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Ultima Manual v2 5i - QB3

1. 83 8 2 3 5 CUSTOM Wavell ictescarcsuveetecscesduadadaavaadideadeatiided roads Cad Pea du dot 83 8 2 4 Use Marked Points During Acquisition eusssesse 84 8 3 ADC InDUE CONO S eege ee Eeer 86 8 3 1 Selecting Input Channels 86 0 23 2 ADC CONMQUIANO Mg EE 87 OG PON CS ANF e EE 89 8 4 1 EE 89 8 4 2 calibrauoN Eeler 89 8 4 2 1 Manual GaliDralON MEN 97 8 4 2 2 Automate C lee e NEE 97 So Ee leie Bea EE 98 8 5 1 Calibration Files and General Controls 99 8 5 2 Marking points Basic Controls eeeeeeeeeeeeeeseee 101 8 5 3 Hne CTIDIVON EE 103 8 5 4 Wap PING SClUD EE 104 8 5 9 Functional Map Display earestiente ENEE EEA 109 Page 5 of 125 Ultima Manual ver 2 5 8 5 6 Miscellaneous ei de E 111 8 5 Use Marked Points During Acquisition ssee 112 9 ACGUISIION SCCN ANOS EE 114 9 1 TriggerSync Electrophysiology Acquisition 115 9 2 Line scans amp Electrical recordimg 116 9 2 1 Line scanning with electrical recording amp stimulation 116 92 2 VIEWING LINE Scan dala cse ree ere e re ror bo eve ox ete xe vog 116 9 3 X Electrical recording amp Uncagmg 118 9 4 Line scans Electrical recording amp Uncaging 119 9 5 Non Line Scan Images Electrical Recording amp Uncaging 120 96 Functional Map R
2. eeeseeeeeeeeenne 40 1 2 2 2 Optical ZOOM EE 40 12 2 A wie ee 40 7 2 3 Scanning amp Image Window Tools 41 7 2 3 1 HIVE SCA SOD e E 41 1 2 3 2 SMIJES CAN EE 41 I Klees UE 41 7 2 3 4 Average SUI sais toss ccc EE EE 41 7 2 4 Shutter and Imaging Mode a aannannnnnnnnnnennnnnnnnnnnnnnnrnrrrrnnrnnrnnnnnnne 41 7 2 4 1 Hard Shutter ClosediCpnen ona AA 41 1 2 4 2 Soft Shutter Closed Open cccccssssscoussurecsesursesccursureceesans 42 7 2 4 3 Scan Mode Galvo or AO 42 1 2 4 4 Label and Objective Lens Gelechon 42 1 2 4 5 Label Gelechon esera T 42 1 2 4 6 ODIGCIVO LENG ud Mud uut odds adds tud qu utu e denda adds 42 LLD Pan and Scan Rotation Tools 43 7 2 9 1 Pan Control and XY Stage Control 43 7 2 9 2 SCAN TROLAU ON EE 43 1 2 5 3 XY oade ein de BE 44 7 2 6 Laser PMT DAQ WEE 44 7 2 6 1 E Ic Fr ege 44 7 2 6 2 mE uber 44 7 2 6 3 BXeXc mec 45 Feet Vadit t 45 EE SOMOS AER 45 7 2 8 1 Z MOO CONTO EEN 46 7 2 8 2 Z Seres e E e EE 46 7 2 8 3 Collecting a Z series over a large depth 47 LeS ENEE 49 7 2 9 1 T series definitions lseesseeseeseeseeeeneenne e 49 7 2 9 2 Collect a Basic Tesenes 50 7 2 9 3 Collect a Complex T series ccccceccseeceeeeeeeeeteeeeeeeeeeeeenaes 51 KLI AV EE a 52 1 210 1 XY Stage dellnillOlis eoe eo eo oo eoe ei eo tei 52 ZIT MISC cean ee 53 f2 12 SCAN Tute AE ee EE 54 71 3 Praineview Toolba
3. Page 29 of 125 Ultima Manual ver 2 5 safe enclosure around the instrument to ensure optimal performance as well as to provide a safe working environment for the users of this instrument Note Only four detectors many be used simultaneously 4 5 3 1 Top Mounted detectors The Ultima system comes with either dual or quad non descanned top mounted detectors They may be either the standard side on multialkali PMTs or optional GaAsP detectors The emission pathway for both options is similar and is depicted in Figures 4 10 and 4 11 In both cases the fluorescence from the sample is reflected off the primary dichroic in the turret through the IR blocking filter and through the various dichroics and barrier filters to the PMTs Barrier Filters From Laser Primary Dichroic PMT Dichroic IR blocking Filter Sample Figure 4 8 Emission Light Path for dual channel detectors Barrier Filters From Laser Barrier Filters Primary Dichroic PMT Dichroic Dichroic IR blocking Filter Sample Figure 4 9 Emission Light Path for quad channel detectors 4 5 3 2 Sub Stage detectors Some systems come with the optional pair of sub stage multi alkali PMTs These detectors are mounted below the stage and to the left of the microscope Page 30 of 125 Ultima Manual ver 2 5 4 5 3 3 Dodt Gradient Contrast detector some systems come with the optional transmitted light Dodt gradient contrast
4. A PC is provided by Prairie Technologies which contains the three National Instruments PCI cards software and drivers necessary to control the Ultima system 4 2 2 PMT dual preamplifier amp HV control unit O PMT High Voltage 1 PMT High Voltage 2 O Batt On HV 1 Protect HV 2 C C Only Sensor AMan gt Off Override Man O a PRAIRIE TECHNOLOGIES DUAL PREAMPLIFIER amp HV CONTROL Figure 4 2 Dual Preamplifier amp HV control unit The PMT high voltage HV is controlled via the PMT control unit mounted in the instrument rack shown in Figure 4 1 Each unit controls two PMTs If your system has more than two PMTs you will have additional units in your instrument rack On the left side is the MASTER On Off switch This control is used to turn both PMTs on or off The unit remains on unless the main power switch located on the backside is set to the Off position The PMT high voltage may be controlled either via the computer or manually using the HV adjustment knob Set the C C Man switch to C C computer controlled or Man manual as you prefer Normally the system is configured to use the C C mode of operation If set to Man the HV 1 knob adjusts the high voltage supplied to PMT 1 The voltage is displayed in the red LED and the HV 2 knob adjusts the high voltage supplied to PMT 2 In either computer controlled or manual mode the LEDs will display the
5. Show Marked Point Hold and Move Time is turned on then overlaid on the largest graph will be an additional graph in the Marked Point Graph Color indicating when each marked point will occur during the acquisition Page 84 of 125 Ultima Manual ver 2 5 This overlay graph will indicate when the next marked point is being moved to with a spike on the graph To interpret the graph when the acquisition begins the pointing device will already be positioned at the first marked point The pointing device will stay at that location for the amount of time specified in the Mark Points dialog by adding the time for Marked Point Hold Time ms and the Marked Point First Point Delay ms parameters The first spike on the graph indicates that the pointing device is moving to the second marked point The time necessary to move the pointing device is specified in the Mark Points dialog by the Marked Point Move Time ms parameter The pointing device is then held at the location of the second marked point for the time specified in the Mark Points dialog by the Marked Point Hold Time ms parameter The second spike on the graph indicates that the pointing device is moving to the third marked point The time necessary to move the pointing device is specified in the Mark Points dialog by the Marked Point Move Time ms parameter The pointing device is then held at the location of the third marked point for the time specified in the Mark Poi
6. Photoactivation The Photoactivation PA tool is a function which enhances the functionality of a Z series acquisition by allowing the user to apply masks at desired z positions These masks can be on specific z slices or can be applied to the whole stack T To use the PA tool a Z series must first be acquired as described in Section 2 8 While still in the playback window click the PA button This will open a toolbar on the right side of the Image Window Click on New Set By using the scroll buttons at the bottom of the window and the buttons on the right the mask region s are defined These are the regions that are scanned by the laser beam Regions that are not masked are not scanned The selected regions are shown as translucent green areas on the image as shown in Figure 7 25 When all masks are defined close the playback window Click Exit D D H 1 Emsa D ET aj H Bb Uaraser cl m k beep un Ges dera kb Trs E Ser Overig D ea a a a e ke em EUER fo Tekma V eu iuei EX EE Geert e OCE NCAA OLEI COC Die Re eres ODDO 00S DODO OE i I ue Figure 7 27 PA Masks and Acquired Z series with masks To acquire a Z series using these masks it will be necessary to set up a T series See Section 7 2 9 being sure to check the PA box It is important to make sure the Save Images option is also checked Following acquisition the playback window will again open for review of the
7. Tolerance and Connectivity controls until the Processed Image shows a valid representation of the spot from the Original Image window This process is strictly empirical there are not pre determined values that will automatically generate a good Processed Image Third specify the Number of Frames to Average the options are 1 2 4 or 8 If the image is noisy sometimes averaging 2 or more frames together will result in a cleaner image Page 93 of 125 Ultima Manual ver 2 5 gt Fourth define a size factor to be used during the calibration process During this setup part of the Automatic calibration process the size of the spot is determined Then during the actual calibration the size is used as a filter on the image if more than one spot is found This size factor allows the operator to specify a value that will be applied to the measured spot size as a plus minus value to produce a range of sizes that would constitute the correct spot For example if the spot in the Processed Image window is very small on the order of a few pixels then a small value of say 5 should be set in this field If however the spot is large on the order of hundreds of pixels then this field should be set to a larger value such as 50 Either press the OK button to proceed to the next step or press the Back button to return to the previous step Step 5 Defin
8. Trigger Cycle Wait for input trigger before processing line Trigger Each Image Z series Wait for trigger before each image or Z series Cycle Completion Function not used at this time Add New Place a new basic cycle at end of series that can be modified for Z series PA BOT trigger Insert New Insert a new basic cycle in series at position pervious to marked line that can be modified for Z series PA BOT trigger Clear All Function not used at this time Remove Selected Removes selected lines from T series Add Insert TriggerSync Exp When a TriggerSync is running these buttons become enabled When included in a T series they will cause TriggerSync to perform an Acquire operation as defined in the current XY Stage ROl Label Select Apply to all subsequent relevant lines in the T series until changed See Section 7 210 for XY Stage Section 7 4 5 for ROls and Section 7 2 11 for Labels Save Path Base Directory A default path and base directory is set up for saving data This is C Documents and Settings WPrairie with a Base Directory name that includes the type of data acquired the data of acquisition and a file number that is sequentially incremented If a different file name or location is preferred click Browse next to Save Path to change this information 7 2 9 2 Collect a Basic T series A Basic T series is one in which an image or Z series is collected at a desired interval gt Press the Ad
9. 1 003e 2 If the file contains more than one column of numbers only the first column shall be used The field Number of Elements Needed indicates the number of elements the operator created file should contain This value is a function of the Acquisition Time and the Output Rate For example if the acquisition time is 500 ms 0 5 s and the output rate is 10000 samples per second then the number of samples needed in the file would be 5000 0 5 s x 10 000 samples per second 5000 samples The field Number of Elements in File indicates the number of elements that were actually in the file 0 00 Keett O Z Figure 8 14 Custom Waveform To load a user defined file press the Browse button and using the pop up file dialog select the appropriate file If the file contains too few elements then the remaining acquisition time will be padded with the Holding Potential value If the file contains too many elements then the sequence will simply be truncated at the point where the acquisition time is attained 8 2 4 Use Marked Points During Acquisition The lower left hand corner of the dialog is labeled Use Marked Points During Acquisition Figure 8 15 Use Marked Points During Acquisition If a list of Marked Points has been created and the Use Marked Points During Acquisition control in the Mark Points dialog has turned on then these controls would be active If the control
10. Click Save Reference Image Click Acquire to mark points VV Y VY V WV Figure 7 32 Mark Points grid Note Information on marking points in TriggerSync can be found in Section 8 5 Page 73 of 125 Ultima Manual ver 2 5 8 TriggerSync Software TriggerSync is the Prairie application that integrates electrophysiology recording with image collection From TriggerSync the user can define output signals for stimulation of a sample preparation or to control external equipment such as shutters or pumps JriggerSync also allows the user to collect image and line scan data and to use this information in the selection of discrete locations where photolysis is to be performed E Tools CLOSED Figure 8 1 Main TriggerSync window 8 1 Main Operational Modes The TriggerSync program operates in two modes When the application is first started by default it is in acquisition mode While in this mode the user may define various acquisition parameters such as input and output settings calibrate the pointing device and mark points After performing an acquisition the program is in the post acquisition mode While in this mode the user can not change acquisition parameters but can manually scroll through the acquired data performing basic viewing functions such as changing the vertical and horizontal scaling It is important to remember that by default as soon as an Page 74 of 125 Ultima Manual ver 2 5 acq
11. If during the calibration process the software can not successfully determine the location of the spot in the image it will pop up a dialog that will allow the operator to select the spot image location manually The software will then continue and attempt to find subsequent spot image locations automatically For the Automatic point calibration procedure to work it is necessary for the operator to tell the program where the spot image is in the field of view and then to set several controls that help the program to characterize the spot image gt First acquire an image of the spot by pressing the Acquire button located below step 3 Depending upon the system configuration it may be necessary to open a hard shutter in front of the spot laser In the main Page 92 of 125 Ultima Manual ver 2 5 window of TriggerSync there is a Shutter F1 control in the lower left hand corner that may be used to open close the hard shutter in front of the spot laser If the system configuration includes a Pockels controller in front of the spot laser there is a Laser Intensity control in the area below step 3 Use this control to increase the laser intensity until the spot can be seen in the image If it is necessary to physically move the spot location use the four blue buttons located to the left of the Acquire button The four blue buttons are oriented in a diamond pat
12. ms 2228 224 Framerate fpa D 4438 T 134 30 151 88 G H 2 Acquisition took 2 61 s Figure 7 18 Scan information Page 54 of 125 Ultima Manual ver 2 5 7 3 PrairieView Toolbar Options The Prairie View toolbar contains many resources and settings not shown on the main window They are described briefly below 7 3 1 File File Tools Window Display Load Image s e Load Configuration save Configuration Load ROI Definitions Save ROI Definitions save ROI Reference Image Load T Series Settings Save T Series Settings Load amp 3 Stage Locations Save NV Stage Locations e Preferences Exit e Save ROI Reference Image marked on it e Load Save T series Settings one that has just been set up Load Images Loads saved image files Load Configuration Loads system configuration file Resets PrairieView control settings of the laser PMT scan rate scan rotation etc to values previously saved Save Configuration Saves system configuration file Saves PrairieView control settings of the laser PMT scan rate scan rotation etc to values previously saved Load ROI Definitions Loads previously saved region of interest settings Save ROI Definitions Saves defined region of interest settings Saves reference image with saved ROIs Loads previously saved T series or saves e Load Save XY Stage Locations Loads previously saved XY stage locations or saves
