targeting of ele ments and defined positions Check Point Load Position and speed for automatic laser function LPC the defined dots for this func tion are targeted sequentially at the set speed For this movement type as was described for Arrow you can set much higher speeds than for other movement types
191. o close the Element properties window click OK 73 Changing Colors and Line Thickness If you have the Element List window open A Change Edit Change e Click in the Element List window on the ele ment you want to name or annotate e Click on the Change icon button or Click on menu item Change in the Edit menu The Element Properties window will open e Enter your name in the Name field or your comment in the Comment field e To close the Element properties window click OK 74 7 8 Changing Colors and Line Thickness You can change the colors and line thicknesses with which the elements and laser dots are dis played on the monitor The new settings apply to new elements drawn thereafter The changed set tings are retained after the program is closed down If you restore the factory defaults refer also to chapter 3 5 Restoring Settings all the changes you made to color and line thickness set tings will also be lost For elements that have already been drawn colors and line thicknesses can also be changed later Info Select the colors so that they contrast strongly with your sample This way your elements will be more readily recognizable 7 8 1 Selecting Colors for Elements to be Drawn New PALM RoboSoftware includes a Color Palette and a window in which you can select the colors you want available There are 18 colors and three shades of gray besides black and wh
192. o not need to save your el ements manually every drawn element is saved immediately and automatically in the database To load elements from the database Enter Select Data DATA File Enter Select Data e Click on Enter Select Data in the File menu in either the main window or the Ele ment List window or click on the DATA icon button The Please select or enter data window opens Please select or enter data Slide1 Slide2 Collection Device Data Source Data Label Liver Type Description Specimen Data Label Description Holder Label Holder Type Experiment Data Label Description Blue labeled fields are mandatory You must enter all blue fields to enable a position For working e If you are using a slideholder that can accom modate more than one slide select the tab for the slide for which you wish to load elements In the Source Data Specimen Data and Ex periment Data sections of the selection lists in the Label fields you will find all the entries that you have written to the database refer to chapter 7 1 2 Working in Database Mode e Open these selection lists one at a time start ing at the top and select the desired entry e Close this window by clicking on OK Your elements are displayed on the monitor and in the Element List window PALM RoboSoftware 3 1 0106 EN Exporting and Importing Elements 8 4 Exporting and I
193. o your sample PALM RoboSoftware 3 1 0106 EN 10 3 Selection of Automatic Laser Functions You can select the automatic laser functions in the PALM RoboSoftware main window and in the Ele ment List window chapter 7 3 The Element List Window includes instructions for opening this window In both windows you are offered the Cut Tools and a menu command Apart from that you can establish how often the selected laser function should be applied to an element Note If your system is equipped with a PALM Robo Mover and if you want to use the functions to position the caps automatically you have to select the desired laser function in the PALM RoboMover window there you must start these functions too refer to the Manual of the PALM RoboMover To select an automatic laser function using a menu command e Click in the PALM RoboSoftware main window or on item Laser in the menu bar in the Ele ment List window The Laser menu will open in both cases Among other items this includes a list of available laser functions Laser Cut JaintCut Cl losetCut LPC Autol PE Close Cut Autol PE Robal FC ASutoCircle e Click on the required laser function A tick beside an entry shows which laser function is currently selected Info The number of operations cannot be set by a menu command Carry out this setting as de scribed below in Cut Tools in the main win dow or in the window Element Li
194. objective down as far as possible e Click on the Check Point icon button in the Tool Bar or press key combination Ctrl K or e Click on menu item Goto Check Point in the Move menu The following message will appear Clear Path way Objective must be down e Click on OK in the message window The message will disappear and the stage will be returned to the position it was in before it was au tomatically moved to target the Check Point 47 The Load Position 5 6 The Load Position When the stage is in the Load Position you can easily change the slide and the slideholder PALM RoboStage Type II only Caution Set the objective to the lowest possible posi tion before you move the stage to or from the Load Position If the objective is set too high the stage will collide with the it This can cause serious damage to the objective and to your sample if you have purchased a system with motorized microscope the objective is moved automatically To move the stage to the Load Position G Load Position Move ue Goto Loadposition e If you do not have a motorized microscope Move the objective down as far as possible e Click on the Load Position icon button in the Tool Bar or Click on menu item Goto Loadposition in the Move menu The stage is moved to the Load Position The PALM Robo Select Slideholder window will open PALM Robo Select Slideholder The loadposition i reached
195. omize Element List 64 User Interface 15 Cut 95 D Database Mode change database 10 change to File Mode 10 Information Center 8 load elements 84 PALM RoboSoftware 10 save elements 84 save images 90 Delete elements all 68 any 69 last drawn 69 Deselect elements element list 68 Detail scan 51 go to overview scan 51 149 Index Double Beam system 145 Draw elements AutoCircle 61 circle 60 dot 60 line 60 line freehand 59 measuring line 80 rectangle 60 Reference Point 59 resume 70 text 61 Element List customize display 64 display of the element types 64 information of the elements 62 open 62 overview of functions 65 Element types AutoCircle 58 dot 58 line 58 Reference Point 58 ruler 58 text 58 Elements add comment 73 add elements to a group 79 center 66 change color 77 change comment 73 change correct shape 71 copy 72 create new set 56 definition 145 deselect monitor 67 display 65 draw 58 edit 70 export 85 group 78 hide 65 import 85 load 83 merge 71 naming 73 number 73 print 86 process 68 remove from a group 79 reposition 70 reposition Scroll function 41 restore 69 72 150 resume 70 rotate 72 save 81 select within a group 79 set color 74 set color for new elements 75 set dot thickness 76 set line thickness 74 76 settings for saving File Mode 81 show anchor points 70 stretch 72 stretch and rotate 72 switch on a
196. on depending on which value you wish to enter or click as often as necessary on the arrow button below the slider or click once and hold the mouse button pressed down until the required value is displayed in the field between the slider and the arrow button Info Please note that the values for Cut and LPC can only be changed separately if you have de activated the combination as was indicated earlier in this chapter PALM RoboSoftware 3 1 0106 EN e Where appropriate select the other tab and carry out the process as just explained e To confirm your changes click OK The window will close To set laser energy and laser focus with menu commands or with keyboard shortcuts Energy Power more Fage Up Energy Power less Fage Down Focus up Home Focus down End Note With menu commands or keyboard shortcuts you change the settings for the laser functions that are set in Laser Tools You also change the settings when the laser settings are not dis played however you then have no control over the changes that have been carried out There fore before you use these commands switch to the Laser Tools display and select the laser function for which you want to change the en ergy and focus as described earlier in this chap ter Setting with menu commands e Click on items Energy Power more or Energy Power less in the Laser menu to increase or reduce the laser energy e Inthe Laser menu c
197. ong defined lines Start and end dot of a line element are connected by a straight cut line It remains a joint between start and end dot With this function geometrical figures can be cut The cut area remains fixed within the tissue The entire area can be catapulted later by a single laser pulse With this function only the cells upon which you have drawn a Dot element are catapulted into the collecting vessel AutoLPC With this function the contents of a defined area of glass mounted samples are catapulted into your cap The area is therefore not catapulted in one piece but with several laser pulses The original structure of the material is not retained when us ing this function 95 Laser Functions Both this function and the following one Close amp Cut AutoLPC can only be used for glass mounted samples e these functions cannot be used if the sample is placed on a membrane LPC membrane With glass samples only a limited amount of cell material per laser pulse can be catapulted out with one laser shot Larger areas have to be catapulted with multiple laser pulses The exact position of the laser pulses is calculated by PALM RoboSoft ware you have to define only the border line of the area and the distance LPC distance between the laser pulses as well as their orientation diag Onal or parallel The required distance between the single laser pulses depends on the qualities of the tissue Close
198. ons function enables series of samples especially those whose structures only become visible following a specific treatment such as coloring to be processed significantly more quickly only a single sample needs to be treated accordingly You then only need to draw the ele ments for this one sample and then transfer them to the other samples Through the adaptation when transferring elements your elements will also be placed on the other samples on the desired spots Note that the adaptation can only function satis factorily if the relative position of the structures on the various samples is identical If the tissue block out of which you cut the samples contains a diag onal structure for example and the other struc tures are vertical the differences cannot be automatically compensated for You have the pos sibility to correct such variations by hand after the transfer procedure To keep any necessary correc tions small it is recommended that the samples are processed in the same sequence as when they were cut out of the tissue block and to transfer each of the elements from one sample to the next 1 gt 2 2 gt 3 if necessary after manual cor rection The sample for which you draw the elements is re ferred to below as the source sample The sample to which the elements are transferred is referred to as the target sample You can carry out the transfer and adaptation with the PALM RoboSoftware the adaptation if re
199. oportions of the elements are not retained when stretching the elements To rotate elements lines rectangles ellipses AutoCircle K Ctrl key Pointer Tool e Select the element you want to rotate e Drag the Pointer on one of the squares at the corners of the selection rectangle e Press and hold the Ctrl key The cursor changes to a circular arrow e Pull with depressed mouse button The selected element is rotated The square oppo site to the selected square serves as point of rota tion 72 To rotate and stretch elements lines rect angles ellipses AutoCircle ls Ctrl key Shift key Pointer Tool e Select the element you want to stretch and rotate e Drag the Pointer on one of the squares at the corners of the selection rectangle e Press and hold the Ctrl and Shift keys The cursor changes to a circular arrow e Pull with depressed mouse button The selected element is rotated and stretched The Square opposite to the selected square serves as a bench mark To restore a changed element Edit Restore e Select the element you want to restore e Click on menu item Restore in the Edit menu The last changed element will be restored Note If you have joined two elements and if you use the function Restore for the new developed element the original element with the lower number is restored The original element with the higher number cannot be restored 7 7 3 Copying Ele
200. or focus setting 140 switching the lamp on and off 139 142 Work Position meaning 141 Microscope Tools 139 Microscope Window 140 Mode 18 3D Mode 127 Cursor Mode 40 Stage Mode 39 Naming element 73 Navigator launching and exiting 49 load scanned image 52 overview scan 50 save scanned image 52 152 O scan a detail 51 scanning sample 50 show elements 51 Optovar 143 Overview scan P go to detail scan 51 PALM Robolnstructor 18 Position marking text element 61 Position stage 39 center elements 66 centering tool 41 in Stage Mode 39 keyboard 41 return from Check Point 47 return from Load Position 48 scroll 40 scroll bar 41 set speed 43 stop movement 42 target Check Point 47 target Load Position 48 with mouse in Stage Mode 39 with mouse Scroll 40 Preview of UV laser functions 98 Print elements 86 Program Q exit 10 functionality 7 general help 18 installation 9 layout 13 operation 17 PALM Robolnstructor 18 software modes 18 standard configuration 13 start 10 Quadratic Attribute 60 80 R Reference 146 PALM RoboSoftware 3 1 0106 EN Reference Position 3D Offset define 122 meaning 146 target 123 Reflector Offset adjust 136 reset 137 Restoring deleted elements 69 Ruler 80 S Save elements 81 Save images 89 90 automatic during laser function 90 Copyright Note 87 create folder 88 manual 89 90 select folder 88 specify file name
201. or instructions on how to proceed PALM RoboSoftware 3 1 0106 EN Filter Colors and Names list Information about the existing filters up to six if a Ludi fil ter wheel is installed up to eight if a micro scope filter wheel is installed You can make or change any entry yourself in the Color column refer to chapter 12 8 2 Specifying Names for the Fluorescence Excitation Filters for instructions on how to proceed If you have installed a microscope filter wheel the names that were specified in the control program for the microscope appear in the Name column If you have installed a Ludi fil ter wheel this column is empty 12 8 2 Specifying Names for the Fluorescence Excitation Filters You can specify names for your installed fluores cence excitation filters e g color names If you Subsequently select an excitation filter the names you specified will appear in the selection list Setup Filter e Inthe Setup menu click on menu item Filter The Fluorescence Filter window will open e n the table double click in the row of the filter for which you want to change the color informa tion contained in the Color column e Enter the desired name for the filter e Repeat for all filters which you want to rename e To confirm your change click OK The window will close PALM RoboSoftware 3 1 0106 EN Fluorescence Filter 12 8 3 Selecting a Fluorescence Excita tion Fil
202. or transfer Note ring the elements to further samples When creating the reference elements choose exactly the same sequence as for the source sample Otherwise the references will not be correctly collated e Ifyou are using a multiple slideholder then you should click on the tab associated with your tar get sample in the Element List window Slide 2 in the example image shown below e Group the three elements and allocate the group a name e g Slide 2 PALM RoboSoftware 3 1 0106 EN 113 The Serial Sections Function Element List File Edit Move Laser D Ars pt Lt cu y Slide Slide2 Slides Summary Show Types Area um 11 Freehand 138521 sli E 12 Freehand 128639 sli 2 13 Freehand 135713 sli 14 slidez Group e Select the three drawn elements and click on the menu item Define Group Reference Fig ure in the Edit menu not applicable if you have drawn elements of the Reference Point type This opens the Groups window e Choose the group to which the selected ele ments belong in the example Slide 2 and click on the OK button to close the window The PALM RoboSoftware creates three new ele ments of the Reference type for the target sam ple Element List File Edit Move Laser D ars a C1 fcu Slide Slide2 Slides Summary Show Types ie 11 Freehand Ref 1 138521 sli a 12 Freehand Ref 2 128639 sli A 13 Freehand Ref 3 135713 sli i4 slide
203. osi tion for the IR laser beam If you are working with a PALM MicroTweezers Single Beam System with out 3D Laser Beam Positioning the Reference Po sition is not important to you except as an indicator of the IR laser focus However the Reference Position assumes greater importance in the system with 3D Laser Beam Po sitioning If you have moved the IR laser beam to any position during your work you can instruct the system to return it to the Reference Position This function is useful if for example you have moved the beam into an area outside the image section displayed on the monitor and you are perhaps no longer certain of the exact location of the beam You can change the Reference Position at any time If you have purchased a system with two 3D movable IR laser beams you can define a separate Reference Position for each beam Note The IR Laser Marker must always indicate the IR laser focus of the laser beam Otherwise the IR laser will not yield satisfactory results Therefore you should never place the position of the IR Laser Marker anywhere except on the IR laser focus To define a reference position for the IR laser beam e Insert the slide that you colored with the black felt pen into the stage you can also use a sam ple that you no longer need for your work e Switch the IR laser on e Set the power so that you can see the effect of the IR laser beam e g an eroded site on the colored slide e
204. ot which should be displayed on the monitor The stage will be positioned on the desired spot Info You can position the stage by clicking on the proof scan To do this change to the tab Scan and select Slide Section or Section 1 PALM RoboSoftware 3 1 0106 EN Analyzing Images Using the Cellenger Software 6 4 Analyzing Images Using the Cellenger Software The Cellenger software enables images of sam ples to be analyzed using sets of rules that have been defined in advance and also allows elements to be generated This helps with routine operations to automate the selection of the areas of interest Info You can only avail yourself of this analytical facility if your system includes the Cellenger software Please contact P A L M Microlaser Technologies GmbH if you wish to upgrade your system e Scan the section of your sample that you wish to analyze as described in chapter 6 2 Scan ning a Sample with PALM Navigator e Select the Cellenger Analyze tab PALM Navigator Navigation Scan Cellenger Analyze Axiovision Analyze Minimum area Analyze Ruleset v so Funktionellen Regelsat Slide Minimum brightness 100 Delete elements Maximum brightness A 150 Analyze AOI M Use autofocus suspend scan for focus e From the Analyze Ruleset selection list choose the set of rules you wish to use for anal ysis A number of sliders for setting various parameters now app
205. ou can and must disable Stage Mode and Freeze Mode yourself To use the other movement methods you must be in Cursor Mode the modes are automatically entered as soon as you select the appropriate functions and automatically exited at the end of the movement task You can immediately interrupt the movement of the stage for example in case of an emergency The speed with which the stage is moved can be programmed separately for each movement type PALM RoboSoftware 3 1 0106 EN 5 1 Movement Types 5 1 1 Positioning the Stage with the Mouse Stage Mode In Stage Mode you control the movement of the stage synchronal with the mouse Each mouse action is translated into a movement of the stage You can perform movements in any required direc tion If you are using an objective that inverts the im age a mouse movement to the right in Stage Mode will appear on the monitor as a movement to the left If you select Invert Stage Motion the stage will be moved in the opposite direction to the mouse movement In this case the motion of the image on the monitor is in the same direction as the movement of the mouse To invert stage movement Mowe Invert stage motion e Click on menu item Invert stage motion in the Move menu or open the context menu by clicking the right mouse button and select menu item Invert Stage motion A tick beside this menu item indicates that the function is enabled Info You must invert
206. ou do not have to accept this specification You can also save your settings later in another folder You can also create a new folder when saving specify whether the settings should be saved automatically when the program Is closed To specify a folder for saving the settings Setup PALM Robo e Inthe Setup menu click on menu item PALM Robo The Preferences and Configuration window opens The Configuration tab will be displayed Saving Settings Folder E Documents and Settings adminMy Docume The name and full path of the currently set folder appears in the Folder field of the Saving Set tings section Now you can either specify a folder via the Folder field or via the button to the right of the field or create a new folder If you want to specify a folder in the Folder field e Enter the name and full path of a folder in the Folder field Info You can also enter the name of a folder that does not yet exist It will be created automat ically when you exit the window If you wish to select a folder using the button e Click on the button The Setting Folder window opens A directory tree structure is displayed in the window and the currently set folder appears underneath in the Folder field e Select the desired folder or create a new folder e Close the Setting folder window by clicking on OK You are back in the Configuration register in the Preferenc
207. our line e Click on the point where you want to start your line and hold the mouse button down e Move the cursor with the mouse button held down the line is displayed e Release the mouse button if you want to set a corner point and press it again when you want to continue drawing your line The continuation of the line is displayed e Double click on the dot where you want to end your line or e Click once on the dot where you want to start your line e Click once on the point at which you want to set a corner dot e Double click on the dot where you want to end your line 7 2 5 The Rectangle Tool mj Rectangle Tool E Quadratic Attribute ea Centric Attribute 60 Use the Rectangle Tool to draw rectangles The dot at which you begin your drawing becomes a corner of the rectangle e Select the Rectangle Tool in the Graphic Toolbar If you select Quadratic Attribute as well the drawing function is limited to squares If you select Centric Attribute as well the dot at which you begin your drawing becomes the center point of the rectangle or the square e Move the cursor to the dot where you want to begin your drawing e With the mouse button pressed down drag the rectangle or square out until it is the desired size and then release the mouse button 7 2 6 The Circle Tool Dl Circle Tool Hl Quadratic Attribute ey Centric Attribute Use the Circle Tool to draw circles or ellipses The dot at w
208. ping the left mouse button depressed pull the border to the required width or e double click on the column separator line in the table header The column width is set to the smallest width that still allows all entries to be read To change the sort sequence e click on the title of the column you want to use as the sort criterion Click on the column title again to reverse the sort sequence A triangle with the apex pointing up or down ap pears in the table header for the corresponding column This indicates that the entries in this col umn are sorted in ascending or descending order PALM RoboSoftware 3 1 0106 EN Displaying and Hiding Elements 7 3 4 Functions in the Element List Window Creating a new set of elements File Mode Saving elements Loading saved elements Export information of the elements Import information of the elements Printing of elements to a file or on a printer Selecting and deselecting elements Changing properties of the elements Deleting single elements or all elements only elements that have not yet been processed with the laser can be deleted Updating the Element Numbering Forming and editing groups of elements Centering elements on the monitor Working with the Serial Sections function re fer to chapter 10 9 The Serial Sections Func tion Selecting elements for laser functions Selection of laser function Starting the laser