13. File Edit Operate Windows Help 12 gt Update 1 0 e l 75 0 i i 1 500 000 1000 00 d 500 000 1000 00 e pen Before Starting Data Collection lose After Data Collection Complete tart Stimulus Control with Data Collection l i e 500 000 1000 00 r3 100 0 00 25 0 50 0 50 0 00 i 75 0 1 500 000 1000 00 r 500 000 1000 00 r3 IK DACO Out z i e r3 r3 r3 r3 ue L ue r3 Nk r3 r3 r3 ue r3 ii Slo Gu phil r3 r3 Le r3 i e pli r i t 500 000 1000 00 I I I 200 000 400 000 600 000 800 000 1000 00 es j QF TES Figure 8 8 Acquisition and DAC Output Setup Page 78 of 125 Ultima Manual ver 2 5 8 2 1 Acquisition Setup pen Before Starting Data Collection lose After Data Collection Complete Start Stimulus Control with Data Collection Figure 8 9 Acquisition Setup Number of Cycles Number of Cycles indicates the number of acquisitions to perform when the Acquire button is pressed Cycle Period Cycle Period specifies the amount of time in milliseconds for each cycle This value must be at least 1 ms longer than the Acquisition Time Acquisition Time Acquisition Time specifies the amount of time that data should be acquired when the Acquire button is pressed Acquisition Rate Acquisition Rate specifies the rate at which the inputs should be sampled durin
14. J 256 J 512 J 1024 2048 verras aam b Figure 7 3 Scan resolution tools 7 2 1 1 Image Size Image size is a definition of how the collected data is acquired and displayed In the case of 512 x 512 for example data is acquired at 512 x 512 bins with each bin sampled according to dwell time 7 2 1 2 Pixels Per Line amp Lines Per Frame Pressing the green bar to the left of the Image Size window section activates this mode The Pixels Per Line function allows the user to define the number of pixels to dwell upon in each horizontal line X direction of the image Preset values are offered as a convenience Values may also be selected by means of the slider tool or by typing directly into the highlighted field The minimum value allowed is displayed to the left of the highlighted field The Lines Per Frame function allows the operator to define the number of lines in the Y direction in each image Preset values are offered as a convenience Values may also be selected by means of the slider tool or by typing directly into the highlighted field Note Be aware that it is possible to choose configurations in which the pixels are not square This may effect how the images are displayed by third party software Page 39 of 125 Ultima Manual ver 2 5 7 2 2 Zoom and Dwell time Tools Dwell Time Per Pixel us 3 3 DG e be DR DG 0 15 1 00 Figure 7 4 Zoom and dwell time tools 7 2 2 1 Dwell Time Per Pix
15. a line is 3 Page 103 of 125 Ultima Manual ver 2 5 After selecting the desired number of points the operator may either press the Add to List button or the Replace List button If the Add to List button is pressed the new marked points that form the defined line will be added to the current list of marked points This allows the operator to string together a series of marked points that might consist of randomly specified locations along with one or more straight line segments and so on If the Replace List button is pressed then any previously defined marked points will be deleted and the new list of marked points will only consist of those from the line definition 8 5 4 Mapping Setup This section of the Mark Points dialog contains controls to help the operator create a grid of marked points Currently this option only works with the Mark Point Using Red Blue Cursor control set to the Red option While this grid of marked points may be used in any experimental way that any of the other marked points may be used within the TriggerSync program a grid of marked points provides a new data acquisition and analysis option This new option is referred to as Functional Mapping To create a grid of marked points Position the red cursor in the image window to indicate the location of what would be the upper left hand corner of the grid Press the Mark First Corner button Positi
16. be indicators for all of the marked points This file will use the specified file name and have AllPoints appended to the name If Option C is selected then a separate file will be saved with the current image and each of the marked points individually superimposed on the image These files will use the specified file name and have Pointn appended to the name where n would go from 0 to the number of marked points If Option D is selected then a text file is created that contains two columns of numbers The first column is the x cursor position and the second column is the y cursor position for each marked point This file uses the specified file name with a dat extension 8 5 3 Line Definition This section of the Mark Points dialog contains controls to help the operator create a straight line of marked points Currently this option only works with the Mark Point Using Red Blue Cursor control set to the Red option To create a line of marked points first place the red cursor in the image window where you wish to start the line Then press the Mark 17 EndPoint button Next place the red cursor in the image window where you wish to end the line Then press the Mark 2 EndPoint button Below the Mark 2 EndPoint button is a control labeled Number of Points Use this control to specify the number of points you wish to create on this line including the two endpoints The minimum number of points on
17. be used Below the four blue buttons are four fields that indicate the location of the current pointing device The bottom most two fields indicate the absolute position of the pointing device in the x and y dimension The number that is displayed within these fields is dependent upon the pointing device The top two fields indicate the relative position of the pointing device with regard to the absolute position For a galvanometer based pointing device the values displayed in these fields are in volts For a motor driven pointing device the values displayed in these fields are in motor steps Whenever the position of the selected pointing device is modified using the previously described blue buttons you will see the blue cursor in the image window move and the values displayed in the relative position and absolute Page 100 of 125 Ultima Manual ver 2 5 position fields will change as well In addition any action that results in a new physical position for the selected pointing device will cause the blue cursor in the image window to move to indicate where the pointing device is currently located as well as cause the values displayed in the relative position and absolute position fields to be updated To the right of the relative position fields is a yellow button Reset Relative When this button is pressed the values in the relative position fields will be reset to O 8 5 2 Marking points Basic Controls Immediate
18. control output signals will happen and then after the acquisition and stimulus control has completed the shutter will be closed In option B first the acquisition and stimulus control will start then the shutter will be opened the shutter will remain open for the amount of time specified by Open Shutter Time and then close The acquisition time is determined by the setting of Acquisition Time In option C first the acquisition will start then the shutter will be opened and the stimulus control will begin the shutter will remain open for the amount of time specified by Open Shutter Time and then close The acquisition time is determined by the setting of Acquisition Time Open Shutter Time Open Shutter Time specifies the value for the amount of time in milliseconds that the shutter should be opened based upon the setting for the Open Close Shutter control 8 2 2 Stimulus Control Depending upon the system configuration the operator has the ability to program a maximum of from 2 to 8 analog output signals Along the left hand side of the dialog there is a section labeled Stimulus Control Pulse Train Figure 8 10 Stimulus Control Page 80 of 125 Ultima Manual ver 2 5 The entire right half of the dialog is used to provide graphical display of each of the possible DAC output waveforms independently and then the bottom most graph the largest displays the DAC output waveforms overla
19. desired location the Upper Left Corner press the OK button to proceed to the next step or press the Back button to return to the previous step Page 94 of 125 Ultima Manual ver 2 5 Step 6 Define Upper Right Hand Corner When this step is reached an image window will be displayed with the label Upper Right Hand Corner Image In this step the operator must manually position the spot in the upper right hand corner of the area to calibrate gt Manually move the spot location using the four blue buttons located to the left of the Acquire button The process is to move the spot left or right using the Blue buttons and then to use the Acquire button to capture a new spot image You will notice that during this step the Blue buttons that control movement of the spot up and down have been desensitized This forces the operator to only make left or right movement corrections when defining the upper right hand corner location relative to the upper left hand corner location Once the spot is in the desired location press the OK button to proceed to the next step or press the Back button to return to the previous step Step 7 Define Lower Right Hand Corner When this step is reached an image window will be displayed with the label Lower Right Hand Corner Image In this step the operator must manually position the spot in the lower right hand co
20. erger OPIN EE 46 Z Sernes Cal CUO EE 47 Senes e E 49 PV OVA CAD WEE 52 SLES cial e EE o3 Scdti ITO e DEE 54 TMG MACE VVINIG OW EE 58 Brightness Over Time windows ccccccceceseecaeeeeeeeeeeeeeeseeeseeeaes 61 Line Scan Definition tool eseesesseeeeeennrne 62 Liniescan Eent tel EE 63 ne Scan Data CIS EE 63 ESSE AME VIO WOE o nediwetitddt areae use nu ad fcrt trn or Gt fere refte 63 Beie leg Ee 65 Define an ROI and ROI image cccccccsecceeeceeeceeeeeeseeeseeeseeees 67 PA Masks and Acquired Z series with masks 69 um Line Profile Measurement windows esses 70 MIP Maximum Intensity Projection wipdow sanoanonnnnnnnnnnnnnnnn 71 Image Window and Mark Points WINdOW s nannanonnannnnennnnnnnnnnnnn 12 MarK FONS MNE E 13 MAKR ONIS ele WEE 13 Page 123 of 125 Figure 8 10 Figure 8 11 Figure 8 12 Figure 8 13 Figure 8 14 Figure 8 15 Figure 8 16 Figure 8 17 Figure 8 18 Figure 8 19 Ultima Manual ver 2 5 Figure 8 1 Main TriggerSync wipdow nenne 74 Figure 8 2 Analyze Acquired Data mode cccccccecceceeeeeeeeeeseeeeeeaeeseeeaeeees 15 Figure E 1 TIGOCI SYNC ACUE oneng EE 75 Figure 8 4 View Channel Sensitivity cccccccsecceecceeeeeeeeeeseeseeeseeeseeeaeesaees 76 Figure 8 5 Change Scale amp Auto Gcale cc eccceccceccseeceeece secs eeseeeseeeeeeseeeeaes 76 Figur
21. first simply stop TriggerSync and then re execute it prior to proceeding In general the basic procedure is to enable the appropriate scan mode from within PrairieView then to configure the electrophysiology and photolysis parameters within TriggerSync and finally to initiate data acquisition Following are several examples that illustrate common acquisition scenarios The intent of this section is to provide step by step sequences that may be followed in order to collect various types and combinations of data Detailed instructions on the commands listed are NOT included below It is assumed that the operator is already familiar with the individual commands as described elsewhere in this manual Page 114 of 125 Ultima Manual ver 2 5 9 1 TriggerSync Electrophysiology Acquisition gt Click on Acquisition and DAC Output Setup o Confirm or define stimulus control and acquisition parameters Enable the desired ADC input channels on main TriggerSync window gt Press Acquire in the main TriggerSync window Page 115 of 125 Ultima Manual ver 2 5 9 2 Line scans amp Electrical recording To record with TriggerSync during a line scan start TriggerSync while Prairie View is running Using PrairieView choose the appropriate scan pan and zoom settings to position the image of the specimen Press the Line Scan button S and position the line scan where desired Press Begin Linescan 9 2 1 Line scanning with elec
22. length of scan acquisition e LUT Look Up Table Used to adjust the display intensity scale for an image zu r rc CH CH CH CT C CO a e ROI Region of Interest Used to define a region of interest from which to obtain an image e Snap Camera Icon Saves the current image displayed in an Image Window e pm Line Profile Measurement Used to calculate the FWHM le t Page 58 of 125 Ultima Manual ver 2 5 e PA Photoactivation Used to apply masks to specific slices in a Z series e MIP Maximum Intensity Projection Displays the maximum intensity profile for a position in the z plane at a user selected x y coordinate point e MP Mark Points Allows the user to perform a marked points experiment in PrairieView Note To use the BOT LS LUT ROI and MP functions Live Scan must be Stopped and a single scan acquired before selection these functions To use the PA function a Z series must be defined Page 59 of 125 Ultima Manual ver 2 5 7 4 1 Selecting an Input Channel Ch1 Ch2 Ch3 Ch4 The top four buttons on the left hand side of the Image Window indicate which input channel is to be used to collect data for that window These input channels are generally PMTs but can also be from another detection device such as a Dodt detector Only four input channels are available at any given time By convention Ch1 represents PMT 1 on the PrairieView Main Window Ch2 represent PMT 2 etc
23. masked regions image data Page 69 of 125 Ultima Manual ver 2 5 7 4 8 um Line Profile Measurement The um tool displays a plot of intensity along a user defined line on each of the active channels When the um button is clicked a line appears on the image window and a Line Profile window opens By dragging the ends of the line in the image window it can be positioned across the area of interest By moving the white and dark blue marks along this line in either window the area of interest can be more sharply defined for FWHM calculations To calculate the FWHM of the area between these cursors choose the desired channel under Select Ch and click FWHM When the MIP mode is active See Section 7 4 9 the Z Mode option on the Line Profile window can be used to acquire a profile or calculate FWHM along the z axis at a point in X Y as defined in MIP Line Profile Intensity GA ce ce e n3 ce e e 1 0 60 Microns um New FWHM El Line sm nm Z Mode Calculate FwHM Channel 2 v 1 0 10000 0000 0000 0000 Figure 7 28 um Line Profile Measurement windows Page 70 of 125 Ultima Manual ver 2 5 7 4 9 MIP Maximum Intensity Projection The MIP tool displays the maximum intensity profile for a position in the z plane at a user selected x y coordinate point To use the MIP tool a Z series must first be acq
24. of scan area that is non linear is used to indicate the percentage of the total defined scan area in the x or y dimension that corresponds to the non linear region For example if non linear movement is in the x axis and the image is 512 pixels in the x axis and it is noticed that the non linear region extends 50 pixels in on either side then the value for the control Percentage of outside edge of scan area that is non linear would be 10 50 is approximately 1096 of 512 After these values have been set press the Calibrate button to begin the calibration Page 96 of 125 Ultima Manual ver 2 5 As the calibration proceeds there are a number of fields directly below the Calibrate button that indicate the status of the calibration The first two fields indicate the current point number being calibrated relative to the total number that need to be calibrated The third field indicates the elapsed time since the Calibrate button was pressed 8 4 2 1 Manual Calibration If the Manual calibration method had been selected in Step 4 1 then starting at the first calibration point a new dialog will appear that shows the image of the spot and just two buttons A field below the image window in this dialog indicates which calibration point is currently displayed e g 1 of 4 gt Place the red cursor in the image window over the center of the spot in the image window Use the tools on the lower left hand
25. of the image window to zoom the image window to provide for greater accuracy in placement of the red cursor gt Once the cursor is placed press the Accept button After pressing the Accept button the software will move the pointing device to the next location acquire a new image and then display it in this same window and wait for the operator to place the red cursor over the spot and once again press the Accept button This process will repeat for all of the points in the calibration setup or until the Abort Calibration button is pressed Note If the operator presses the Abort Calibration button then this dialog will be dismissed and the calibration procedure will terminate 8 4 2 2 Automatic Calibration If the Automatic method had been selected in Step 4 2 then starting with the first calibration location the software will move the pointing device to the desired location acquire the spot image and then attempt to automatically find the spot in the image If the spot is found then the application will automatically proceed to the next location and repeat the steps This will continue until all of the calibration locations have been reached The Automatic Calibration will pause in its operation if it is unable to find the spot or if it finds more than one spot in the image If this occurs a new dialog exactly like the one described in the previous section on manual calibration appears Thi
26. of the motor driven pointing device To use this feature gt Nudge the pointing device to the desired location gt Acquire an image of the pointing device gt Move the red cursor from its original position and center it on the spot from the pointing device gt Press the Optimize button This process should eliminate small movement errors that sometimes occur with motor driven pointing devices 8 5 Use Marked Points During Acquisition This feature allows for very high speed positioning of the pointing device for this reason this feature is only available if the selected pointing device is driven by galvanometers This feature allows the operator to position the pointing device at multiple different locations during a single acquisition cycle This also means however that the electrical data collection for these multiple locations will be strung together in a single acquisition cycle This is distinct from other acquisition modes that use the marked points where there is a complete cycle or more than one of data acquisition for each marked point gt If the operator wishes to perform an acquisition where all of the marked points will be visited in a single cycle then set the control Use Marked Points During Acquisition to On gt The control Marked Point Hold Time ms is used to specify how long the pointing device should stay at each marked point during this special acquisition mo
27. time JriggerSync is executed the last list of marked points will still be present in the program As locations are marked there is a control immediately below the Mark Point button that contains a list of the current marked points Immediately to the left of the control is the string Move to As more locations are marked this control will automatically update The operator may use this control to immediately move the selected pointing device to any of the locations that have been previously marked To do this simply click on the control and select the desired point number As the pointing device is moved you will notice the blue cursor in the image window move to the point location just selected Also the values for the Page 101 of 125 Ultima Manual ver 2 5 absolute position will be updated and the values for the relative position will be zeroed Below the Move To control is a button labeled Move to Next Point F11 Just as the Mark Point control had F9 as a hotkey for its action this control uses F11 as a hotkey By pressing this button or F11 the software will automatically select the next point in the marked points list from the current value For example if Point 4 is the current value for the Move to control and the operator presses the Move to Next Point button the Move to control will increment to Point 5 and the blue cursor in the image window will move to the new point locat
28. 00 4 Record Current Location User Defined Shift All Locations Goto Position Set Origin 0 0 xz 32 89 100 43 2283 40 e Goto Origin Load XY Stage Locations Save XY Stage Locations None Figure 7 16 XY Stage tab 7 2 10 1 XY Stage definitions Clear All Function not used at this time Remove Selected Removes selected lines from T series Record Current Location Used to record current stage position Shift All Locations Moves all listed locations by a defined distance Move to Selected Next Previous Moves stage to indicated location Go To Position Drop down list allowing user to select desired position for move stage Load XY Stage Locations Used to load previously saved stage positions Save XY Stage Locations Used to save stage positions This is necessary to choose desired location in a T series experiment Step Size Allows user to define distance to move stage per arrow click Page 52 of 125 Ultima Manual ver 2 5 Set Origin 0 0 Sets current x y location as the origin Go To Origin Returns stage to x y position defined as origin 7 2 11 Misc Under the Misc tab is the label function which gives the user the flexibility to define and save a set of operational parameters for imaging control including laser power PMT settings channels and pixel dwell time It is also possible to set up label groups for more complicated or multi laser applications Laser
29. Channel Colors To turn on the channel simply click on the channel button Grayscale 000 Pseudocolor 001 f lau Range Check 002 To select a display color for a channel right click on that BEL iD 1 Ca 405 i Bl Cascade Blue 423 channel button and a dropdown menu with choices will E oos appear It is important to note that if a solid color is selected for each input channel then multiple channels may be selected for simultaneous display within a single Image Window Additionally the same input channel may be displayed in multiple windows if desired EB 5 Please note that the gray scale and pseudo color options ES Lucifer Yelow 533 may not be combined with multiple channels in one Image 7 Rhodamine 123 533 Wi ndow E FA phycoerythrin 578 C SNARF ACID 580 ES Chramomycin amp 3 590 JC High Can 590 E Lissamine Rhodamine 590 es Ethidium Bromide 535 D XRITC op J BaBa 3 602 C Calcium Crimson 611 P Texas Red 615 C Propidium ladide 617 EN sHARF BASE 630 C Fura Red IAU Ea Acridine Orange ANA 650 T aminoactinomycinD 655 Mate E Allophycocyanin 667 B crs 670 perCP b80 Page 60 of 125 Ultima Manual ver 2 5 7 4 2 BOT Brightness Over Time Pressing the BOT button located on the left hand side of an Image Window enables the measurement of pixel intensity over time In the Brightness Over Time window press Add N
30. HV of the PMTs The PMT HV control unit also has overload protection circuitry designed to prevent damage to the PMTs caused by exposure to an overly bright light source The switch in the center of the unit labeled PROTECT OVERRIDE is Page 17 of 125 Ultima Manual ver 2 5 used to either enable this feature in the PROTECT mode or disable it the OVERIDE mode This mode only responds when the PMT becomes saturated In order to reset the PMTs reduce the cause of the saturation the laser power may be too high the PMT HV may be set too high the room lights may be on etc and then toggle the switch by moving it to OVERRIDE and then back to PROTECT A second form of protection circuitry uses a photodiode sensor to monitor the ambient room lighting When the SENSOR ON switch is in the up position the PMTs will be set to 0 V when the room lights are turned on If the room lights are then switched off the PMTs are turned back on This mode of protection is only activated by external light sources Important Note The red LED display calibration buttons arrow and Reset Enter buttons located directly below the LED displays should not be used These controls are used to calibrate the LED displays They are factory set to display the correct high voltage values for each PMT but have no effect on the voltage itself DO NOT CHANGE THESE SETTINGS AS HE CALIBRATION IS DIFFICULT AND TIME CONSUMING If the display c
31. Important When an image is not visible or suddenly disappears it may be due to this PMT HV safety feature Page 36 of 125 Ultima Manual ver 2 5 7 PrairieView Software Prairie View is the software that controls all of the scanning and image collection functions of the Ultima Image size scan rate pan zoom scan rotation PMT settings laser wavelength and power level file saving and naming are all set and easily controlled using PrairieView This acquisition software also offers a number of features to meet most imaging needs including scan rotation line scan brightness over time BOT region of interest ROI Z series photoactivation marked points and T series 7 1 PrairieView Main Window Once the Ultima and the laser have been powered up the operator is ready to start PrairieView by clicking the icon located on the desktop The main program screen and one Image Window will appear as shown in Figure 7 1 EP IG E imagn Window 1 Es Iss Viren e Heim e Pre Fer Line II LweSca Kew Window p the i IY Sege Som seine HAE me eed E 4 B eer SE gn vem J ee tre fo ool 29 si cen Pate o Scanine Petiod mat 3120 Frame Penod ma 1557 440 Fraser i2 0 62 be ep ya zw FU SSS 1 01 De Ve 1 EG al LX e D 1 Anpan Figure 7 1 PrairieView Main Window Page 37 of 125 Ultima Manual ver 2 5 7 2 Main Window Controls
32. Most of the controls of PrairieView are accessed through the Main Control Window Prairie View v2 5 0 0 File Tools Window Display Help e scanning amp Image Window v mm e Resolution Shutter Controls Tools Label Select Current Settings 204842048 Objective Lens GR Zoom and Oe Ge mi Pan and Scan Dwell Time Rotation Tools BM i D Tools Ce re a n 0 Laser PMT DAQ Z Series T Series 2 P Laser PC Oz XY Stage Misc 1 P Laser PMT TaserLine TT xj gr ai Hr care Cemez T A 4 b Intensity SE A H Controls C see 1 EIN IK Ei WE C senes T PMT Voltage K Die mj Hh and DAQ Gain CGestzer 0 ATE Controls Sr Glas we i Cites 0 A xb xj Hr Laserline al HU ESP WE CGestzer T zl HE x Hr Z amp T series Acquisition Status Scanline Period ms 3 120 Frame Period ms 1597 440 Framerate fps 0 626 Sca n amp F ra m e Rate nfo GER Y 0 Z 0 FOV 5124512 1 Del Dun Prog ram Messages Figure 7 2 PrairieView Main Screen Page 38 of 125 Ultima Manual ver 2 5 7 2 1 Scan Resolution Tools The green vertical bar on the left of this section allows the user to switch between the standard square image scan choices and independent selection of the pixels per line and lines per frame Prairie View v2 5 0 0 i Prairie View v2 5 0 0 File Tools Windows Display H File Tools Window Display F Al Jr 128 J 256 J 512 J 1024 2048 Dee CR KI 2048x2048 128
33. PMT DAG Z Series T Series 2P Laser PC Oz wY Stage Mise Single Scan Esbocuments and Settings userhdy Docum Ee Single lmage 10032006 1620 4222 Labels amp Label Groups T I FAT CAE des EET DAD Gainls Current_Setk SC e 2 H HIC Fale True 0 61000 0 0 0 0 2020101 71 rd Update Label Delete Selected Delete Al babel o label2 Label3 Labeld E 38BH E E E Add Label Update Label Delete Selected Delete All Label Group Group Values Label Group Groups Figure 7 17 Misc tab To create and use a label gt Press the Add Label button A row named Label 001 with current settings will be created gt To change the name of the label double click on the box with Label 001 in it and rename as desired To change any of the settings associated with the label first adjust the setting in the appropriate place for example an increase in laser power would be done in the Laser DAQ PMT tab It is then necessary to click the Update Label Values button gt Selection of a specific label is done by clicking on a label listed under the Label Select drop down menu Page 53 of 125 Ultima Manual ver 2 5 7 2 12 Scan Information At the bottom of the PrairieView Main Window are some boxes containing scanline and frame rate information as shown in Figure 7 17 It also gives current stage location information Scanline Period ms 4 352 Frame Pernod
34. Page 25 of 125 Ultima Manual ver 2 5 In systems with a single galvo pair used for both scanning and uncaging the scanning inputs are connected to the X Input and Y Input BNC connectors and the uncaging inputs are connected to the X2 Input and Y2 Input BNC connectors The Switch BNC input is driver by the TriggerSync software to select between the scanning and uncaging control signals In systems with separate pairs of galvos for scanning and uncaging each pair uses the X Input and Y Input signals from its own Galvanometer Control Box Two amber colored LEDs on the front of each box illuminate to indicate a shutdown state for the X and Y galvos which occurs briefly on power up an afterwards only if the galvo experiences some failure such as being driven too hard so that it overheats The X Feedback and Y Feedback are galvo position feedback signals which are used by Prairie during testing and configuration AOD systems will use the same Galvanometer Control Box In AOD scanning mode the X galvo is held still and used for panning the image while the AOD scans in X Page 26 of 125 Ultima Manual ver 2 5 4 3 Optics A key part of imaging capabilities of the Ultima Multiphoton Microscopy system is the optics used T RE SCATTERED RADIATION MAX AVERAGE OUTPUT POWER 5W EMITTED WAVELENGTHS 650 1100 nm PULSE DURATION 10 300fs CLASS 4 LASER PRODUCT WARNING Never look into the laser beam path The las
35. RI produce 1000 counts per revolution Two of the DCRI switches affect motor behavior they are the Coarse Fine and Normal Alt switches Coarse and fine settings vary depending on the installation typical values might be 1 8 to 1 32 stepping in coarse mode and 1 64 to 1 256 stepping in fin mode Assume a coarse setting of 1 16 stepping and a fine setting of 1 256 stepping in the following explanation In coarse control mode each count from the dials micro steps a motor 1 16 of a step 0 3125 um for the X Y motors and 0 03125 ums for the Z motor In fine control mode each count from the dials micro steps a motor 1 256 of a step 0 020 y for X Y motors and 2 nm for the Z motor When switching from fine to coarse control any motors not already on a 1 16 step boundary first move to such a boundary using 1 256 step micro steps before switching to coarse control The normal alt switch controls when the motors are enabled In normal mode each motor individually disables itself after its most recent motion and enables itself immediately before being ordered to move When disabled the motors do not resist being turned by hand and will not hold the stage stationary if other disturbances occur In alt mode the motor is enabled continually whether or not it is moving X Y fine mode 1000 micro steps per knob turn 1 256 step micro step 1 motor rotation 200 steps 1 mm rotation 19 5 um knob turn with a minimum st
36. TED WAVELENGTHS 650 1100 nm PULSE DURATION 10 300fs CLASS 4 LASER PRODUCT Never look into the laser beam path The laser used in the Ultima system is a Class 4 infrared IR laser It does not have a beam visible to the naked eye The optics used in this system may also cause some back scattered or reflected laser light It is therefore critical to operate this system following all safety instructions and wearing appropriate laser safe eyewear Page 11 of 125 Ultima Manual ver 2 5 CAUTION A Installation of the Ultima Multiphoton Microscopy System To ensure proper installation of this system it must be installed by Prairie Technologies technicians B Do not disassemble Disassembly of this system may result in electrical shock and other hazards including exposure to Class 4 laser radiation C Power supply cords The Ultima system comes with all necessary power cord Do not change or replace them as use of an improperly rated power cord may result in system malfunction or failure D Prevent contact with moisture Moisture contact with any component of the system may result in a short circuit or damage to optical components If water gets into a system component immediately discontinue use of the system turn off power and contact Prairie Technologies immediately E Handle with care This system is designed as a precision optical instrument Each optical and electronic component has been place with great care to as