209. point for the laser action if you have en abled this function refer to chapter 10 5 3 Other General Laser Settings for instructions on how to do this otherwise it remains stationary at the spot where the last laser action took place If you made the corresponding prior setting an image will be saved before and or after processing of each element refer to chapter 9 4 Saving Im ages Automatically during a Laser Function for instructions on how to make these settings If you have not drawn any elements an error mes Sage is displayed If you have only drawn or selected elements on which the selected laser function cannot be used e g LPC for cut lines or Cut for LPC Dots an error message is displayed and no laser function takes place On conclusion of the laser operation your elements are displayed on the monitor again this only takes place during the laser operation if you have made the appropriate settings refer also to chapter 10 5 3 Other General Laser Settings This dis play serves as a check of the laser function Ele ments that have been processed with the laser are displayed on a green background in the Element List window 109 Stop Continue 10 8 Stop Continue In case of emergency you can stop all functions and movements in progress To stop all current movements with one key board shortcut e Press Esc on the keyboard To stop all current movement in main window D S
210. porting and Importing Elements The file contains all the properties of the ele ments that are displayed in the Element List window display color number element type number of laser actions carried out area size comment position and the coordinates of their anchor points Print Elements prints your elements on a printer or generates an Excel import file You can open such a file with Microsoft Excel or with another corresponding program The file con tains all element properties as you have defined with printing of the elements refer to chapter 8 5 Printing Elements PALM RoboSoftware 3 1 0106 EN Info When you save images you can specify that they are to be saved along with superimposed elements refer to chapter 9 Saving Imag es However it is always only an image of the elements that is stored and you cannot load or import it back into the PALM RoboSoftware window again 8 1 Settings for Saving Elements in File Mode To save elements you can specify a folder When you save your settings later this folder will be selected automatically However you do not have to accept this specification You can also save your elements later in another folder You can also create a new folder when saving determine whether an element file which has already been saved but subsequently changed is to be saved automatically when you exit the program or that the changes made are to be rejected The
211. pter 5 3 Setting Speed for instruc tions on how to proceed Speed for the Position movement type To center an element by moving the stage To center an element you can also scroll over the Sample or position the stage with the arrow keys refer to chapter 5 Positioning the Stage for in struction on how to do this However these meth ods can only help the process if you know where your element is PALM RoboSoftware 3 1 0106 EN Selecting and Deselecting Elements 7 6 Selecting and Deselecting Elements You can select elements either on the monitor or in the Element List window Which method you should choose depends on the next operation you want to do with the selected elements If you want to edit elements as described in chapter 7 7 2 Editing Elements you have to select them on the monitor For all other operations with elements you can select them on the monitor or in the Element List window It is often more simply to do this in the Ele ment List window because here you have an overview of all drawn elements Note Note that the elements selected on the mon itor are not selected in the Element List win dow and vice versa With both methods you can select a single ele ment multiple or all elements 7 6 1 Selecting and Deselecting Ele ments on the Monitor On the monitor selected elements are displayed with a surrounding white rectangle At all corners and in the center
212. quent catapulting with the LPC laser function AutoCircle Tool Use this tool to mark areas in your sample which you can then catapult Un like the Dot Tool the AutoCircle Tool is used to draw a circle with a variable radius With the AutoCircle laser function the area is cut out along the circumference of the circle and cata pulted subsequently Ruler Use the Ruler to measure zonal lengths The zone is shown as a line Text Tool Text With the Text Tool you can identify individual areas of your sample so that for example they can be easily located again and annotated These comments are then displayed at the specified position on the monitor Reference Point Tool Use this tool to define ref erence points For example you need them for the Serial Section function refer to chapter 10 9 The Serial Sections Function Ruler Text and Reference Point type ele ments have no effect on the laser functions The elements are displayed on the monitor as col ored lines dots or areas You can preset the colors and line thickness or the dot sizes for display and you can change them later Refer to chapter 7 8 Changing Colors and Line Thickness for instruc tions on how to do this Note It is sometimes not possible for the laser to process elements located very close to the edge of the slide If you draw elements here they will be deleted automatically by the PALM RoboSoftware When instal
213. r Pressure Catapulting LMPC This technology combines microdissection in which the samples are cut out with zero contact with Laser Pressure Catapulting LPC Laser Pressure Catapulting LPC With a single laser pulse objects marked with an LPC dot are aimed and catapulted into a cap Load Position Stage Position of the stage on which the slide holder or slide respectively can be changed The stage can be moved to the load positon and back by a menu command only Stage II Load Position Focus Default focus settings of the microscope which can be set by a menu command motorized micro scope only lowest possible position of the objec tive Before the stage is moved to its Load Position or to the Check Point the Load Position of the ob jective must be set to avoid damage to the objec tive or the stage and the slide respectively 146 Reference Position IR Laser The Reference Position of the IR laser is the point at which the laser beam hits the sample The as sociated Laser Marker must be referenced to this point When you have set the position for the Laser Marker you have also defined its Reference Posi tion The Reference Position assumes particular impor tance with a PALM MicroTweezers System with 3D Laser Beam Positioning With this configuration you move the laser beam to the Reference Position with a menu command Reference Stage PALM RoboSoftware uses the Reference Position of the sta
214. r 10 5 Specifying Laser Settings and chapter 11 Tweezers Op tion Settings for saving elements chapter 8 1 Set tings for Saving Elements in File Mode Settings for saving images chapter 9 Saving Images PALM RoboSoftware 3 1 0106 EN 3 1 Launching Settings If your system does not include database function ality you will always be working in File Mode In this case there is no need to configure the launch settings If your system does include database functionality you can choose whether to work in Database Mode or File Mode The PALM RoboSoftware allows you to specify which mode the program should Start in File Mode the program will start up in File Mode database functionality is not available Database Mode with selectable database when the program is launched you can select which database you would like to use or whether you wish to work in File Mode Database Mode with pre selected database you can specify a database for PALM RoboSoft ware to open automatically when it is launched Info The procedure for creating a database Is de scribed in the Information Center Manual To specify which mode PALM RoboSoftware should start up in Setup PALM Robo e Inthe Setup menu click on menu item PALM Robo The Preferences and Configuration window opens The Configuration tab will be displayed Operating Mode C File mode only l Database mode with selectable DB
215. ram Layout and Operati0DM 0oo ooooooooonnaa 13 2 1 The PALM RoboSoftware Layout o a 13 2 2 Customizing the User INt6rtaceOs star dd er ey See ae ae ee ee ea ae 15 2 3 ODC ATION wm ae frase eS oe ao rd ae ae Be ee ee A ee ee ae 17 2 31 MOUSE Lata a care ae a E dd o a ee A 17 2 3 2 Key DOTA SOM CUS cuca ad Bee WEA Bee oe RRS a ee ea ee A 17 o COMO MCU Ao 2 yaa A ae ue aa AN 17 234 SOnRWare MONOS id 24th 4 oe eee eee o ae ee e eo oe ieii 18 2 4 Help and Information about PALM RoboSoftware o o e es 18 2A ONIS NECIO e rai aa a be A AS A ae 18 2A2 TRE PALM RODO AStrUctor cs ca dol le a sh del dai da E ti ed op ean 18 2 4 3 Program and Manufacturer minie cia ar Aa a a a 19 DG SYSECMiINIOMIMATIONS er car lo leido ral MB ee la a ees GE de 20 2 4 5 PALM RODOWINTONM AION sy vicio a as a ek A A A a id A 20 2 5 Sta US DIS lays e rr la or Ao ata dd e or ees cece reco da 21 3 Settings ptas dos ace ta td a as as eee 23 3 1 MAUACMING SENOS i sto o paa a E A e AA 23 3 2 Serting SIMGCNOIGSE iy acs ia a add tee a ae e we ae ee 24 3 3 Turning on Automatic Focusing for Elements o o a 25 3 4 Saving ana Loading Seting asar ar eo A 25 3 5 Restorind SS CttndS mdd A A A De ee E Ai 27 3 6 Unlocking Add On Software COMpONentS a 27 3 7 Incorporating an Imaging Editing PrO9raM o o o o e e 28 3 8 nora Ter Alar stats Su a E a paa SR AA a ee
216. re or Energy Power less in the Laser menu to increase or reduce the IR laser power e Inthe Laser menu click on items Focus up or Focus down to set the IR laser focus higher or lower Setting with keyboard shortcuts e Use the Page Up and Page Down keys to raise or lower the IR laser power e Press Home or End to increase or reduce the value for the IR laser focus The values that are currently set are displayed in the Laser Tools 121 Determining Suitable IR Power and IR Focus Settings 11 3 Determining Suitable IR Power and IR Focus Settings The settings that you require for various samples will vary to some degree In order to determined the optimum power and focus settings you must experiment on similar samples with different set tings The factory defaults may serve as Suitable starting points for your experiments If in doubt start at very low power and adjust upwards grad ually until you obtain the desired effects The IR laser focus can be set using a slide for ex ample with an area you have colored with a black felt pen With sufficient power a hole appears in the paint film which should make the effect quite simple to assess The more precisely the focus is set the smaller the hole should be 122 11 4 Defining a Reference Position When you aligned the IR Laser Marker to the laser focus as described in chapter 4 3 Setting the La ser Marker you also defined the Reference P
217. re use When your system is first set up all the software components that you have purchased are already unlocked If you purchase additional components later you will receive a license key that can be used to unlock them To unlock add on software components P About Help fF About PALM Robo e Click on the About icon button in the Tool Bar or click on About PALM Robo in the Help menu This opens the window About PALM Robo License License Key 0425 3530 D4AC8 E BBO Enter Hew License e Click the Enter New License button in the License area A window will open up e Enter your new license key then click the OK button The new license key is displayed in the License key field in the About PALM Robo window the new software component can now be used Info If you enter an invalid license key by mistake your previous key will be entered automatically 2 Incorporating an Imaging Editing Program 3 7 Incorporating an Imaging Editing Program You can incorporate an image management and editing program e g Adobe Photoshop or ACD Systems ACDSee in such a way that it can be launched from within the PALM RoboSoftware This type of software will referred to as Imaging Soft ware from now on You only must define the pro gram path in the PALM RoboSoftware Info The image processing software is not a PALM product Neither does P A L M supply such software To define the