37. To uncaging shutter Analog outputs For Experiments Figure 4 5 NI BNC 2110 4 2 4 Software communication through NI hardware The software used for imaging PrairieView and the triggering software TriggerSync communicate by passing messages to each other using a TCP IP protocol In order for PrairieView to recognize that TriggerSync is running TriggerSync must be started or restarted after PrairieView is in operation Hardware synchronization of image collection and electrical recording is achieved by sharing a TTL trigger signal by the 6052 6110 and 6713 boards the PFIO TRIG1 input on the upper BNC 2090 lower BNC 2090 and BNC 2110 box for the 6052 6110 and 6713 boards respectively PrairieView and TriggerSync are triggered by a falling edge on PFIO TRIG1 4 2 5 Device Control Box and DCRI NOTE The following describes the use of the Mike s Machine motorized X Y stage and an Olympus BX 51 with automated Z axis fine focus control The step sizes for other configurations may vary from what is described below Page 23 of 125 Ultima Manual ver 2 5 The motor system consists of the Prairie Device Control Box the Device Control Remote Interface DCRI with three dials and eight toggle switches the shielded motor cable an RS 232 cable and the motorized stage All three motors have 200 full steps per revolution The XY motors move the stage 1mm per revolution the Z motor moves 0 1 mm per revolution The dials on the DC
38. ZA PRAIRIE TECHNOLOGIES Ultima Multiphoton Microscopy System User Manual version 2 5 PrairieView TriggerSync 3030 Laura Lane Suite 140 shipping P OBox 620677 mailing Middleton WI 53562 0677 USA Phone 608 662 0022 Fax 608 662 0023 Internet support Support Prairie Technologies com www Prairie Technologies com Ultima Manual ver 2 5 Page 2 of 125 Ultima Manual ver 2 5 Table of Contents EE dicic nt nnn Oa A E 8 2 Sale e te EE 9 2 1 Warning and caution symbols used in this manual 9 2 2 Warning labels used on the Ultima Multiphoton Microscopy System 13 P deeem T T UEM 14 It SCOPES oris WANA ass cess cies da ETRAS EVEN E PUR PU CGU UI 14 3 2 Conventions Used in This Manual 14 ou Mur OVE N ee od Rn elo tdv idm ith ttum deas 14 4 dardware dnd Ge de ale 16 MEME e EE 16 4 2 Rack and Other Electronic CGomponents enonneneensnennsnennsnrerrrrerrn 17 4 2 1 ORT X oT 17 4 2 2 PMT dual preamplifier amp HV control unit 17 4 2 3 National Instruments BNC breakout boxes amp boards 18 4 2 3 1 NIOUSZE DOS CI scans ente iota LU MM aM LUN abor kann enn Sere 18 4 2 3 2 KURS Deor Mec 19 4 2 3 3 NI 6110 6115 Board pixel data sampling methodology 19 4 2 3 4 NI BNC 2090 amp BNC 2110 boxes eene 19 4 2 4 Software communication through NI hardware 23 4 2 5 Device Control Box and DCH 23 4 2 6 Prairie P
39. a Manual ver 2 5 NOTE If the TriggerSync program is running Line Scan acquisition will automatically synchronize with the experiment that has been defined in TriggerSync A coordinated recording will be made that synchronizes the electrical recording from TriggerSync with the image data from the PMTs Use the Linescan Viewer program located on the Windows desktop to view both the line scan image and electrical data that has been collected in this manner Page 64 of 125 Ultima Manual ver 2 5 7 4 4 LUT Look Up Table A Look Up Table LUT is the function that is used to color the display of the data to be displayed on the computer screen The images from the Ultima are digitized to 12 bits which means that the input channel data intensity scale ranges from O no signal to 4095 saturated signal In a black and white LUT values of 0 are usually represented as pure black and values of 4095 are usually represented as pure white Since the computer only has 256 grey levels a function or LUT is used to define the display intensity scale If these 256 display grey levels are used to display the full range of 4096 intensity levels then each display grey level is equal to 16 PMT data intensity levels Lookup Tables Track Mode 200000 Drag Cursor Zoom Log Scale l l l l 813 1538 245 3276 4095 Intensity 1 Low Cutott High Cutoff CH Channel 2 100 Bu Cursors deren Reset Display SE Pixel Cou
40. a multiple of 2 2 4 6 8 The spacing between successive points will be dependent upon the size of the specified grid and the number of points specified in the x and y dimension After the number of points in the x and y dimension have been specified the operator needs to select the Map Method Currently three options are available Option A Sequential The order that the marked points will be generated for the grid will a simple progression starting at the upper left hand corner location and increasing across to the upper right hand corner then dropping down and continuing from the left side to the right side and so on Option B Non neighbor The order that the marked points will be generated for the grid will follow a mathematical progression that is designed to keep successive points from being too close to each other The following section defines the method used for computing these locations To eliminate the effects of desensitization of receptors by previous uncaging at nearby pixels it is necessary to pseudo randomize the uncaging positions If the area that you wish to perform the uncaging in is composed of 2Nx 2Ny pixels the position of the scanner X Y at the th uncaging 0 1 A NX Ny is determined as X i Nx mod i 2 Px int mod i 4 Nx 4 Y i Ny int mod i 4 2 int i 4 Nx where Px is for example Px 0 4 2 6 1 5 3 7 when Nx 8 In the statements listed above mod i
41. a wv LS TE 0533 0000 0000 0000 Figure 7 21 Line Scan Definition tool Using the mouse click and hold on either of the outside blue boxes to position the line scan location vertically Use the inner pair of blue boxes to select the lateral range to scan The laser beam will ONLY scan the selected locations thereby reducing the amount of photo damage to the specimen A Linescan Control window appears when the LS button is pressed This window is used to define the number of lines to acquire in succession the number of repetitions to acquire those lines and the period for the acquisition It is also used to indicate any trigger controls and allows the user to select a free draw option by checking the appropriate box Click on Start Linescan to begin the acquisition The acquired data will be displayed below Base Directory Once the line scan is complete this mode can be exited by again pressing the LS button on the Image Window It is possible to look at acquired line scan data by opening the Linescan Viewer program located on the Ultima computer desktop Under File find and open the line scan data of interest An image file will open rotated 90 degrees as shown in Figure 7 21 so that the x axis is defined as time In the Linescan Viewer it is possible to look at the line profile of the image data pixel dwell rate by selecting a position of interest along the line f electrophysiological d
42. alibrations are inadvertently changed they may be re calibrated by referring to the supplied documentation on the Hawk brand meters 4 2 3 National Instruments BNC breakout boxes amp boards There are 3 National Instruments PCI boards in the system PC computer the NI 6052E NI 6713 and the NI 6110 6115 There are also three external rack mounted National Instruments components two BNC 2090 units and a single BNC 2110 The NI 6052E and NI 6713 are connected together internally to share clock signals and other control signals This results in highly synchronized operation The 6052E board is used by TriggerSync to make electrical recordings The 6713 board is used to generate output signals to various components as well as electrical stimuli for experiments The 6110 6115 board is used to drive the imaging galvanometers and to collect PMT data 4 2 3 1 NI 6052E board The NI 6052E board collects all the input signals generated while running experiment This board samples the enabled channels sequentially If only one channel is enabled the collection rate can be as high as 333K samples sec If additional channels are enabled for acquisition maximum number of acquisition channels is 8 the maximum sampling rate is divided down proportionally Signals can be input to this board via the upper BNC2090 box in the electronics rack Page 18 of 125 Ultima Manual ver 2 5 4 2 3 2 NI 6713 board The NI 6713 board generates the output wave
43. arameters in the Confocal software After the image has been acquired it will be automatically displayed in the image window in the Mark Points dialog gt The Nudge F7 button is used to position the selected pointing device at the location of the red cursor in the image window This could be useful for validating the accuracy of the calibration of the pointing device To use this feature Place the red cursor on the location within the image where the pointing device is to be moved Press the Nudge button you should see the blue cursor move and the values in the absolute and relative fields updated as well Make the necessary control changes to actually acquire the image of the pointing device the spot image Acquire an image by pressing the Snap button If the calibration is accurate then the location of the spot in the image field should correspond to the location of the red cursor If your system configuration includes a separate Pockels control on the uncaging laser there will also be slider control in this section labeled Laser Page 111 of 125 Ultima Manual ver 2 5 Intensity The operator would use this control to adjust the voltage of the uncaging Pockels If the selected pointing device is motor driven the Optimize F6 button will be activated for galvanometer based pointing devices this control is desensitized This control allows the operator to fine tune the calibration
44. ata in samples second has also been acquired it is possible to overlay the two sets of data to see how the image data and the electrical data correlate in time Page 62 of 125 Ultima Manual ver 2 5 Linescan Control 512 WIEN 3 024 om he Ee ime ms m Free Draw EE Total Acquisition Time s E Reset E Calculate EE 1 548288 C Zoom e Zoom Zooomr Save Path IC ADocuments and Settings Prairie Browse Se SERRTE RETE Start Linescan s Line Scan 0022007 1454 1 4 Number OF Lines Ext Repetition Trigger Scanline Period ms Display Zoom mag BpEs 4 Input Trigger Figure 7 22 Linescan Control Ee 01x BES erre meme Figure 7 23 Line Scan Data display Ib LinescanViewer 3 0 vi File Display i gt SY Line 1 7 ineScan 10052006 1547 1 Line 1 Line 1 Profile 1 2 Data LineO00001 prm 655 440 0 420 0 400 00 create Coor Lanz Move Profile Up Copy Profile s Move Profile Down to Line s 360 01 380 0 351 6 Delete Selected Profile s 0 0006 0 0250 0 0500 0 0750 0 1000 0 1250 0 1500 0 1750 0 2000 0 2250 0 2500 0 2750 0 3000 0 3250 0 3500 0 3713 pero RS Tei B r ies 2 S ni EF Figure 7 24 Linescan Viewer NOTE If the TriggerSync program is running the operator is prompted to enter a file name for the line scan image and electrical data All the data will be saved once the line scan is completed Page 63 of 125 Ultim
45. ated regions and triggers are possible using the T series tab This allows the user to optimize a complex set of experimental parameters to the acquisition of key data LaserPMT DAQ Z Series T Series 2PLaser PC Oz XY Stage Misc T Series Cycle Data H Images Max E Trigge Senet Period s Speed Time s Seres SERIES Cycle k T H vem EP ITE Add TriggerSyne Esp Add sr Stage huic ST eS UNS ooo lomo liu Update g a save Path 12d T Series Iterations C ADocuments and Settings Prairie 7 Leave hard shutter open e for duration of T Series Base D rectory TSeries 12052006 1230 46 m GE Figure 7 15 T series tab 7 2 9 1 T series definitions Images Z series How many images or Z series are to be acquired at this position Period The length of time it takes to go from start to start in a collection of images or stacks Max Speed A mode which allows images to be acquired continuously there is no live update while this acquisition is occurring Max Speed is available for Z series only with piezos Time Minimum amount of time to acquire the image s calculated by PrairieView Z series Acquire a Z series instead of a single image See Section 7 2 8 PA Use predefined photoactivation PA masks corresponding to a z slice See Section 7 4 7 Page 49 of 125 Ultima Manual ver 2 5 BOT Collect brightness over time BOT data based on predefined regions See Section 7 4 2
46. ation data is dependent upon many system parameters it is useful if the calibration file name incorporated enough information so that the operator can keep the different calibration files separate and easily select the proper file For example if the operator is using a 60x water H2O lens at an image resolution of 512x512 and a zoom of 1 he she might select a calibration file name of Lens60 H20 512x512 zoom1 cal the Cal extension will be automatically appended to the operator specified name Now if the operator wishes to calibrate the same lens at the same image resolution but at a zoom of 2 he she might specify a file name of Lens60 H20 512x512 zoom2 cal Step 4 1 Calibration Method This step allows the operator to specify whether a Manual Default or Automatic calibration should be performed In general it is recommended that the operator select the Manual option The operator can now either press the OK button to proceed to the next step or press the Back button to return to the previous step Step 4 2 Setup for Automatic Calibration If in calibration Step 4 1 the operator had selected the Manual calibration option this step would have been skipped In its current implementation for the Automatic point calibration to proceed successfully the spot image needs to maintain a relatively constant intensity and shape throughout the area of the image to be calibrated
47. ay press the Cancel button If the OK button has been pressed then the operator will be prompted again to make certain that they wish to delete this point If YES is pressed the point is deleted If NO is pressed the point is not deleted Below the Erase Point button is the Show Points button When this button is pressed it will display a new image window with the current marked points locations indicated on the image in the color specified in the Overlay Color control near the bottom right hand corner of the Mark Points dialog Below the Show Points button is the Save Point Images button When this button is pressed it will display a dialog that presents the operator with four check boxes Page 102 of 125 Ultima Manual ver 2 5 A Save Open Image B save Open Image with All Points Marked C Save Individual Open Image with Each Point Marked D Save Cursor Locations The operator may select any combination of these options After selecting the desired options press the Cancel button to exit the dialog or press the OK button to save the desired data If the OK button is pressed the operator will be prompted to select a directory and base filename to use for saving the desired data If Option A is selected then the current image will be saved with the specified file name If Option B is selected then the current image will be saved and superimposed on the image will
48. ays electrophysiology data along with fluorescence line scan profile s Create a profile o Position profile in desired location by clicking in line scan window o Zoom up image window if desired o Copy profiles to other line scans o Create multiple profiles Page 116 of 125 Ultima Manual ver 2 5 Print the graph Export the data Width control sets number of lines to average Move profile up down shifts the profile by the chosen amount gt See the LinescanViewer application documentation for a complete description of how to use this application OOF e Page 117 of 125 VV VVWV Ultima Manual ver 2 5 9 3 Electrical recording amp Uncaging Start Prairie View Start TriggerSync In PrairieView use Live Scan or Single Scan to locate the area of interest Use pan rotate and zoom to orient the target region Load the correct point calibration file for your scan parameters o Open the Mark Points dialog in TriggerSync o Load the appropriate calibration file for the Uncager Calibration File field Click on Acquisition and DAC Output Setup in riggerSync o Confirm or define stimulus control and acquisition parameters and close the dialog Enable the desired ADC input channels on main TriggerSync window Mark Points o Open the Mark Points dialog in TriggerSync o Press Snap in TriggerSync to collect a reference image on which to mark your points o Use any combination of dis