218. rious functions To open the Element List window View Element List FS e Click on menu item Element List in the View menu or click in the Status Bar on the field showing the number of elements Depending on the slideholder used you will find at least two tabs one for displaying totals and one or more for your slide s in the example shown there is one tab each for the 3 slides in a 3 slide holder The window can be resized If not all of the table s columns are in view use the scroll bar to move across the table 7 3 1 Items Shown on the Slide Tab The Show Types field allows you to decide whether all elements or only the elements of spe cific types are to be listed Lines Freehands Dots AutoCircles Rulers Text References etc 62 all Reference ok E ieee OO EOS E 233 4 x 203 7 um 434 9 x 296 3 um 259 4 x 215 4 um 200 0 x 200 0 um 200 0 x 200 0 turn 96 0 x 152 5 um 1429 0 x 453 4 um 77 2 x 94 0 um 15 8 x 16 4 um 15 8 x 16 4 um 200 0 x 200 0 turn 1174 5 x 424 9 um Area um 104912 0 39637 9 1537 8 1085 7 2087 4 1809 1 104510 6 39598 51 104297 8 39742 61 105230 3 39703 2 102825 0 39381 4 104833 0 39801 4 104545 0 39024 4 104371 4 39931 4 104780 4 39449 21 104059 1 39066 4 Information on individual elements in the columns of the table Color color in which the element is displayed on the monitor Nr sequential n
219. ry important functions the PALM Robo Instructor You can also call up information about the program and the manufacturer 2 4 1 Online Help Online Help provides information about the program layout and the meaning of the icon but tons the menu commands and the tools To open the Online Help Help PALM Robo Help Fl e Press Fl or click on PALM Robo Help in the Help menu The Help window opens e Click on the point in the Help window about which you have questions or open the glos sary To navigate you can also use the context menu Open the context menu by right clicking anywhere in the Help window and choose Back or Forward depending on whether you want to page backwards or forwards To close the Help window e Right click on the title bar A context menu will open Click on the Close item here or press key combination Alt F4 The Help window will close 2 4 2 The PALM Robo Instructor The PALM Robo Instructor explains in detail how to carry out the following functions Calibrating the System Defining the Lens Offset Saving elements in a file Retrieving elements from a file Info All of these functions are also explained in de tail in this User Manual chapter 4 Calibrat ing the System and chapter 8 2 Saving and Loading Elements in File Mode PALM RoboSoftware 3 1 0106 EN Help and Information about PALM RoboSoftware To open the PALM Robo Instructor Help PALM
220. s for you to change drawn elements at a later time The options avail able to you depend on the type of the element Figure elements lines rectangles ellipses and AutoCircle elements You can move these elements You can change their shape You can extend lines You can merge two elements into one Dot elements Ruler elements measuring lines and Reference elements You can move these elements Text elements You can move these elements You can change the text You can restore changed elements see section To Restore Edited Elements in this chapter Info Note that you need to select the element you want to edit on the monitor not in the Ele ment List window Elements are treated by PALM RoboSoftware as polygons they consist of straight lines and dots the anchor points Each element consists of at least two anchor points and a line With some functions for editing elements you can work more precisely if the anchor points are visible To show or hide the anchor points of the ele ments View Show Points e Click on menu item Show Points in the View menu The menu command works as a toggle The anchor points are displayed as white squares and are numbered 70 To move elements ts Pointer Tool e Select the element you want to move e Drag the Pointer on the element you want to move The cursor changes to a fourfold headed arrow e With
221. s of laser beams IR 1 and IR 2 Info If you have purchased a Double Beam System with a 3D movable IR laser beam IR 1 is the 3D movable laser beam and IR 2 is the fixed beam If you wish to divide the power equally between the two beams 50 50 e click on the word Balance If you wish to set the power of IR Beam 1 or IR Beam 2 to 100 e Click on item Trl or Tr2 The power of the other beam in each case will then be set to 0 If you wish to set different power values for each of the two beams e Drag the slider in the Balance field or click on one of the arrow buttons repeatedly or click once and hold the mouse button down until the desired values percentage of the total laser power appear in the fields situated between the slider and the arrow keys or click in one of the text boxes enter the required value and press Enter the value in the other text box will be automatically adjusted accord ingly To change the focus e drag the slider in the relevant Focus field or click on the arrow buttons or click and hold the mouse button down until the required value is displayed in the field between the slider and the arrow button or click in the text box enter the required value and press Enter 119 Setting IR Laser Power and Focus To set the IR focus using the mouse f wheel E e Open Laser Tools if it is not already displayed click on the entry Laser Tools in the V
222. s soon as you have launched the program and adjusted the sam ple in the microscope If you have already drawn elements during your current session and you want to process another Sample you must first after saving it if you wish remove the existing element set from the monitor To prepare a new sample for processing in File Mode File New Elements e If you want to save previously drawn elements for later work save them as described in chap ter 8 2 Saving and Loading Elements in File Mode e Click on menu item New Elements in the File menu of the main window or click on item New Elements in the File menu in the Element List window chapter 7 3 The Element List Window includes instructions for opening this window or press the keyboard combination Ctrl N If any elements are displayed on the monitor they will be removed without a request for confirma tion If there are no elements on the monitor the File gt New Elements command has no effect 56 7 1 2 Working in Database Mode In Database Mode after the program has been launched you can initially draw only a single ele ment using the Freehand Tool refer to chapter 7 2 3 The Freehand Tool in order to test the la ser settings for instance To distinguish it from other elements this element is displayed on the monitor with a dashed line Before you can draw elements you must enter var ious details of your sample and your projec
223. saved image if they are dis played on the screen However other features such as Laser Mark ers crosshairs etc are not saved ba Save Image File ET Save mage e Click on the Save Image icon button in the Archive Toolbar or click on menu item Save image in the File menu Your image is saved in the database 90 9 4 Saving Images Automatically during a Laser Function When saving images automatically and in each case immediately before and or after a UV laser function the current microscope image will be au tomatically saved If you are working in File Mode refer to chapter 1 6 File Mode and Database Mode the images are saved in the specified folder refer to chapter 9 2 Specifying Folder to Store Images In If you are working in Database Mode the images will be stored in the database Setup PALM Robo e Inthe Setup menu click on menu item PALM Robo The Preferences and Configuration window opens e Click on the Laser tab W After Cut W After LPC Auto Image W Before Cut W Before LPC The Laser function section contains four Auto Image checkboxes You use these to specify whether the microscope image that is currently displayed on the monitor is to be saved automati cally before or after a laser function You can de cide whether images for laser functions or LPC functions or for both are to be saved e Click in one of the four Auto Image chec
224. selecting Elements on the Monitor or apply another function to position the stage e g the scroll bars refer to chapter 5 1 3 or make sure that the scroll arrow is outside the selection rectangle Note that you may not be able to see the selection rectangle if elements are selected which are outside the current monitor dis play 5 1 3 Positioning the Stage Using the Scroll Bars Scroll bars are located on the bottom and left edg es of the microscope image With these you can move the stage If you want to move the stage gradually e Click once on one of the arrow icon buttons on the scroll bar If you want to move the stage over a larger dis tance e Click once on one of the arrow icon buttons and hold the mouse button depressed until the Stage has reached the desired position or Click on the position cursor on the scroll bar and drag it to the desired position If you want to move the stage in steps of one im age width or height e Click beside the position cursor on the scroll bar 5 1 4 Positioning the Stage with the Keyboard e Press one of the four arrow keys The stage will continue to move until you release the key This allows you to move the stage up down and to the left and right If you press two arrow keys simultaneously e g gt you can also move the stage diagonally PALM RoboSoftware 3 1 0106 EN 5 1 5 Positioning the Stage with the Centering Tool The Centering Tool ca
225. ser Power and FOCUS a a 118 Determining Suitable IR Power and IR Focus SettingS o o 122 Defining a Reference Position ananasa aa a a a a 122 Settings for the FOOLSWItCM a a4 eaa A AAA A eek oe ee wae 123 Activating and Deactivating the IR Laser 0 oo e me 124 Moving Objects In the Z AXIS ss ats o a be hee da ed ee eek a a E te he de A 124 The 3D Laser Beam Positioning o a 125 Setting the Speed of the 3D Laser Beam PoOSItiONINQ o o 2 125 Moving the IR Laser Beam with the Mouse 3D 1 3D 2 Mode 127 Moving the IR Laser beam via the Keyboard o o es 127 PALM RoboSoftware 3 1 0106 EN 5 Contents 12 12 1 12 2 12 3 12 4 12 5 12 6 12 7 12 8 12 8 1 12 8 2 12 8 3 12 9 12 9 1 12 9 2 12 9 3 12 9 4 12 10 13 13 1 13 2 13 2 1 13 2 2 13 2 3 13 2 4 13 2 5 13 2 6 13 2 7 13 2 8 13 2 9 13 2 10 13 3 Working with Fluorescence Option 000 cee eee eee 129 General Introductory REMarkS veda wae tera ee a Ee Oe we ba ee eh Ee ED A 129 Settings for Working with Fluorescence Manual Microscope 0 0 0 00 eee 129 Settings for Working with Fluorescence Motorized Microscope ooo ooo 129 Activating a Fluorescence Settings Record 0 130 FREEZE Modera ot E Hn ee atk eg ee ee ed de Re 130 Opening and Closing the Shutter
226. set e Click on menu item Factory Lens Offset in the Setup gt Calibration menu A prompt will appear Do you really want to reset to Factory Lens Offset for this Lens e Confirm with Yes The default factory settings for the Lens Offset will be restored immediately Note The Lens Offset will also be reset if you restore the factory default settings refer also to chap ter 3 5 Restoring Settings 37 38 PALM RoboSoftware 3 1 0106 EN Movement Types 5 Positioning the Stage PALM RoboSoftware offers various methods of moving the stage with the mouse Stage Mode and Scroll with the scroll bars below and to the right of the microscope image with the keyboard arrow keys via commands that target predefined positions or elements positioning or with the PALM Navigator refer to chapter 6 3 Positioning the Microscope Stage When the automatic UV laser functions are start ed the stage is automatically moved along the de fined cut lines or from dot to dot You can set the speed to suit the various methods of moving the stage refer to chapter 5 3 Setting Speed In order to prevent the stage from being moved in advertently you can freeze its movement in any position i e disable all functions that would cause the stage to move The program distinguishes accordingly between the various movement modes and the Freeze Mode The active mode is displayed on the Status Bar Y
227. settings for saving elements can be car ried out in this way Setup PALM Robo e Inthe Setup menu click on menu item PALM Robo The Preferences and Configuration window opens e Click on the Elements tab Saving Elements Folder Save Element files automatically The name and full path of the currently set folder appear in the Folder field of the Saving Ele ments section Now you can either specify a fold er via the Folder field or via the button to the right of the field or create a new folder 81 Saving and Loading Elements in File Mode If you want to specify a folder in the Folder field e Enter the name and full path of a folder in the Folder field Info You can also enter the name of a folder that does not yet exist It will be created automat ically when you exit the window e Click on OK If you wish to select a folder using a button e Click on the button The Element folder window will open A directory tree structure is displayed in the window and the currently set folder appears underneath in the Folder field e Select the desired folder or create a new folder e Close the Element folder window by clicking on OK You are back in the Elements register in the Preferences and Configuration window Saving Elements Folder dminhMy Documents My PalmDatalElements r W Save Element files automatically e Click on the checkbox in fro