49. crete points lines of points or rectangular arrays of points MODE 1 Run Acquisition at Each Point NOTE The current cycle number and point number can be viewed in the upper right corner of the main TriggerSync window Select Acquire Loop option in main TriggerSync Window This will run the Cycle Number of acquisitions set in the Acquisition and DAC Output oetup dialog at each point of Uncaging MODE 2 Move to Different Points During a Single Acquisition NOTE The Point Number in the upper left corner of the main TriggerSync window does not update when collecting data in this mode Select Use Marked Points During Acquisition at the bottom of the Mark Points window in TriggerSync o Move time is on the order of 0 10 ms o Setfirst point delay time o Verify the Hold and Move times Press Acquire in riggerSync Page 118 of 125 VV VV WV Ultima Manual ver 2 5 9 4 Line scans Electrical recording amp Uncaging Start PrairieView Start TriggerSync In PrairieView use Live Scan or Single Scan to locate the area of interest Use pan rotate and zoom to orient the target of the specimen Load the correct point calibration file for your scan parameters o Open the Mark Points dialog in TriggerSync o Load the appropriate calibration file for the Uncager Calibration File field Mark Points to define locations to be uncaged o Open the Mark Points dialog in TriggerSync
50. d so that experimenting with different Map Methods can be done by simply making the desired change and pressing the Generate Map Points button without having to redefine the bounding corners Page 108 of 125 Ultima Manual ver 2 5 8 5 5 Functional Map Display aew Points Rdv Configuration Help No online analysis Trace Averages Running Average UV Amplitude Analysis Amplitude Analysis using cursors Exponential Fits Fluorescence Quantification Functional Mapping Click on the Functional Mapping selection in the Analysis menu as shown above IS Functional Mapping vi PAE EEE EE PE ERR N DECHE ECH AECH DAN RC LIRE TER EA M N H T T T T T T 0 0000 0 0100 0 0200 0 0300 0 0400 0 0500 0 0600 0 0700 0 0800 0 0900 0 1000 0 1100 0 1200 0 1300 0 1400 0 1500 0 1600 0 1700 0 1800 0 1900 0 1990 b ba ES TEE rat AIF irt vs n Load Current Data Load Saved Data m Esc sm Electrical EletriealMap enter RotatedDown16x16 prm Show Tools Export Data Figure 8 23 Functional Map Display Analysis Page 109 of 125 Ultima Manual ver 2 5 If a functional mapping experiment has just been conducted the most recently collected data will be displayed Otherwise use the Load Saved Data button to read in a data file d sum Electrical Map Export Data gt Use the pair of vertical cursors on the electrical data to define the window in tim
51. d New button A row with default settings will be created gt To select the number of images or Z series to acquire click on the Images Stacks field type the desired number of images to collect and press Enter The default value is 1 so no changes are necessary if only one image or Z series is desired Page 50 of 125 VV VV WV Note Ultima Manual ver 2 5 Click on the Period field type in the time required for this line of acquisition to take Press lt Enter gt Click the Max Speed check box if this is desired Click the Z series check box This will cause a Z series to be collected as currently defined in the Z series tab Section 7 2 8 Verify the Save Path Press Start T series The active T series line is the one marked by an arrow in the leftmost column of the T series cycle data list 7 2 9 3 Collect a Complex T series A Complex T series is one in which numerous steps defining different operations are strung together into a single experiment lt is possible to collect a Z series at a set interval or to collect individual images and Z series alternately For example a user wants to create a complex T series in which there are three steps 1 collection of 10 single images with 30 seconds between each image 2 a waiting period of 4 minutes and 3 the collection of a Z series To create a Complex T series such as this VV VV Oo NN NN V VV Note Press the Add New b
52. d be repeated Time Delay between Pulse Trains ms specifies the number of milliseconds used to separate one iteration of the defined pulse train s from the next iteration of the defined pulse train s To edit the parameters for a given pulse train first select the pulse train to edit using the Pulse Train control Pulse Potential defines the height of the pulse train and Duration defines the amount of time each pulse is held at the Pulse Potential The Number of Pulses defines the number of pulses in the pulse train First Pulse Delay specifies the number of milliseconds from the start of the acquisition until the first pulse starts Inter Pulse Delay indicates the number of milliseconds to separate each pulse 8 2 3 4 G F C General Fluorescence Control When the G F C protocol is selected the controls for the other protocols will be desensitized This protocol simply maintains the specified Holding Potential during the acquisition 8 2 3 5 Custom Waveform Page 83 of 125 Ultima Manual ver 2 5 When the Custom Waveform protocol is selected the controls for the other protocols will be desensitized This protocol allows the a file to be created that contains any desired sequence of voltage values The file should be a text file that contains a column of numbers that represents the desired voltage values The column of numbers can be in standard notations e g 01003 or in scientific notation e g
53. de gt The control Marked Point Move Time ms is used to indicate how long it physically takes for the pointing device to move from one marked point to the next This value defaults to 0 1ms since it takes approximately 100 microseconds for the two galvanometers to move and stabilize between two relatively close locations If the spacing between the marked points Page 112 of 125 Ultima Manual ver 2 5 becomes too large it may be necessary to increase this value to 0 2 or 0 3ms The control Marked Point First Point Delay ms is used to indicate an additional time delay at the start of the experiment in addition to the Marked Point Hold Time before moving to the second marked point As its name implies this value is only applied to the first marked point The control Synchronize Marked Points with Selected DAC Output is used to automatically synchronize the movement from one marked point to the next with a pulse train defined for the selected DAC Output specified by the control DAC Output Typically the selected DAC Output will be used to control a Pockels device on the laser controlled by TriggerSync By turning on this option the software will automatically generate the timing information for the movement between the specified marked points to be properly synchronized with the Pockels control The control Replicate Points when turned on and the control Synchronize Marked Points with Selected DAC O
54. desired file The Pinhole Motion control will only be active on older Prairie systems that include a NeD Pinhole for near field detection To the right of the Pinhole Motion control is a set of four blue buttons and a control labeled steps These blue buttons are used to physically move the selected pointing device The selected pointing device will move an amount specified by steps The value to use for steps is dependent upon the type of pointing device being used For a galvanometer based pointing device the steps field should be specified in millivolts mV So a value of 200 in this field indicates a desired movement equal to 200 mV from the current location For a motor driven pointing device the steps field should be specified in motor steps So a value of 55 in this field indicates a desired movement equal to 55 motor steps from the current location The four blue buttons are oriented in a diamond pattern to indicate the direction the pointing device should move when the button is pressed For example if the top most blue button is pressed the pointing device should move up or towards the top of the current image by the amount specified in the steps field Based upon the accuracy of the pointing device calibration it will most likely not be necessary for the operator to use the blue buttons to position the pointing device manually Typically just the red cursor will
55. desired gain value from the list of choices 7 2 7 2P Laser The 2P Laser tab provides additional options for laser control of the Ultima s laser s including laser selection and wavelength adjustment Laser PMT DAQ Z Series T Series 2P Laser PC Oz XY Stage Mise 2 P Laser Status 2 P Laser Control Wavelengths Step Dat ae um d KR H bo Eck P _CaserSelection Selection Wavelength Sernes Calculator zl Start Wavelength nm Set Start b Define Wavelength nm mw Laser 1 lo lo Stop Wavelength nm Set Stop Bx Error lo Define mis i Laser In fn wee KK C Defi mS AU G lintea m Leave hard shutter open for duration of W avelength 5 eres C ADocuments and SettingsVFrairie Start Wavelength Sernes Base Directory WSeries 12052006 1220 0 4 Figure 7 11 2P Laser Control 7 2 8 Z series Under the Z series tab are the controls that are used to collect stacks of images called Z series or z stacks and to directly control the z motor when searching for a desired focal plane in the specimen This tab allows the user to define start and stop positions step size or number of steps and necessary PMT adjustments Page 45 of 125 Ultima Manual ver 2 5 Laser PMT DAQ Z Series T Series 2P Laser PC Oz XY Stage Misc Z Series Slice Data E Z Series Calculator Z Motor S
56. detector This detector is mounted at the back of the microscope and is used for obtaining DIC like images which are useful for imaging unstained cells for example in electrophysiological experiments involving patch clamping Page 31 of 125 VV VVWV YYY VV VV WV Y VV Y VV VW Ultima Manual ver 2 5 5 Ultima Startup Procedure Following instructions from laser manufacturer put laser in ready state Turn on the electronics rack Check and or install filters and dichroics into upper PMT detection path BG39 Olympus cube and filters Turn on the PC Important Disable anti virus software Close Sub stage detector shutter Sub stage detectors are optional and may not be present in your system Start Prairie View Start TriggerSync if you plan to conduct electrical recording experiments Verify the communication link between PrairieView and TriggerSync A message will appear at the bottom of the Prairie View window stating that the link has been established Open laser shutter in laser software provided by laser manufacturer Push Olympus trinoc plunger in to the Bi position Position Olympus epi dichroic wheel to some open position Transmitted light will turn on automatically Put sample on stage and focus Turn off or COMPLETELY shutter the epi fluorescence mercury lamp house if present on your system The Shutter mechanism provided on the front of the Olympus epi fluorescence illuminator is NOT a
57. e Ext Sensitivity value mV V for each analog output The Ext Sensitivity mV V control is used as a scale factor for the voltage values entered in the stimulus protocols To determine the actual voltage being output use the following equation 1 Ext Sensitivity protocol voltage Output voltage For example in the G F C protocol if an Ext Sensitivity of 20 is used and the Holding Potential mV is set to 10 the actual output voltage will be 0 5 Volts 1 20 10 z 0 5 This scaling is applied to all voltages in all of the available protocols DAC Label This control allows the operator to specify a text string to display over the individual graph window along the right hand side of the dialog For Page 81 of 125 Ultima Manual ver 2 5 example DAC 6 Out is often wired to control the Pockels cell device so the operator might want to set the DAC Label for this DAC to something like Pockels Output 8 2 3 Protocols 8 2 3 1 I V Current Voltage Figure 8 11 I V Setup When the IV protocol is selected the controls for the other protocols will be desensitized The I V protocol allows the operator to divide the acquisition time among various Epochs For each epoch the operator specifies the amount of time and the desired voltage value In addition for epochs B through there is an additional field labeled Increment mVY This field is used when running an acquisition that ha
58. e that corresponds to the data you wish to use in your map Select the arithmetic operator under the Analysis Selection Choices are average maximum minimum and sum Image Option offers two choices o Electrical Map creates a map of the entire rectangular region by means of interpolation to fill in the pixels that were not uncaged directly o Pixel Replacement replaces the individual pixels that were uncaged only with no interpolation Select the Processed Image Color of your choice This is for display purposes only and has no effect on the data The color can also be changed after the map has been generated Process causes the functional map s to be generated The reference image collected with the PMTs will also be displayed for reference along with a calibrated intensity color scale B210 FF Figure 8 24 Functional Map example Page 110 of 125 Ultima Manual ver 2 5 ib Scale Information l 48 0 x Figure 8 25 Functional Map Calibrated Display scale 8 5 6 Miscellaneous Controls Located below the lower right hand corner of the image window is a collection of miscellaneous controls gt When the Snap F3 button is pressed a new image will be acquired If TriggerSync is running in conjunction with a Confocal system then pressing the Snap button will cause the Confocal software to scan a new image using the scan parameters currently defined scan p
59. e 9 6 oequentlal EE Tf PIGUIE 6 72 SAVE E Me ln EE T Figure 8 8 Acquisition and DAC Output Setup ccc cecceecseeeeeeeeeeeeeeeeees 78 POUES O ELE Lei SEUD EE 19 Self UE Oro giro A 80 WME SOU EE 82 PRED ROTER T TR TR 82 Pulse TFA iicestassecasenedaasacaediaeacaas TER cnedeaesasadiaaacaaseaedcaedeaeieaadiaesesaseas 83 Een EE 84 Use Marked Points During Acquisition 84 DAC Out Composite ele BEE 86 PLDC ele EE 86 ADC CONTGUPAT ON EE 87 PONG allOratlOn Bal Ee DE 90 Figure 8 20 Mark Points window eeeseeeeeeeeneneneen nnne 98 Figure 8 21 Show map boundary sessesssssssseseeeeenenn nennen nnn 105 FIgure 0 22 Show trap d e 108 Figure 8 23 Functional Map Display Analysis eese 109 Figure 8 24 Functional Map example nnnnanennnnnnunsnnnrensnnnnnrsnnnrnrnrsrrnrernernen 110 Figure 8 25 Functional Map Calibrated Display scale 111 Page 124 of 125 Ultima Manual ver 2 5 List of Tables Table 1 Maximum NI 6052 and NI 6713 Sample Rates 19 Table 2 Motor Controller Resolution ccc ccccsecceeeseeteeeeeeteeeeeeeeeeeeeeeeeeeeees 25 Table 3 Non Neighbor order of uncaging evample 107 Page 125 of 125
60. e Upper Left Hand Corner As soon as this step is reached an image window will be displayed with the label Upper Left Hand Corner Image In this step the operator must manually position the spot in the upper left hand corner of the area to calibrate gt Manually move the spot location using the four blue buttons located to the left of the Acquire button The four blue buttons are oriented in a diamond pattern to indicate the direction the pointing device should move when the button is pressed For example if the top most blue button is pressed the pointing device should move up or towards the top of the current image by the amount specified in the steps field The selected pointing device will move an amount specified by steps The value to use for steps is dependent upon the type of pointing device being used For a galvanometer based pointing device the steps field should be specified in millivolts mV So a value of 200 in this field indicates a desired movement equal to 200 mV from the current location For a motor driven pointing device the steps field should be specified in motor steps So a value of 55 in this field indicates a desired movement equal to 55 motor steps from the current location The process is to move the spot left and or up using the Blue buttons and then to use the Acquire button to capture a new spot image Once the spot is in the