228. soft ware of which insert you are using The illustrations below show the windows for the Petri dish holder and the 3 slide holder Save elements to file if PetriDish Holder Petri_elements E OK Cancel Save elements to file ES W Slide1 Testslide palm a Iw Slider Testslide2 palm ie I Slides Testslide3 palm W all active Test We Abbrechen PALM RoboSoftware 3 1 0106 EN Saving and Loading Elements in File Mode If you are using a slideholder that can accommo date more than one slide this window can be used to save the elements for one or more slides at the same time The elements for each slide are saved ina separate file so that they can be reloaded sep arately see next section To load an elements file The window also enables you to enter a common name that is used automatically for all files before a suffix is added to produce a unique file name e If you wish to save the elements for all slides Tick the check box all active enter a common file name in the adjacent field to the right Test in the example shown above and then click the OK button e If you wish to save the elements for one or more slides Tick the corresponding check boxes Slidel Slide2 and or Slide3 in the example enter a file name for each and then click the OK button Your elements will be saved in the specified folder chapter 8 1 Settings for Saving Elements in File Mode explains
229. st 97 Preview of the Automatic Laser Functions To select an automatic laser function in Cut Tools e Inthe PALM RoboSoftware main window or the Element List window click on the arrow beside the selection field in Cut Tools A list opens displaying the available laser functions e Select the required laser function with a mouse click To set the number of operations in Cut Tools 1 RoboLPC e In Cut Tools either in the PALM RoboSoft ware main window or the Element List win dow double click in the checkbox beside the selection box for the laser functions or simply click on the checkbox and mark the available entry by dragging the mouse over it with the left button pressed down or simply click on the checkbox and delete the existing entry Del or Backspace key e Enter the required number of operations 98 10 4 Preview of the Automatic Laser Functions The display of the elements on the monitor de pends on which laser function you have set It serves as a preview of the laser functions Cut a a Cut lines element no 12 in the illustration above are displayed with the line thickness you specified refer also to chapter 7 8 Changing Colors and Line Thickness Elements of type Dot nos 13 and 14 are shown as unfilled circles elements of type AutoCircle no 15 are indicated by a thin circular line and an unfilled circle in the middle Close amp Cut am C
230. stem with an UV and IR laser click the UV button The Laser Tools for the UV laser will be displayed The current settings are displayed in the fields of the Energy and Focus blocks All values are set in analog form e Drag the slider or click on the arrow buttons or click and hold the mouse button down until the required value is displayed in the field between the slider and the arrow button PALM RoboSoftware 3 1 0106 EN Specifying Laser Settings To set the UV laser energy and UV focus in the PALM Robo Laser Settings window Laser Energy amp Focus UY Laser FY e Click on the Energy Focus UV Laser entry in the Laser menu or press F9 on the keyboard PALM Robo Laser Settings window opens It con tains a selection box for the magnification and two tabs for making energy and focus settings On each tab there are sliders and arrow keys for set ting the parameter for the cutting and catapulting functions PALM Robo Laser Settings Ea Cancel Values for Lens 5x r Energy Eocus Cut lt gt LPC Energy Energy Cut LPC Auto iw Change 100 Cut Delta LPC Delta e Check that the magnification of the objective set on the microscope is also shown in the Val ues for Lens selection box and correct the set ting as necessary e Choose the tab for the parameter you want e Pull the slider for the Cut function or the LPC functi
231. t With out this data your elements cannot be saved in the database When this data is entered distinctions are made between three hierarchical levels which you might use in the following way see illustration overleaf Source represents the tissue sample that you wish to process Specimen represents a tissue sample that is placed on the slide for processing Experiment represents the specific examina tion that you intend to perform on the tisuue Sample If you would like to create a new Set of objects for the same slide you should enter a new experi ment If you would like to work with a different slide you must enter the required data in the Specimen section If you would like to work with a different tissue sample you must describe it in the Source section PALM RoboSoftware 3 1 0106 EN Creating a New Set of Elements To prepare a new sample for processing in Database Mode Enter Select Data DATA File Enter Select Data e Click on Enter Select Data in the File menu in either the main window or the Ele ment List window or click on the Enter Select Data icon button The Please select or enter data window opens Info If you are using a multiple slideholder a sepa rate tab will be displayed for each slideholder position as well as a tab for entering data for the catapulted samples When you open this window the tab for the currently active slide is
232. t any time To reset the Reflector Offset Setup Calibration d Factory Retlector 0ffset e Click on menu item Factory Reflector Offset in the Setup gt Calibration menu A prompt will appear Do you really want to reset to Factory Reflector Offsets e Confirm with Yes The default factory settings for the Reflector Offset will be restored immediately Note The Reflector Offset will also be reset if you re store the factory default settings refer also to chapter 3 5 Restoring Settings PALM RoboSoftware 3 1 0106 EN 137 138 PALM RoboSoftware 3 1 0106 EN The Microscope Tools 13 Control Microscope Zeiss Axiovert 200M If you have purchased a system with a motorized The Microscope Tools enable you to make the microscope you can also control settings via PALM most frequently used settings the Microscope RoboSoftware For that the Microscope Tools in Window offers additional setting options the main window as well as the Microscope Win dow are available You can open it by menu command or via an icon button If you are using an ApoTome on your microscope its current status can be displayed on the monitor 13 1 The Microscope Tools 4 setting the focus to any position Focus Light This parameter can also be set using the mouse keeping the right mouse button pressed roughly set the focus with the Autofocus 8 mouse wheel fine adjustment can be per Sata 3200K 9 formed wi
233. t save them separately from the images On the other hand saved images can no longer be reloaded into the PALM RoboSoftware window However the supplied PALM RoboSoftware pack PALM RoboSoftware 3 1 0106 EN age also includes a program which you can use to observe and process your images the PALM Infor mation Center Instructions for working with the Information Center are the subject of the pro gram s user manual You can integrate an image processing program referred to in this document as Imaging Soft ware of your choice e g Adobe Photoshop or ACD Systems ACDSee into the PALM RoboSoft ware this can be done in such a way that you can launch the imaging software from within the PALM RoboSoftware Refer to chapter 3 7 Incorporating an Imaging Editing Program and chapter 1 8 2 Imaging Software for instructions on how to do this 9 1 Inserting a Copyright Note You can specify a Copyright Note that will appear on the image on the monitor and on each stored image if you have opted to show it this option can be switched on and off using the menu command View gt Copyright Setup PALM Robo e Inthe Setup menu click on menu item PALM Robo The Preferences and Configuration window opens e Click on the Images tab Copyright Info Copyright P 4 L M Microlaser Technologies e Enter the desired text into the field Copyright Info and close the window by clicking OK 87 Specifyi
234. t take place in the RoboLPC function as the sample pos sibly will not remain fixed within the tissue when cut 105 Finding Suitable Laser Settings 10 6 Finding Suitable Laser Settings Perfect cut and catapult results can only be achieved with optimal parameter settings The op timal settings are not however the same for all samples You must select the best suited settings for each type of sample by experimentation To achieve cut lines that are as fine as possible you should enter the lowest possible energy set ting with which you can still achieve the required cutting effect sometimes multiple cutting along the same line with lower energy achieves better results than a single cut with higher energy In addition to that the laser focus should be at the same level as that of your sample With a higher energy setting you cannot just cut within the laser focus but also above or below If the laser focus happens to be in the glass smaller or larger glass segments are catapulted out The catapult function requires a different energy and focus setting to that of the cut function For catapulting the laser beam is extended to an ap propriate value de focused You can select sepa rate values for the cut and for the catapult function or combine the values for the cut and catapult function When you then set the values for en ergy and focus for a function these values are automatically changed for the oth
235. t the Time Lapse function manually tr Start Time Lapse manually e Click on the Start Time Lapse manually icon button To cancel the Time Lapse function During video recording and the storage sequence a STOP icon button appears on the Tool Bar mi Stop e Click on the STOP icon button The Time Lapse sequence is canceled 93 94 PALM RoboSoftware 3 1 0106 EN 10 Cutting and Catapulting 10 1 Laser Functions With the PALM MicroBeam you can cut on the mi croscope and catapult parts of the sample De pending on the objective of your work you will need variable options of this function or combina tions thereof PALM RoboSoftware gives you the following automatic laser functions was iit eae ee T TE D e 2 With this function the laser cuts your sample along pre drawn lines Figure elements Lines rectangles and circles so that a clearly split line between required and non required material is generated In the cut area all biological material is destroyed by ablation Thus a pure sample preparation can be carried out without fear of contamination Close amp Cut With this function the laser also cuts your sample along defined lines Unlike with the Cut function however the start and end dot of a line element are connected here by a straight cut line PALM RoboSoftware 3 1 0106 EN Laser Functions JointCut As with function Close amp Cut the laser cuts your sample al
236. tations and with all graphic tools you can switch the display of the Scroll Rectangle on and off using menu command View gt Scroll Rectangle Outside the boundary of the Scroll Rectangle you can also draw with all graphic tools in Freeze Mode and with all graphic tools except the Freehand Tool in Cursor Mode Ifyou are in Freeze Mode and move outside the Scroll Rectangle with the mouse while drawing the stage does not follow the movement How ever you can continue to draw even beyond the border of the visible image You can check the result by exiting Freeze Mode again and moving the stage so that your element is displayed in its entirety on the monitor However this practice of drawing outside the border in Freeze Mode is not to be recommended because it means that you have no control over your drawing Ifyou are in Cursor Mode and you move outside the Scroll Rectangle with the mouse while drawing the stage follows the movement auto matically for all tools except the Freehand Tool for instructions on how to draw outside the Scroll Rectangle with the Freehand Tool refer to chapter 7 2 3 The Freehand Tool Select a low scrolling speed for the stage so that the movement of the image on the monitor can be easily followed with the naked eye 7 2 2 The Reference Point Tool ta Reference Tool Reference Point With the Reference Point Tool you define reference points on your sample For example you need them for the
237. tep 1 of 2 Click Cursor at actual 3D 1 Position e Click on the actual position of the laser beam with the mouse The following message will appear Step 2 of 2 Move this point to screen center and click again e The program will switch automatically to 3D 1 Mode Use the mouse to move the laser beam to the crosshairs at the image center and click the left mouse button The cursor will reappear the program automati cally switches out of 3D 1 Mode The following message will appear Calibration finished This message will disappear automatically after a few seconds If you have purchased a system with two 3D mov able laser beams calibrate the second laser beam IR 2 in the same way as for IR 1 Note The calibration always applies only to a magni fication that has been set before the calibra tion If you intend to work with various magnifications you must recalibrate for each change of magnification 35 Resetting Calibration 4 6 Resetting Calibration You can reset the calibration to the factory default setting at any time To reset the calibration Setup Calibration j Factory Calibration e Click on Factory Calibration in the Setup gt Calibration menu A message appears Do you really want to reset to Factory Calibration for this Lens e Confirm with Yes The manufacturer s default calibration is restored immediately 36 4 7 Adjusting the Lens Offset If you have drawn
238. ter The fluorescence excitation filter can be selected separately via the drop down list FL Setup Filter e Inthe Setup menu click on menu item Filter e The Fluorescence Filter window will open Actual Position 3 rhod Rhodamin ki e Open the drop down list Actual Position and select the desired position In the drop down list all possible filter settings are listed If you have entered names for the fil ters as described in chapter 12 8 2 Specifying Names for the Fluorescence Excitation Filters the names will be listed otherwise they will be numbered from 1 to 6 The filter wheel will move automatically to the selected filter position 133 Editing a Fluorescence Settings Record 12 9 If you need certain settings and sequences for flu orescence more frequently you can save them as a settings record You can create and save as many of these records as you wish You can assign a name to each setting These names will then appear in the Fluorescence Tool in a selection list in the FL field With this selection Fluorescence Fluorescence Names and used Functions Editing a Fluorescence Settings Record list you can then activate a saved settings record as described in chapter 12 4 Note In the Basic configuration with the manual microscope these functions are not active Mo hne fiter tute Feflcton Laver_ Licht Lazer 1 Laser DAPI 2 DAFI FITC 3 FITE T
239. th a keyboard shortcut or a menu command Move Setup Speed Fe e Click on Setup Speed inthe Move menu or press F8 on the keyboard The PALM Robo Speed window will open e Select the tab associated with the movement type for which you want to specify an increment the last two tabs are used for setting the speed of the 3D Laser Beam Positioning for the IR laser refer to chapter 11 8 1 Setting the Speed of the 3D Laser Beam Positioning Speed Delta 12 for Faster slower functions e Enter an increment in the Speed Delta for faster slower functions field e Close this window by clicking on OK 45 The Reference Position of the Stage 5 4 The Reference Position of the Stage Inthe drawing plane PALM RoboSoftware requires a Reference Position for calculating the coordi nates of your elements This reference point with its coordinates 0 0 is stipulated by the PALM RoboSoftware With each start of the program the stage is moved to the end positions in x and y di rection This position is defined as reference point Info The Reference Position is retained for as long as the controller unit remains switched on even if you shut down and restart the PALM Ro boSoftware The point of origin is particularly important when you have drawn elements and have saved them for later use The automatic positioning of the stage to the reference point ensures that on re loading your elements they will appear at t
240. th the mouse wheel and the left 10 mouse button held down 10000 pm 100 5 7 setting the focus to Load Position 5 Work Position 6 and Check Position 7 refer to chapter 13 2 3 Defining and Setting Fixed 2 Positions for the Focus N FP have previously moved the stage to the Check Point as described in chapter 5 5 The Check Point Otherwise the objective will col lide with the slide or the stage and may be damaged Caution Set the focus to Check Position only if you 1 8 Switching the microscope lamp on and off B 9 opening and closing the shutter on the microscope transmitted light shutter Em optional 10 setting the color temperature of the micro scope lighting to 3200 K 11 setting the intensity of the microscope lamp Via the Microscope Tools you can make the most frequently used settings directly in the main win dow 1 switching the Autofocus on and off in case the microscope is equipped with this func tion If the Autofocus is switched on with each change of the objective and with each targeting of an element during a laser func tion the image is focused automatically 2 releasing the automatic focusing As soon as you click on the button the image is focused 3 defining the focus setting Work Position refer to chapter 13 2 3 Defining and Set ting Fixed Positions for the Focus PALM RoboSoftware 3 1 0106 EN 139 The Microscope Window 13 2 The Microscope Window
241. the Pointer Tool click anywhere in the ele ment or in the Selection Rectangle if one exists and drag the element to its new position with the mouse button held down e Release the mouse button Info If you have selected multiple elements or an el ement group all selected elements or all ele ments of the groups respectively are moved To continue elements lines rectangles ellipses Note Select the laser function Cut before continu ing the element refer to chapter 10 3 Selec tion of Automatic Laser Functions for instructions on how to proceed Otherwise you will not obtain the desired result D Ctrl key Freehand Tool e Select the Freehand Tool e Press and hold the Ctrl key PALM RoboSoftware 3 1 0106 EN Processing Elements e Move the cursor to the start or finish dot of the element that you want to continue the start and finish dots of an element are indicated by a small square with thick black lines and the same color as the element Note Please take care that a square with thick black lines is displayed A square with thin black lines indicates an anchor point of the element If you continue drawing from one of these points you change the shape of the consisting elements see To change the shape of elements lines rectangles ellipses AutoCircle below in this chapter e Keeping the mouse button depressed draw the desired extension To join two elements lines rectang
242. the entire group refer to chapter 7 7 Process ing Elements edit or delete single elements within a group like ungrouped elements refer to chapter 7 7 Processing Elements center a group of elements on the monitor by a menu command refer to chapter 7 5 Center ing Elements on the Monitor add elements to a group remove elements from a group Info If you want to merge elements into a group add elements to an existing group or remove elements from a group you can use the menu commands in the main window of the PALM Ro boSoftware or in the Element List window af ter selecting the elements Note that you have to use the corresponding menu command in the main window if you have selected the elements on the monitor If you have however selected the elements in the Element List window you need to use the corresponding menu command in this window To merge multiple elements into a group Edit Create Group e Select the elements you want to merge into a group either on the monitor or in the Element List window e Click on menu item Create Group in the Edit menu in the main window or in the Element List window 78 Element List File Edit Move Laser DHEES Slide1 Slide2 Slides Summary SB SO y Show Types all Color Nr Name Type cutshot Area um Grp 1 Freehand 3944 Grp04 E 2 Freehand a 3 Freehand 4 Grp04 Group 3269 Grp04 1445 Grp04
243. ting the Reflector Offset All available elements are automatically selected and surrounded by a selection rectangle the se lection rectangle is indicated by a white line You will receive the following prompt Step 1 of 2 Click Selection Rectangle e Click on any dot within the selection rectangle and hold the mouse button down Info If you have drawn elements that are currently positioned outside of the monitor section some or all of the borderlines of the selection rectan gle will also be outside the monitor section In this case the border lines are not visible Note If you click inside the rectangle and inadvert ently do not hold the mouse button down the selected elements will not be moved to the cor rect position In this case reset the Reflector Offset as explained below and start the opera tion again You will receive the following message Step 2 of 2 Now move the Figure to the new position e Move the element or elements holding down the mouse button so that your reference ele ment is matches the respective structure of your sample When you have moved the win dow to the desired position release the mouse button Then even elements that are outside the current monitor section will be moved The following message will appear New Reflector Offset is set This message will disappear auto matically after a few seconds You can restore the default factory settings for the Reflector Offset a
244. tings for the Footswitch For the footswitch you can specify whether the IR laser is only to remain active while the switch is being depressed or whether it is to function as an On Off switch Info You can only use the footswitch when you are in 3D Mode or Stage Mode refer to chapter 11 8 2 Moving the IR Laser Beam with the Mouse 3D 1 3D 2 Mode and chapter 5 1 1 Positioning the Stage with the Mouse Stage Mode To set the function of the footswitch Footer IR Footer Hold Switch e Inthe Setup menu click on menu item PALM Robo The Preferences and Configuration window opens Select tab Laser This is divided into a number of sections There are two radio buttons in the bot tom Footer section e Click on the Hold button if you want the foot switch to function as a pressure switch the IR laser will remain activated as long as the foot switch is depressed or click on the Switch button if you want the footswitch to function as an On Off switch the IR laser be switched on or off each time the Switch is pressed 123 Activating and Deactivating the IR Laser 11 6 Activating and Deactivating the IR Laser You can specify whether the IR laser is activated and deactivated by the computer or by the footswitch The footswitch can only be used in 3D Mode or Stage Mode chapter 11 8 2 Moving the IR Laser Beam with the Mouse 3D 1 3D 2 Mode explains how to switch into 3D Mode and chapt
245. tivate one of the saved flu orescence settings records as described in chapter 12 4 OPEN the selected filter will be targeted then the shutter will be opened CLOSE the shutter will be closed then the selected filter will be targeted The shutter then remains closed CLOSE OPEN the shutter will be closed then the selected filter will be targeted The shutter is then opened again Light p don t care don t care OFF OW The setting for Light controls the microscope illu mination not the fluorescence lamp available with the motorized microscope only e Close this window by clicking on OK An item now appears in the Fluorescence window with your settings e Repeat in the same way if you want to create more settings records e Close the Fluorescence window by clicking on OK The window will close your new items will be saved 135 Adjusting the Reflector Offset 12 9 2 Changing a Settings Record You can change a settings record at any time To do so proceed as for creating a new record e Inthe Setup menu click on menu item Fluorescence This opens the Fluorescence window e Double click on the row in the table containing the settings record that you want to change or click once in this row and then click on the Change button Change This opens the Fluorescence Entry window e Make your changes as described above and click on OK to close th