61. e elt Ee TT 122 9 6 1 Display and analysis of Functional Maps 122 r eg IQUE Sz r m 123 ISU OF ADIOS EE 125 Page 6 of 125 Ultima Manual ver 2 5 This page intentionally blank Page 7 of 125 Ultima Manual ver 2 5 1 Preface Every effort has been to ensure that the data given in this document is accurate The information figures tables specifications and schematics contained herein are subject to change without notice Prairie Technologies Inc makes no warranty or representation either expressed or implied with respect to this document In no event will Prairie Technologies Inc be liable for any direct indirect special incidental or consequential damages resulting from any defects in its documentation Page 8 of 125 Ultima Manual ver 2 5 2 Safety Precautions 2 1 Warning and caution symbols used in this manual The Ultima Multiphoton Microscopy system is designed with the utmost safety of the user in mind However improper use or failure to follow safety instructions may result in personal injury and or property damage Please read this manual before operation to ensure proper use of the system oafety instructions in this manual are accompanied by the following symbols to highlight their significance Please pay attention to the instructions highlighted by these symbols Disregarding the instructions accompanying this warning symb
62. e its own unique calibration file Close imaging beam shutter Start PrairieView start TriggerSync Verify the communication link between PrairieView and TriggerSync A message will appear at the bottom of the Prairie View window stating that the link has been established Turn off channels 1 amp 2 Turn on channel 3 Set to 4ms line Open Uncaging laser shutter in TriggerSync Perform Point Calibration as described in Section 8 4 VV VW VV VV N 6 4 Resetting the PMT High Voltage Important The PMT high voltage will automatically turn off if an overload condition is detected The system is designed with two levels of PMT protection First is the photodiode that is located in the PMT high voltage power supply This senses when the room lights are on and automatically turns the PMT high voltage to O V The second protection is built in to the signal detection electronics When and excessively bright signal is detected the PMT high voltage is again set to 0 V To reset the PMT high voltage power supply gt Correct the condition that caused the overload condition to occur gt Turn the Protect Override toggle switch on the front of the PMT high voltage power supply momentarily to Override gt Turn the Protect Override toggle switch on the front of the PMT high voltage power supply back to Protect This will cause the PMT high voltage power supply to return to its pre overload values
63. e performed inthe 512x512 mode with optical zoom setto 1 0 and rotation at 0 Objective Name Power Foy Width FOV Height Scan Mode Calibrated 1 default objective 1 115 5593699 115 5593699 DualScan True z default objective 1 111 7504639 111 7504639 OualScan True Add New Objective Enter calibration distance here um 50 0 E Remove Selected Objective Calibrate Selected Objective Select Add New Objective Fill in the information for objective name power and NA Enter the calibration distance obtained by drawing a horizontal line from edge to edge on the calibration object Page 56 of 125 Ultima Manual ver 2 5 Click on Calibrate Selected Objective The FOV Width and Height field will now contain values and the Calibrated field should say True Close this window All calibrated objectives shown in this window will also appear under Objective Lens the PrairieView Main Window VV NN 7 3 3 Window Opens a new Image Window Functions the same as the New Window button 7 3 4 Display Display Help e Ghost This utility that causes the Main j Ghost Mace Control Window to appear translucent t IINE on may be useful when combined with a dark SOW Seve Ear desktop background color and operating scale Bar Options the system in a darkened room Overlay Color ws e Show Scale Bar Places a scale bar on Overlay Font the Image Window Channel Enabled Color e Scale Bar Options Al
64. e scan Page 28 of 125 Ultima Manual ver 2 5 head is used to move it in and out of position When it is in position the Ultima operates as a laser scanner system When it is retracted a normal CCD camera customer supplied may be used with standard trans illuminated or epi illuminated microscopy techniques The mirror is in a mount manufactured by Prairie It is also possible to mount a dichroic mirror in this position Contact Prairie for more details The Beam Combining Dichroic is a 760nm dichroic used to combine the two 2 photon laser beams so that one is used to uncage and the other for imaging The mirror is in a mount manufactured by Prairie g A has e E Mm MSS T T3 Ex o 7 imm 2 ei i i ining sue RR m 9 Shutter EZ Beam Com Dichroic E Li E Camera Port Mirror Dichrok Dichroic Fa i Figure 4 7 Ultima scan head dichroic locations 4 5 2 Microscope The Ultima Multiphoton Microscopy System is built around a modified upright microscope The manuals associated with this microscope have been provided to the user Please refer to these manuals for specific information about the microscope components 4 5 3 Detectors CAUTION PMTs are extremely sensitive to light Care should be taken to eliminate all sources of stray light from reaching the PMTs as this will dramatically reduce the sensitivity of the system Prairie Technologies provides a light tight and laser
65. ect Sequential File vi Select the base Filename Select the First value to to be used in the sequence be placed after the Filename First File DOOO1 dat Select Directory to save sequential Files lt Not 4 Path E Figure 8 6 Sequential File 8 1 2 4 Save Experiment A little further to the right is a button labeled Save Experiment When this button is pressed the operator will first be prompted as to whether the experiment should be saved in TriggerSync format this includes all operating parameters as well as the data or to Export format this includes only the data which is a format readable by a spreadsheet program such as Microsoft Excel Figure 8 7 Save Experiment Page 77 of 125 Ultima Manual ver 2 5 8 2 Acquisition and DAC Output Setup In the upper left hand corner of the main window of TriggerSync is a button labeled Acquisition and DAC Output Setup Pressing this button will bring up a dialog that the operator uses to control various input and output parameters to be used during a riggerSync acquisition While this dialog is open the operator may not adjust any parameters in TriggerSync or any other TriggerSync dialog that are not part of this dialog Acquisition and DAC Output Setup F When this dialog is opened the Mark Points dialog will automatically be closed and the Acquisition and DAC Output Setup dialog opens Acquisition and DAC Output Setup
66. eing performed this button changes to read Stop Scan Clicking a second time causes the scan to stop and the laser beam to be shuttered so that it no longer scans across the specimen 7 2 3 2 Single Scan When Single Scan is clicked the laser is scanned once across the specimen with the result displayed in the image window s The laser is shuttered following collection of the image 7 2 3 3 New Window Clicking on New Window will cause another image window to appear on the desktop It is possible to have multiple image windows open at one time The operator can for example assign a different PMT to separate image windows if desired 7 2 3 4 Average Sum During data acquisition the underlying sampling rate is 1 sample per 0 4 us for a galvo system This means that for a 4 us dwell time the system is either averaging or summing 4 us 0 4 us 210 samples per bin 7 2 4 Shutter and Imaging Mode 7 2 4 1 Hard Shutter Closed Open Although software controlled for routine imaging it is possible to manually open and close the hard shutter to allow access to the beam by TriggerSync or other interfaced applications Page 41 of 125 Ultima Manual ver 2 5 7 2 4 2 Soft Shutter Closed Open Although software controlled for routine imaging it is possible to manually open and close the soft shutter This is most useful when the mechanical movement of the hard shutter causes disruptive vibration during electrophysiological exp
67. el Controls the dwell time that the laser beam is at each pixel location in the image with values in microseconds us The minimum value allowed is displayed below and to the left of the slider Increasing pixels per line Zooming or rotating may decrease the minimum dwell time possible 7 2 2 2 Optical Zoom Controls the size of the area of the specimen that is being scanned An optical zoom 2 will cause the microscope to scan an area that is 72 the width and 72 the height of a scan at a zoom 1 Optical Zoom does not affect the number of pixels in the image It is possible to increase the true optical resolution of the system by using this function The Reset button causes the zoom to return to the default value 1 The minimum value allowed is displayed below and to the left of the slider Note It is not possible to set the optical zoom to less than 1 7 2 2 3 Window Size There are four options under Window Size Fit is a default to 512 x 512 1 1 means that one display pixel is equal to one acquisition pixel Smaller decreases the image size by approximately 10 Larger increases image size by approximately 10 Page 40 of 125 Ultima Manual ver 2 5 7 2 3 Scanning amp Image Window Tools Single Scan Jee Soft Shutter Figure 7 5 Scanning tool S in 7 2 3 1 Live Scan Stop Scan To start the Ultima system scanning click on Live Scan While a live scan is b
68. en the TJriggerSync application is first started If the Disable Analysis control is checked then this is the only mode of operation for TriggerSync until the Disable Analysis control is unchecked This is the operational mode that TriggerSync must be in for the operator to set the various acquisition parameters calibrate the pointing device and create a list of marked points Page 75 of 125 Ultima Manual ver 2 5 8 1 2 Analyze Acquired Data On This mode is intended for post acquisition evaluation of the data This includes analysis of the most recently acquired data as well as analysis of previously acquired and saved data When the Analyze Acquired Data mode is on several menu options are disabled as shown in Figure 4 Also most of the controls on the main TriggerSync window will also not operate although they may not be disabled like the menu options In addition when Analyze Acquired Data mode is on some additional controls will become visible in the main TriggerSync window 8 1 2 1 View Channel Sensitivity At the top center of the window just left of the buttons that are used to select the desired input channels is a control labeled View Channel Sensitivity This control specifies which input channels vertical scale y axis values will be displayed along the left hand side of the graph area The values for the channels minimum maximum and middle will be displayed in the same color as that specified f
69. ent the target region Load the correct point calibration file for your scan parameters o Open the Mark Points dialog in TriggerSync o Press Snap in TriggerSync to collect a reference image on which to mark your points o Load the appropriate calibration file for the Uncager Calibration File field Mark Points to define locations to be uncaged o Use the Mapping Setup section of the Mark Points dialog to create a grid of marked points To perform a Functional Map acquisition the marked points list must only consist of a single grid of marked points as defined in the Mapping Setup procedure Click on Acquisition and DAC Output Setup in riggerSync o Confirm or define stimulus control and acquisition parameters then close the dialog Enable the desired ADC input channels on main TriggerSync window Turn on the Functional Map acquisition mode by clicking on the Functional Mapping checkbox along the right hand side of the main TriggerSync window Press Acquire in TriggerSync Save the Functional Map data 9 6 1 Display and analysis of Functional Maps For a description of the display and analysis options for Functional Map data see the TriggerSync documentation Section 8 5 5 Page 122 of 125 Figure 3 1 Figure 3 2 Figure 4 1 Figure 4 2 Figure 4 3 Figure 4 4 Figure 4 5 Figure 4 6 Figure 4 7 Figure 4 8 Figure 4 9 Figure 7 1 Figure 7 2 Figure 7 3 Figure 7 4 Figure 7 5 Figure 7 6 F
70. ep size of 19 5 nm For the Z axis the figures are one tenth those above only 0 1 mm motor rotation Z coarse mode is 31 25 um per knob turn 31 25 nm minimum step Z fine mode is 1 95 um per knob turn 1 95 nm minimum step For all axes the amount of motion for a single micro step indicated above is an ideal estimate The actual amount of motion will vary from micro step to micro step due to internal factors within the motor drive electronics inaccuracies and friction within the mechanical hardware Also backlash will introduce positioning errors each time the direction of motion is changed The most accurate positioning occurs at each whole step as the motor naturally wants to sit at this position and the electronics are not trying to balance the rotor between two phases as they are required to do when micro stepping Page 24 of 125 Ultima Manual ver 2 5 312 5 um 19 5 um 312 5 um 19 5 um Z 3125yum 1 95 um Table 2 Motor controller resolution The Device Control Box contains power supplies three micro stepping motor drivers and the logic for responding to commands from the DCRI or from the RS 232 input and for controlling the motors through the micro stepping motor drivers Three LEDs on the front of the Device Control Box provide basic status information The leftmost LED indicates power on the center LED is on whenever the controller has moved any of its three motors within the previous half second The righ
71. er beam at first pinhole Use adjustments in sidecar to center spot on objective lens target Stop iris down only to the size of the ring in the objective target window Leave iris in this position to minimize back reflections into upper PMTs Close measurement and automation Return Uniblitz shutter switches to their normal positions Restart system software 1 2 Uncaging Beam Alignment in Scan Head Important Re aligning the uncaging laser will introduce an offset into all of the spot calibration files gt VVVVVV WV YYY YYY VV WV Close Prairie View Close TriggerSync Remove sample Raise objective lens all the way up Install objective target in turret Open measurement and automation toolbox on windows desktop Under Devices and Interfaces o Traditional NI DAQ devices Select PCI 6713 Select Test Panels e Use analog output and DC voltage e Set channels 5 and 7 to 0 00 Volts e Be sure to press update channel after selecting each channel Close shutter for imaging beam Open shutter for uncaging beam Verify that the image of the X axis galvo mirror is centered on objective target ring Use adjustments at top of tower to center beam at first pinhole Use adjustments in sidecar to center spot on objective lens target Stop iris down only to the size of the ring in the objective target window Leave iris in this position to minimize back reflections into upper PMTs Close measurement and automation Return Un
72. er used in the Ultima system is a Class 4 infrared IR laser It does not have a beam visible to the naked eye The optics used in this system may also cause some back scattered or reflected laser light It is therefore critical to operate this system following all safety instructions and wearing appropriate laser safe eyewear 4 4 Laser Light Path After exiting the laser the beam of light travels to a Pockels cell modulator assembly through a set of mirrors which is an electro optical modulator used for beam attenuation At this point via a pick off window approximately 4 of the laser beam is sent to a power meter so that laser power can be monitored After passing through another set of mirrors and an interlocked electronic safety shutter the beam passes into an enclosed beam steering periscope assembly which steers the aligned beam into the side car and then into the scan head As it enters the scan head the beam passes through an alignment iris and into the scan head a self contained unit not accessible to the user where it passes through one or more matched pairs of galvanometers optics and optical components suitable for rater scanning a microscope image when coupled to an appropriate objective lens The beam exits the objective The beam is then scanned across the sample as a means of fluorescent excitation Page 27 of 125 Ultima Manual ver 2 5 4 5 Ultima Scan Head Ultima Scan Head Light Path Top View DO eiis shu