246. top Move ein Stop ESC e Click on the Stop icon button in the Tool Bar or click on Stop in the Move menu To stop all current movement in the Element List window D Stop e Click on the Stop icon button in the Icon Bar Info You can still use the icon button and the menu command when the Element List window is open All current operations are stopped immediately To continue after a Stop lad Continue More mp Continue e Click on the Continue button in the Tool Bar or click on Continue in the Move menu The program will start the laser function with the element that was selected when the function was interrupted 110 10 9 The Serial Sections Function With the function Serial Sections you can trans fer elements that you have drawn for a sample to other samples This method saves time when pro cessing consecutive tissue samples The elements only need to be drawn for one tissue sample and are then automatically or if required manually transferred to subsequent tissue samples When you transfer the elements they are adjusted in such a way that the corresponding structures in the target sample cover them exactly You can also mount the samples at any angle on the slide Any differences in the positioning will be compen sated for in the course of the adaptation Make sure however that on assembly the samples are not stretched or contracted The Serial Secti
247. ts E main window x Delete all unprocessed elements Element List window Edit E Delete all unprocessed elements Ctrl y e Click on the Delete all unprocessed elements icon button or click on menu item Delete all unprocessed ele ments in the Edit menu in the main window or in the Element List window or press key combination Ctrl Y A prompt appears Do you really want to delete all Elements e Confirm the prompt by clicking OK Any elements that have not yet been processed by the laser are deleted PALM RoboSoftware 3 1 0106 EN Processing Elements Info If you are working in File Mode refer to chapter 1 6 File Mode and Database Mode you can also delete all elements by clicking on New EI ements on the File menu In this case any elements that have already been processed by the laser will also be deleted If you are working in Database Mode and cre ate a new record group refer to chapter 7 1 2 Working in Database Mode or select a new record group see 8 3 Saving and Loading El ements in Database Mode your elements will likewise disappear from the screen They are however saved in the database and can be re trieved at any time To delete the last drawn element Delete last element Edit ED Delete last element Back5p e Click on the Delete last element icon button or press the backspace key on the keyboard or click on menu item Delete last ele
248. tting will be re tained after you exit and relaunch the program The size of the Scale Bar is dictated by the PALM RoboSoftware depending on the magnification set ting this cannot be modified Note The scale is dependent on the calibration re fer to chapter 4 Calibrating the System ite Customizing the User Interface To show or hide the Copyright Note View Copyright e Click on menu item Copyright in the View menu A tick on the left of the menu entry indicates that the Copyright Note is displayed This setting will be retained after you exit and relaunch the program Info Don t forget that the Copyright Note can only be displayed if you have defined it in advance this procedure is explained in chapter 9 1 Inserting a Copyright Note In this way you can switch the display of a Laser Marker on or off Laser Marker View Lazer Marker LU Lazer A 1 Laser lA 2 Laser e Inthe Laser Marker menu click on menu item View Laser Marker A submenu opens containing a list of all available Laser Markers e Click on the name of the Laser Marker you want to show or hide These settings will be retained after you exit and relaunch the program To change the appearance of a Laser Marker Laser Marker Layout LaserMarker UY Laser IR 1 Laser IR 2 Lazer e Inthe Laser Marker menu click on menu item Layout Laser Marker A submenu opens containing a list of
249. tting you want for the respective part which is to be select ed automatically when PALM RoboSoftware is launched or closed Every field contains a drop down list containing all the possible settings for the respective microscope part In addition each in cludes an entry don t care If you select this en try the setting that is currently active at the time PALM RoboSoftware is launched or closed will be the one that is retained Don t Care If all the current settings are to be retained when PALM RoboSoftware is launched or closed e Click on the Don t care button under Star tup or Shutdown All entries in the fields for Startup or Shutdown will be set to Don t care If certain settings for the individual parts of the microscope are to be made automatically when PALM RoboSoftware is launched or closed e Open the respective drop down list Reflector Condenser Filter Shutter Reflected Transmit ted Lamp Baseport Sideport and select the desired setting of course the selection options for launching and closing of PALM RoboSoftware are the same Info The entries in the lists are made in the control program for the microscope apart from shut ter and lamp and can only be changed there If only the entry lt none gt is available in a list this means that the corresponding part is not motorized in your system and therefore cannot be set via PALM RoboSoftware e To close the Microscope Setup w
250. u can activate it by clicking on Scroll Rectangle in the View menu Scrolling with the mouse also works when the Scroll Rectangle is not displayed As soon as you move into the area outside the Scroll Rectangle the cursor becomes a broad white arrow e Press and hold the mouse button down The stage is moved in the direction of the arrow You can also change the direction of movement during the movement itself by moving the cursor from an image edge towards an adjacent corner or vice versa e Release the mouse button when you have found the required area of your sample and positioned it in the correct place The stage stops moving The Scroll function is only active as long as the cursor is outside the Scroll Rectangle Movement of the stage is therefore also stopped when you drag the cursor into the Scroll Rectangle with the mouse button depressed PALM RoboSoftware 3 1 0106 EN Movement Types Note With the Scroll function you can move elements while simultaneously positioning the stage If you have selected elements or groups of ele ments on the screen and the scroll arrow is within the selection rectangle these elements or groups of elements will also be moved By this method you can move elements or groups of elements over greater distances quickly If you do not want to move elements or groups of elements deselect all elements and groups of ele ments refer to chapter 7 6 1 Selecting and De
251. u to record video sequences save series of images You can specify the time interval between images and the period over which images can be saved If you are working in File Mode refer to chapter 1 6 File Mode and Database Mode the images are saved in the specified folder refer to chapter 9 2 Specifying Folder to Store Images In If you are working in Database Mode the images will be stored in the database You can either activate the Time Lapse function manually or define an event to activate the func tion automatically The necessary settings can be configured in the Time Lapse Configuration window or on the Time Lapse tab in the Preferences and Configu ration window The available settings are identical in the two windows To open a window for setting the Time Lapse parameters TH A Time Lapse Setup PALM Robo e Click on the Time Lapse icon button The Time Lapse Configuration window opens Or e Inthe Setup menu click on menu item PALM Robo The Preferences and Configuration window opens e Click on the Time Lapse tab 91 Saving Images and Videos Using the Time Lapse Function 4 Time Lapse Configuration Time Lapse Experiment Mame TimeLapse Sequence Description File prefix TimeLapse SequenceO1_ State planning _ show loo rile Time Lapse Definition data all time data fintervals and durations in minutes Interval Duration e
252. ultiple elements ts Shift key Pointer Tool e Select the Pointer Tool in the Graphic Toolbar e Click with the mouse on the first element you want to select e Press and hold down key Shift click on all other elements you want to select as well To stop selection of elements Edit DeSelect All Ctrl e Click on menu item DeSelect All in the Edit menu or press key combination Ctrl A or If it is not yet active select the Pointer Tool in the Graphic Toolbar and click anywhere outside the selection rectangle A elements will be deselected Info If elements outside of the image section dis played on the monitor are selected it is possi ble that you do not see the selection rectangle as it is larger than the monitor In this case you cannot deselect the elements with one mouse click Info If you want to deselect only single of multiple selected elements first you have to deselect all elements then select the desired elements again 67 Processing Elements 7 6 2 Selecting and Deselecting Elements in the Element List Window The selected elements in the Elements List win dow are highlighted in blue To select or deselect all elements Edit Select All DeSelect All e Click on menu item Select All or DeSelect All in the Edit menu or To select or deselect single elements e If you want to select a single element click on the corresponding entry in the table of the El
253. umber of the element Name name of the Elements you can enter and edit the name via the menu command Edit gt Change Type element type Line Freehand Dot Auto Circle Ruler Text Reference etc cut shot number of cut and shot laser functions performed Area m2 area of the elements of type Fig ure and AutoCircle for elements of other types an area value has no meaning Grp name of the group if the element belongs to an element group Comment possibly a comment entered by you for element types Reference Reference is automatically entered here You can edit this entry via the menu command Edit gt Change Hx W height and width of the elements Position coordinates of the element Selected elements are shown on a blue background while elements that have already been processed by the laser are shown on a green background PALM RoboSoftware 3 1 0106 EN The Element List Window 7 3 2 Items Shown on the Summary Tab Element List File Edit Move Laser OD htt amp ras Slide Slide2 Slides Summary Figures Figurels Dotis AutoCircie s AutoCircie s 303325 am gt y lButoCircle Show Types all Show summary For over all 11409 1 Circle 198245 1 Rectangle 1 Circle 2 FreeHands 31224 6244 The Show Types field allows you to decide Number of elements number of elements whether all elements or only the elements of sp
254. ur sample is displayed Sharply e Click on menu item WorkPos in the Focus gt Define menu To set the focus to one of the default settings Focus o CheckPos SP WorkPos Sa LoadPoz e Click on the desired menu item in the Focus menu CheckPos WorkPos or LoadPos The focus is set to the corresponding position Caution Set the focus to Check Position only if you have previously moved the stage to the Check Point as described in chapter 5 5 The Check Point Otherwise the objective will col lide with the slide or the stage and may be damaged 141 The Microscope Window 13 2 4 Setting the Aperture Diaphragm The aperture diaphragm can be adjusted with the motor The speed of the adjustment is selectable Speed apo e Move the slider in the Aperture row to the desired position to the left slower to the right faster e Click on the left or right arrow key in the right border of the line to close or open the aperture diaphragm The aperture diaphragm will be adjusted In the field on the left the current position is displayed Aperture 0 00 13 2 5 Switching the Lamp On and Off Lamp Lamp On O Lamp Lamp Off Lamp Switch Lamp ON Lamp Switch Lamp OFF e Click on the Lamp icon button or click on the required item Switch Lamp ON or Switch Lamp OFF in the Lamp menu 13 2 6 Setting the I ntensity of the Lamp Light o 1 100 HH 4 gt
255. ut LPC aD 30 Arrow Speed setting for SD Mouse with Lens 5x Lun Sec actual Speed 1581 Speed Delta 1 for faster slower functions The PALM Robo Speed window will open In the lower section of the window you will see tabs for setting the speed of the different movement types These last two tabs control the speed settings for the IR laser 3D Laser Beam Positioning Set speeds for both movement types in the same way Select the tab of the movement type for which you want to change the speed 3D or 3DArrow Enter the desired value with the slider or enter the desired value in the Actual Speed field Close this window by clicking on OK To change the speed of the Speed Tools tool Speed Stage Urn Sec we i mes alo Open the Speed Tools tool if it is not displayed click on the menu item Speed Tools in View gt Menu or press key combination Ctrl E Click on the arrow beside the field under Selected Speed 126 A list of the different movement types will open e Select the movement type for which you want to change the speed 3D or 3DArrow e Enter the desired value with the slider or click on one of the arrow buttons below the Slider repeatedly or click once and hold the mouse button down until the desired value is reached or enter the desired value in the field below the Slider To change the speed with menu commands or with ke