73. eriments Hard Shutter Soft Shutter Figure 7 6 Shutter and imaging mode 7 2 4 3 Scan Mode Galvo or AOD This button allows the user to select between the standard galvo imaging mode and the optional AOD imaging mode 7 2 4 4 Label and Objective Lens Selection Label Select Current Settings m Figure 7 7 Label and objective lens selection 7 2 4 5 Label Selection Label selection allows the user to select previously defined and saved experimental protocols These protocols include saved settings for active channels PMT selection and DAQ gain laser power settings Setting up labels is covered in Section 7 2 11 7 2 4 6 Objective Lens Objective Lens allows the user to select a previously calibrated objective setting for an objective used on the system The procedure for calibrating objective lenses can be found in Section 7 3 2 1 Note Proper objection selection is important as it is used in determining pixel size Failure to select or use a properly calibrated objective will result in invalid measurements Page 42 of 125 Ultima Manual ver 2 5 7 2 5 Pan and Scan Rotation Tools 7 2 5 1 Pan Control and XY Stage Control Pan Control Control Sr Stage Control Current Locabion T a a aa Dr a Move to Previous 4 1600 b gt zee ele _FinePan Pari po 00 t E emu Sei Figure 7 8 Pan Control amp XY Stage Control Below the Pan Control banner are two sets o
74. es Place the red cursor in the image window at the location of the desired upper left hand corner gt Press the Mark First Corner button gt Next only move the vertical line of the red cursor to the location of the second corner this will guarantee that this line is oriented with the image x axis gt Press the Mark Second Corner button gt Lastly only move the horizontal line of the red cursor to the location of the third corner this will guarantee that this line is oriented with the image y axis gt Press the Mark Third Corner button Once the three corners of the grid have been defined the Show Map Boundary button becomes active If the operator presses this button a separate image window will appear with a box drawn over the section of the image showing the grid boundaries The color of the box is determined by the color specified in the Overlay Color control just below the Show Map Boundary button This allows the operator to visualize where exactly the grid will be created Figure 8 21 Show map boundary Page 105 of 125 Ultima Manual ver 2 5 Next specify the number of marked points to create in the x and y dimension of the grid These values are set using the X Dimension and Y Dimension controls Currently the number of marked points in the x dimension must be a multiple of 4 4 8 12 16 and the number of marked points in the y dimension must be
75. es By entering values for any three of these values the fourth value will be calculated automatically The Page 46 of 125 Ultima Manual ver 2 5 Define Calculate radio buttons indicate the three values that the operator must define as well as the one that is calculated by the system Senes Calculator Start Position urn 0 00 Define atop Position um Set Stop Limits 1 00 Define Step Size um DU A Define Goto Mid Pos Humber OF Slices ko e Calculate Adjust PHT Figure 7 14 Z series calculator To collect a Z series in which the system calculates the number of slices images to collect gt VV Vv VV V WV 7 2 8 3 Use the al and v arrows to move the z motor to the correct focal plane for the starting position of the Z series Press Set Start Limits Use the and v arrows to move the z motor to the desired focal plane for the ending position of the Z series Press Set Stop Limits Enter the desired Step Size if different from the distance displayed Click on the field type the new distance press lt Enter gt Confirm that the Save Path and Base Name are correct This defines where the images will be saved Press Start Z series Acquisition of the Z series can be monitored in the window below the Start Z series button Collecting a Z series over a large depth When collecting a Z series over a large depth it is sometimes desirable
76. evice Using the Select Device to Calibrate control select the desired pointing device gt Press the OK button to proceed to the next step Step 2 NeD Pinhole Option If the specified device in step 1 is Uncager then this step will be automatically skipped and the procedure will jump straight to step 3 If however the NeD Pinhole device was selected in step 1 then the operator must specify which NeD Pinhole location to perform the point calibration for The NeD Pinhole device consists of three locations that may be selected from Iris Pinhole and Confocal Pinhole The operator can now either press the OK button to proceed to the next step or press the Back button to return to the previous step Step 3 Calibration File Name In this step the operator must specify the directory and file name to use for saving the calibration information To select the directory and file name gt Click on the Browse button and then use the pop up dialog to select the directory and enter the file name to use After the Save button in the pop up dialog is pressed the selected directory path and file name will be shown to the left of the Browse button Page 91 of 125 Ultima Manual ver 2 5 The operator can now either press the OK button to proceed to the next step or press the Back button to return to the previous step Hints on Naming the Calibration File Since the calibr
77. ew to add regions for BOT data acquisition Using the mouse draw boxes in the regions of interest up to 32 Press Start to begin acquisition The graph in the Brightness Over Time window displays the average intensity over time of the outlined regions Hold CTRL and drag to Hold SHIFT and Left Click to zoom in _ SHIFT and Right Click to oom out Hold SHIFT and drag a box to z 4000 Image Window 1 Intensity i 1 i 10 Ue 14 22 Seconds Brightness Over Time Regions Ka lay Channel Pasition Width Height Area CH Wl Oo 5775 148 53 784 X 506 Y 40 0000 0000 0000 0000 0 0 um 2 vi Chi 358 106 114 187 21318 Blue 3 vi Chi 116 338 228 84 19152 Brown r Regions Graph Options Add New EUM Graph Width Seconds Frames Frames Seconds Frames Frames Remove Selected Move All 30 0 Seconds Clean Fixed Full Ful Save Path D Start with EE C Documents and Settings Prairie Input Trigger start Base Directory r Update Display Brightness OverTime 10052006 1644 3324 Save Graph Figure 7 20 Brightness Over Time windows Page 61 of 125 Ultima Manual ver 2 5 7 4 3 LS Line Scan Pressing the LS button located on the left hand side of an Image Window enables the line scan definition tool T 4 i G A h d 1 pith 9 D H P 4 d j 4 3 an J d d AP Ki d da a d s
78. f icons On the left is an indicator that shows the relative position and orientation of the scanned area A red and yellow box indicates the scanned area the yellow side is by definition the bottom side This box will change its size as a result of the Optical Zoom function described above Similar changes will be displayed when the image is panned or shifted laterally and when the scan is rotated The Coarse Medium Fine button controls the feel or resolution of the pan controls The image may be panned left right up or down To do so click on the arrow indicating the direction to pan the image Clicking the 0 in the center causes the pan to reset to the middle of the field of view 7 2 5 2 Scan Rotation scan rotation is controlled by means of the slider or values in degrees may be typed directly into the highlighted field Rotation is possible from 180 to 180 Figure 7 9 Scan Rotation Page 43 of 125 Ultima Manual ver 2 5 7 2 5 3 XY Stage Control The XY Stage Control allows the user to move the stage in the x y and z directions It also allows the user to recall saved locations The step size is shown in the center of the arrows and has a default of 10 um This can be adjusted in the XY Stage tab see Section 7 2 10 7 2 6 Laser PMT DAQ This tab located at the left center of the Main Control Window enables adjustments that control the laser power to reach the s
79. forms for the uncaging galvos the Pockels cell and the stimulus signals maximum number of stimulus channels is 5 used in experiments This board can generate a waveform at 1M samples sec if only one channel is enabled The maximum sampling rate drops as more channels are enabled DACSOUT and DAC OUT are set aside for driving the uncaging galvos the BNC2110 box has been modified for these two signals with the addition of voltage dividers that convert the 10V output range of these two signals to roughly 2 5V in order to increase the pointing resolution of the uncaging galvos in the region of use The Pockels cell signal currently DACGOUT although it can be changed is similarly passed through a voltage divider to achieve better resolution in the 0 1 4V range of use except in this case the voltage divider is contained in a separate box external to the BNC2110 and the divide ratio can be adjusted if necessary by turning the screw of a potentiometer ime FP PPP channels sees sample sec for NI 6052 Max output 500K 333K 250K 200K 167K 143K 125K samples sec for 6713 Table 1 Maximum NI 6052 and NI 6713 sample rates 4 2 3 3 NI 6110 6115 Board pixel data sampling methodology The NI 6110 6115 board generates the output waveforms for the imaging galvos and collects the PMT data used to generate images There are four 12 bit input channels on the 6110 6115 board Ultima systems that have the 6110 board
80. g the acquisition To avoid aliasing issues the Acquisition Rate should be at least twice the anticipated rate of signal change the operator wishes to detect Shutter Acquisition Delay Shutter Acquisition Delay indicates the amount of time to wait after opening the shutter before starting the acquisition This parameter could be of use when working with a slow mechanical shutter where the operator wants to guarantee that the shutter is fully opened before collecting the electrical data Output Rate Output Rate controls the step size of the output DAC signals during the acquisition Essentially by having a larger Output Rate value there is finer resolution in the voltage to time relationship on the output DAC signals Open Close Shutter Open Close Shutter specifies when the shutter should be opened and closed relative to the data acquisition There are three possible options A Open Before Starting Data Collection Close After Data Collection Complete Start Stimulus Control with Data Collection Page 79 of 125 Ultima Manual ver 2 5 Li B Open After Starting Data Collection Close After Open Shutter Time milliseconds Start Stimulus Control with Data Collection and C Open After Starting Data Collection Close After Open Shutter Time milliseconds Start Stimulus Control after Shutter is Opened In option A the shutter will be opened first then the acquisition and stimulus
81. hat is non linear x and y are only used when the imaging device has non linear movement Note The standard imaging galvanometers that are used exhibit linear motion in the region that Prairie uses them so typically all four of these fields would be set to O If non linear behavior is found in either the x and or y axes of the imaging galvanometers these fields could be used to provide for more accurate calibration of the pointing device Note If non linear behavior is found it would most likely be in the x axis the fast scan axis The control Percentage of Points to place in the non linear region is used to indicate what percentage of the total number of points in the x or y dimension to allocate to the non linear region For example if non linear movement is in the x axis and the operator had indicated that 20 points should be used in the Number of Points control for the x dimension and 40 is entered into the control Percentage of Points to place in the non linear region for the x dimension then of the 20 points 8 points 4096 of 20 will be in the non linear region on both the left and right side So of the 20 total points 16 points will be in the non linear region and 4 points will be in the linear center section This will result in a higher density calibration in the non linear region and should result in higher calibration accuracy of the pointing device in that region The control Percentage of outside edge
82. ibed below Because this window is an interface between the PrairieView and TriggerSync programs it is necessary to set the Mark Points parameters using a Mark Points Wizard which is accessed by clicking on the Configure button Grr Oooo G f Figure 7 30 Image Window and Mark Points window To mark a point Cllick on New Point Select the point of interest in the dialog box by clicking will highlight line Move point to desired location on image Click Configue to set Mark Points parameters Click Save Reference Image Click Acquire to mark points VVVVV WV Page 72 of 125 Ultima Manual ver 2 5 To mark a line of points gt Cllick on New Line gt Select the line of interest in the dialog box by clicking will highlight line Enter number of points in X Point Density field Move line to desired location on image Click Configue to set Mark Points parameters Click Save Reference Image Click Acquire to mark points VV VV WV Figure 7 31 Mark Points line To mark a grid of points gt Click on New Grid gt Select the grid of interest in the dialog box by clicking will highlight line Enter number of points in X and Y Point Density fields Move grid to desired location on image by dragging center point Resize and rotate grid by dragging corners Click Configue to set Mark Points parameters
83. iblitz shutter switches to their normal positions Restart system software Page 34 of 125 Ultima Manual ver 2 5 6 2 Spot Detector Alignment Important This is normally a one time adjustment The only time this will need to be performed is if either of the galvos is replaced a mirror in the scan head is moved or the beam combining dichroic in the scan head is replaced VVVVV VVVVVVVV V V VV WV Put reflective chrome slide on stage Start PrairieView Enable channel 1 in PrairieView Start TriggerSync Verify the communication link between PrairieView and TriggerSync A message will appear at the bottom of the PrairieView window stating that the link has been established Close uncaging shutter in the TriggerSync Mark Points window Image the slide with imaging beam Activate mark points screen Make sure image in mark points screen is updating Manually toggle shutter on uncaging beam to burn small spot on chrome slide Put red TriggerSync cursor over mark on chrome slide Do not use nudge button Turn off channel 1 and turn on channel 3 Close shutter on top of Ultima Open uncaging shutter in TriggerSync Activate focus Move uncaging spot to be coincident to red TriggerSync cursor Close uncaging shutter in TriggerSync Open shutter on top of Ultima Turn off channel 3 and turn on channel 1 Page 35 of 125 Ultima Manual ver 2 5 6 3 Calibrate Point Image Important Each objective lens will requir
84. id on one another for all of the DAC outputs that have been turned on The label on this composite graph includes the names of the analog outputs that are currently turned on In the top center of the dialog are two buttons Update and Accept Done As the operator makes changes to the various fields in this dialog the graphical displays will only be updated when the Update button is pressed After all desired changes have been made press the Accept Done button to close the dialog DAC Out Select To program an analog output first select the desired output using the DAC Out Select control The labels in this control correspond to the labels on the BNC connectors on the BNC 2110 box or BNC 2090 box if no BNC 2110 is present Output Signal After selecting the desired output channel use the Output oignal control to turn this analog output on off when performing an acquisition Protocol Protocol control allows the operator to select a pattern or Protocol for the selected analog output The operator may select from the following Protocols V Ramp Pulse Train G F C and Custom Waveform These protocols are described in more detail in Section 8 2 3 Graph Color This control allows the operator to specify the color to use for displaying the graph both in its own graph window and in the composite graph window in the lower right hand corner of the dialog Ext Sensitivity The operator may specify a separat