256. ut lines element no 12 in the illustration above are shown with the line thickness specified by you in addition a connecting line is shown between the start and finish dots of elements of type Figure Elements of type Dot nos 13 and 14 are shown as unfilled circles elements of type AutoCircle no 15 are indicated by a thin circular line and an unfilled circle in the middle PALM RoboSoftware 3 1 0106 EN JointCut yy Cut lines element no 12 in the illustration above are shown with the line thickness specified by you in addition a connecting line with a remaining small space is shown between the start and finish dots of elements of type Figure Elements of type Dot nos 13 and 14 are shown as unfilled cir cles elements of type AutoCircle no 15 are in dicated by a thin circular line and an unfilled circle in the midale LPC Dot elements are displayed as filled circles with the diameter that you have set nos 13 and 14 in the picture above Elements of type Figure are indicated by a thin line elements of type Auto Circle are indicated by a thin circular line and an unfilled circle in the middle PALM RoboSoftware 3 1 0106 EN Preview of the Automatic Laser Functions AutoLPC a m The positions of the laser pulses are displayed as filled circles with the diameter that you have set element no 12 in the picture above The ele ment s borderlines appear as thin lines elements
257. utter on the filter wheel via a menu command Setup Shutter e Inthe Setup menu click on menu item Shutter The Fluorescence Shutter window will open The window contains three fields displaying a number of buttons Shutter Type field information about the manufacturer Shutter Error field if there are problems with the shutter messages will be displayed here Actual state field this indicates whether the shutter is currently open or closed Buttons for opening and closing the shutter and to close the window PALM RoboSoftware 3 1 0106 EN 131 Activating and Deactivating the Timer 12 7 Activating and Deactivating the Timer PALM RoboSoftware includes a timer for your use Use the timer to set the shutter to close again automatically after a period defined by you when you have opened it Note In the Basic configuration with the manual microscope the function Timer is not active e Timer e Click on the Timer icon button in Fluores cence Tools This opens the Shutter Timer window Shutter Timer Cancel Timeout Clase Shutter automatically after Seconds 3 4 H This contains a checkbox for activating and deac tivating the timer and an input field for setting the time for which the shutter is to remain open A tick in the Close shutter automatically after checkbox indicates that the timer is activated e Click in the checkbox to add or remo
258. values to be displayed or click on over all if total values for all slides are to be displayed together Total values for the selected slide or for all slides together are displayed 7 3 3 Change Items Shown To specify which element types are to be listed Show Types Freehands Lines Rectangles Circles Dots AutoCircies e Click on the arrow to the right of the Show Types field A list containing all the element types will open e If you want to enable or disable a listing of all element types in the table click in the All checkbox or click in the checkboxes in front of the types you want to include or exclude from the list A check in the checkbox indicates that the respec tive element type will be listed in the table e Click on the arrow to the right of the Show Types field to close the selection list Only the selected element types continue to be displayed Info Enabling or disabling the listing of element types does not affect the display of the ele ments on the monitor in any way Refer to chapter 7 4 Displaying and Hiding Elements for instructions on how to show or hide these numbers 64 To change the table format of Element List window To change the column width e Move the cursor in the table header to the col umn dividing line on the right of the column the width of which you want to change The cursor changes to a double headed arrow with a diagonal line e Kee
259. ve the tick Enter the entire period for which the shutter is to be open in the Seconds field e Click in the Seconds field to enter the time or click on the left or right arrow key beside the field to shorten or extend the time e Close the window by clicking on OK 132 12 8 Fluorescence Filter The Fluorescence Filter window contains infor mation about the installed filter wheel and the installed filters You can also make changes to the settings there Note In the Basic configuration with the manual microscope the function Fluorescence Filter IS not active 12 8 1 Information About the Installed Filter Wheel Setup Filter e Inthe Setup menu click on menu item Filter Fluorescence Filter Filter Type Ludi Filter Error Actual Position 12 gt Filter Colors and Hames DoubleClick Color to change The Fluorescence Filter window will open This window contains information about the installed filter wheel and the installed filters You can also make changes to the settings here Filter Type field Information on the manufac turer of the filter wheel Ludl or microscope Filter Error field This is where error mes Sages are displayed in the event of problems with the filter Actual Position field This shows which filter is currently in the fluorescence beam path You can also select the desired excitation filter here refer to chapter 12 8 3 f
260. vement type e Enter the desired value with the slider or click on one of the arrow buttons below the slider repeatedly or click once and hold the mouse button down until the desired value is reached or enter the desired value in the field below the slider PALM RoboSoftware 3 1 0106 EN Setting Speed To change the speed with menu commands or with keyboard shortcuts Move Speed slower Ctrl 5 Speed faster Ctrl F e Ifyou want to reduce the speed press key combination Ctrl S or click on menu item Speed Slower in the Move menu e If you want to increase the speed press key combination Ctrl F or click on menu item Speed Faster in the Move menu Info Changes with keyboard or menu commands al ways refer to the type of movement that is cur rently selected in the Speed Tools tool Therefore activate the tool display before car rying out any speed changes with keyboard shortcuts or menu commands Otherwise you will not be able to see which movement type the changed speed setting will affect If a movement is in progress the selection in Speed Tools is automatically changed to the current movement type and the speed chang es you make are automatically applied to the current motion You can specify a value increment by which the speed is increased or decreased when you make this change via key combinations PALM RoboSoftware 3 1 0106 EN To enter an increment for changing the speed wi
261. vers If you double click in the field the States win dow will open Amongst other things this win dow displays the coordinates of all units refer to chapter 2 5 Status Display PALM RoboSoftware 3 1 0106 EN Customizing the User Interface 2 2 Customizing the User I nterface Info Please note that the following possible set tings described here may not be available depending upon the configuration of your PALM MicroLaser System PALM RoboSoftware offers a number of ways to customize the appearance of the program window to meet your requirements You can change the size of the program win dow The aspect ratio of the microscope image remains unaltered You can display or hide all icon bars tools and the Status Bar You can reposition all icon bars you can re arrange the respective positions of adjacent icon bars both horizontally and vertically You can display or hide the Crosshairs the Laser Markers the Scroll Rectangle the Scale Bar and the Copyright Note You can change the appearance of the Laser Markers These changes are retained after the program has been restarted You resize the program window in the same way as in all Windows programs To display or hide a bar or a tool using menu commands View Menus j Tool Bar sl e s Es Image Processing Tool Archive Tools Fluorescence Tools Lens Tools Laser Tools Ctrl L Speed Tools Ctrl E Microscope Tools Scrol
262. y select the element prop erty to which the list should be sorted You can select whether the file should contain ele ment summaries Print Summary records the number of the elements and the sum of the sizes of their areas for each type and color to the file Print Totals records the total number of ele ments and their total area to the file If you wish check the print Summary and print Totals boxes e Inthe Print to section you should specify whether you want to output the elements ona printer or save them in a file e Click on OK If you have chosen File the Print Elements to File window will open e Entera name for your file select a folder if nec essary and click on OK Your elements will be printed to a file PALM RoboSoftware 3 1 0106 EN Inserting a Copyright Note 9 Saving Images You can save the portion of the stage that is cur rently displayed on the monitor You can save in three ways manual saving every time you enter the corre sponding command the current image will be saved For instructions on how to do this refer to chapter 9 3 Saving Images Manually automatic saving every time you release a laser function an image will be saved for each element that has been processed with the laser before and or after processing Enabling and disabling automatic saving is described in chap ter 9 4 Saving Images Automatically during a Laser Function T
263. yboard shortcuts Move Speed slower Ctrl 5 Speed faster Ctrl F e If you want to reduce the speed press key combination Ctrl S or click on menu item Speed Slower in the Move menu e If you want to increase the speed press key combination Ctrl F or click on menu item Speed Faster in the Move menu Info Changes with keyboard or menu commands al ways refer to the type of movement that is cur rently selected in the Speed Tools tool Therefore activate the tool display before car rying out any speed changes with keyboard shortcuts or menu commands Otherwise you will not be able to see which movement type the changed speed setting will affect If a movement is in progress the selection in Speed Tools is automatically changed to the current movement type and the speed chang es you make are automatically applied to the current motion You can specify a value increment by which the speed is increased or decreased when you make this change via key combinations PALM RoboSoftware 3 1 0106 EN The 3D Laser Beam Positioning To enter an increment for changing the speed with a keyboard shortcut or a menu command e Click on Setup Speed inthe Move menu or press F8 on the keyboard The PALM Robo Speed window will open e Select the tab for the movement mode for which you want to specify an increment 3D or 3DArrow Speed Delta 12 for faster slower functions e Enter an in
264. z Group 15 Reference Ref 1 sli 16 Reference Ref 2 sli 17 Reference Ref 3 sli 114 Step 5a Transferring elements to the target sample automatic adaptation Now transfer the elements which are to be pro cessed with the laser from the source to the target Sample The reference points on the source sample will then be aligned with those of the target sam ple e Select the group of elements on the source Sample and the group of reference elements of the target sample in the example Slide 1 and Slide 2 Edit Match Serial Section Group e Click on menu item Match Serial Section Group in the Edit menu Match Serial Section Groups Select Source Group Mame nr Grp Comment Select Destination Group Name nr Grp comment Slide1 4 Slide1 4 slide2 14 a ee ee gt Delete source group after matching elements into destination group The Match Serial Section Groups window opens e If you are working with PALM RoboStage I or with PALM RoboStage Il you have inserted the target sample in the same position that the source sample occupied before then you Should check the Delete source group after matching elements into destination group box As in these cases the elements are allocated to the same slide position they would appear double if you didn t check this box e Click on the group in the table at the left of the window which you want to transfer to the other sample

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