85. ifferences in data recorded on those input channels can be more easiy compared The next field down is labeled as Manual Sensitivity mV unit Just as the Ext sensitivity value for the output protocols was a scale factor for the output voltages the Manual Sensitivity value is a scale factor applied to the input voltages Also just as there is a separate Ext Sensitivity value for each output channel there is a separate Manual Sensitivity value for each of the input channels Mathematically the data is scaled using the following equation displayed value input value 1000 Manual Sensitivity For example if the input voltage is 0 5 volts and the Manual Sensitivity is set to 20 then value displayed on the graph will be 25 0 0 5 1000 20 The second from the bottom field is labeled Channel Names The string that is entered here will be displayed over the corresponding input button on the main TriggerSync window For example the third input ACH2 might be connected to a patch amplifier so the operator might enter a label such as amp in the Channel Names field for the third input The bottom most field is labeled Channel Units The string that is entered here will be displayed to the right of the color box for the corresponding input button on the main 7riggerSync window For example the third input ACH2 might be Page 88 of 125 Ultima Manual ver 2 5 connected to a patch amplifier so the
86. igure 7 7 Figure 7 8 Figure 7 9 Figure 7 10 Figure 7 11 Figure 7 12 Figure 7 13 Figure 7 14 Figure 7 15 Figure 7 16 Figure 7 17 Figure 7 18 Figure 7 19 Figure 7 20 Figure 7 21 Figure 7 22 Figure 7 23 Figure 7 24 Figure 7 25 Figure 7 26 Figure 7 27 Figure 7 28 Figure 7 29 Figure 7 30 Figure 7 31 Figure 7 32 Ultima Manual ver 2 5 List of Figures Ultima and Ultima IV in vivo system without light boxes 15 Ultima System with light box and beam cover 15 Ulttrga electronies e 16 Dual Preamplifier amp HV control unt 17 NI BNC 2090 boxes A top amp B bottom 21 Close up of NI BNC 2090 boxes 22 INT BNCOC Z MIU 23 Ultima Scan Head light path 28 Ultima scan head dichroic ocatons 29 Emission Light Path for dual channel detectors 30 Emission Light Path for quad channel detectors 30 Prairie View Main Window ccccccceccseeeeecceeceeeceecaeetaueeeeeseeeaeeeaes 37 Prairie View Main Gcreen enne nnne nnn 38 Scan TESOIUTON Be Le EEN 39 ZOOM ANG GWEll TIME de Le EE 40 SCADA OO EG 41 Shutter and imaging mode srncie a e 42 Label and objective lens selection eesssssessse 42 Pan Control amp XY Stage Control 43 Scan ROON E 43 Laser PMT DAQ CONTOS EE 44 2P ASE CONTO eenegen EENEG 45 Z Seres e E 46 e
87. illuminator of the microscope Optional sub stage detectors may also be present located to the left of the microscope base below the stage CAUTION PMTs are extremely sensitive to light Care should be taken to eliminate all sources of stray light from reaching the PMTs as this will dramatically reduce the sensitivity of the system Prairie Technologies provides a light tight and laser safe enclosure around the instrument to ensure optimal performance as well as to provide a safe working environment for the users of this instrument Figure 3 2 Ultima System with light box and beam cover Page 15 of 125 Ultima Manual ver 2 5 4 Hardware and Electronics 4 1 Overview The Ultima Multiphoton Microscopy system contains many electronic and optical components In this section pertinent information regarding the electronics rack shown in Figure 4 1 and key stage and imaging electronics will be described Pockels Cell Controller Device Controller E Galvanometer Flat Panel Displays Controller Keyboard amp Mouse Tray T BNC Breakout Boxes E PMT High Voltage amp a Preamp Power RB Supplies PC Computer ve I Pockels Cell Controller ee Main Rack Power Switch Figure 4 1 Ultima electronics rack Page 16 of 125 Ultima Manual ver 2 5 4 2 Rack and Other Electronic Components The rack contains the majority of electronics necessary to operate the Ultima system 4 2 1 PC
88. in Linescan riggerSync will perform an experiment as you have defined it while line scan data is collected Page 119 of 125 VV WV wv Ultima Manual ver 2 5 9 5 Non Line Scan Images Electrical Recording amp Uncaging Start PrairieView Start TriggerSync In PrairieView use Live Scan or Single Scan to locate the area of interest Use pan rotate and zoom to orient the target region Load the correct point calibration file for your scan parameters o Open the Mark Points dialog in TriggerSync o Load the appropriate calibration file for the Uncager Calibration File field Mark Points to define locations to be uncaged o Open the Mark Points dialog in TriggerSync o Press Snap in TriggerSync to collect a reference image on which to mark your points o Use any combination of discrete points lines of points or rectangular arrays of points Click on Acquisition and DAC Output Setup in 7riggerSync o Confirm or define stimulus control and acquisition parameters and close the dialog Enable the desired ADC input channels on main TriggerSync window Move to Different Points During a Single Acquisition NOTE The Point Number in the upper left corner of the main TriggerSync window does not update when collecting data in this mode Select Use Marked Points During Acquisition at the bottom of the Mark Points dialog window in TriggerSync o Move time is on the order of 0 10 0 20 ms o Setfi
89. ion just selected Also the values for the absolute position will be updated and the values for the relative position will be zeroed When the end of the marked point list is reached it will automatically wrap around and start over with Point O To the right of the controls to mark points is the Erase All Points button When this button is pressed all previously marked points are deleted Other actions besides pressing the Erase All Points button will also result in all marked points being deleted If the NeD Calibration File or the Uncager Calibration File is changed any previously marked points will be deleted In addition within the Mapping Setup section of the Mark Points dialog when the Generate Map Points button is pressed any previous marked points will be deleted this will be discussed in more detail in that section Also within the Line Definition section of the Mark Points dialog when the Replace List button is pressed any previous marked points will be deleted this will be discussed in more detail in that section Below the Erase All Points button is the Erase Point F10 button This control is used to delete a single marked point When this button is pressed a dialog will appear asking the operator to select the marked point to delete The operator merely selects the desired point and presses the OK button If the operator does not wish to delete a point he she m
90. is mode the red cursor within the image window is used with the various marked points generation tools When the Blue cursor option is selected the blue cursor within the image window is used with the various marked points generation tools When the Blue cursor option is selected the selected pointing device must have its physical position changed for example using the four blue buttons next to the Pinhole Motion control to indicate the position to use in conjunction with the various marked points generation tools The NeD Calibration File field is used to indicate the calibration file to use for computing the marked point positions when the NeD Pinhole device is being used with a confocal image When the NeD Pinhole is being used with a digital camera image the necessary calibration numbers are stored in a configuration file ControlNeD ini To select a calibration file click on the Browse button next to the NeD Calibration File field and select the desired file Page 99 of 125 Ultima Manual ver 2 5 The Uncager Calibration File field is used to indicate the calibration file to use for computing the marked point positions when the Uncager device is being used with a confocal image or with a digital camera image This calibration file is one of the files generated in the Point Calibration dialog To select a calibration file click on the Browse button next to the NeD Calibration File field and select the
91. lows user to select position of scale bar in Image Window as well as units of scale bar e Overlay Color Font Allows user to select overlay color font in Image Window 7 3 5 Help Under the Help option the user will find supporting material regarding the Ultima including a copy of the user manual and software revision notes Page 5 of 125 Ultima Manual ver 2 5 7 4 Image Window Images acquired on the Ultima are displayed in PrairieView Image Windows It is possible to have multiple windows open at once Each Image Window is identified by a label in the banner section with a number e g Image Window 1 All open windows will be updated when the Live Image or Single Scan buttons are pressed Paes H OGOD DOO DOOR 00 urs Figure 7 19 The Image Window Note Some consideration should be paid to the number and size of active Image Windows that are opened as the system may become sluggish when attempting to update too many windows simultaneously Located on the left side of the Image Window are buttons for the following controls with more detailed descriptions following e Ch1 Ch2 Ch3 Ch4 The channel buttons indicate which input channel is being used to collect data for that image window BOT Brightness Over Time Used for measurement in average pixel intensity of user defined regions to be monitored over time Dm a CA e BOT e LS Line Scan Used for definition of line scan and
92. ly below the pointing device location information are the basic controls for marking points moving between marked points deleting marked points displaying marked points and saving marked points information The button labeled Mark Point F9 is the basic control used for marking a location on the image for further study using the selected pointing device The F9 in parenthesis indicates that there is a hotkey on the keyboard linked to this control So the operator may press the F9 key and it will result in the same action as if the Mark Point F9 button itself had been pressed The actual location that will be marked when the Mark Point button is pressed is dependent upon the setting of the Mark Point Using Red Blue Cursor control previously described If this control is set to the Red cursor option then the operator merely places the red cursor in the image window at the desired location to mark and presses the Mark Point button If this control is set to the Blue cursor option then the operator must physically move the location of the pointing device using the blue buttons or the Nudge F7 control discussed later to the desired location and then press the Mark Point button As you can see using the Blue cursor option is much slower which is why it is recommended to use the Red cursor option The list of marked points is maintained even after the TriggerSync program has quit running The next
93. n a color box next to one of the input buttons a color selection dialog will appear that will allow you to select the color associated with that input signal The selected color will be the color of the button when it is depressed selected as well as the color of the graph data for that input 8 3 2 ADC Configuration The ADC Configuration dialog contains a variety of parameters that improve the TriggerSync dialog readability and control the scaling of the acquired data To open the ADC Configuration dialog click on the menu option Configuration and then click on the option ADC Configuration This dialog allows the operator to control several parameters for each of the inputs There are eight columns of parameters one column for each of the eight inputs that are available Ix ADC Configuration 1 1 vi File Edit Operate Project Windows Help gt ADC Configuration Automatic Gain Sensing Q Q C GC CH C F F F E F F F F vemax gn apmo Spo pz Tab apmo ao om ymin ss n apo H658 H948 o pod OC henaa cenetista Apap Sepp rl Spyro ppc SERRE e e Channel Hames am o an o pen am o an ACHE o ACHE o pm Channel Units mo mw au mo m bw mv pv E Figure 8 18 ADC Configuration The first three parameters Automatic Gain Sensing Y max and Y min are only used after data has been collected an acquisition has been performed within Trigge
94. n adequate shutter as it leaks light from the lamp house that will flood the PMTs Turn Epi dichroic wheel to Position 1 This automatically turns off the Olympus transmitted light source and directs signal to the upper PMTs Pull Olympus trinoc plunger out the LSM position To use sub stage detectors if present o Turn on sub stage High Voltage Power Supply o Plug appropriate sub stage detector PMT cable into PMT junction box o Install the same filters and dichroics into sub stage detectors as are in the upper detectors o Slide sub stage dichroic in position o Open sub stage condenser iris o Position sub stage condenser o Open sub stage shutter Turn off the room lights Note the laser power reading on the power meter adjust if necessary using the Pockels cell control in PrairieView Use Prairie View to open one or more imaging windows Press Live Scan button This starts the scanning and opens the laser shutter Turn up the PMT high voltage until you see your image Page 32 of 125 Ultima Manual ver 2 5 6 Alignment and Calibration Procedures 6 1 Beam Alignment Important The optical path for the laser beam has been carefully and precisely aligned Re aligning the laser beam may be necessary only under the following circumstances and should be performed only by a factory trained and authorized technician 1 The direction of the laser beam out of the laser changes 2 A mirror on the tabletop is adju
95. nt 8 E 1 638 2457 3276 4095 Intensity 1 Low Cutoff High Cutoff 4094 Channel 3 Reset Display 813 1538 2457 3275 4095 Intensity 1 Low Cutoff High Cutoff CH Channel 4 LU Cursors Te E D Heset Reset Display es D 8 J 638 2457 3276 4095 Intensity 1 Low Cutoff High Cutoff 4094 Figure 7 25 Lookup Table Page 65 of 125 Ultima Manual ver 2 5 Since fluorescent images very often contain a large number of dark pixels it is sometimes desirable to adjust the range of the LUT to exclude some of the dark pixels thereby allowing the LUT to display smaller changes in the data intensity scale There are three options for looking at intensity data in the LUT Drag Cursor allows the mouse to be used to adjust the high and low ranges of the LUT used for each channel by dragging the yellow lines The Low and High Cutoffs give the range defined by the yellow lines Pan X and Zoom X let the user moving the graph to more carefully ascertain the pixel intensity characteristics of the input channel data Log Scale allows meaningful information about the intensity scale to be viewed for images with a large dynamic range Page 66 of 125 Ultima Manual ver 2 5 7 4 5 ROI Region of Interest It is often useful to scan only a selected region of the field of view to optimize image speed Using the Region of Interest ROI tool it is possible to define a small portion of the sample t
96. nts dialog by the Marked Point Hold Time ms parameter This process continues for all of the marked points This graphical display allows the operator to configure the other output channels so that the desired event s will occur relative to the pointing device movement For example the operator has 1 Created a list of five marked points in the Mark Points dialog 2 In the Mark Points dialog the operator has set Use Mark Points During Acquisition to On 3 Marked Point Hold Time to 100ms 4 Marked Point Move Time to 0 1ms 5 Marked Point First Point Delay to 0 If in the Input and DAC Out Setup dialog the operator wishes to expose the sample for 25ms for example by changing the Pockels output voltage from 0 to 5 volts after moving to a new location and the data collection at that point should continue for another 75ms before moving to the next point and repeating the process To set up the proper pulse train on the Pockels output line The Pulse Potential would be set to the appropriate value based upon the Ext Sensitivity setting to get a 5 volt output Setthe Duration to 25 ms gt Set the Inter Pulse Delay to 75 1ms 75ms of additional data acquisition time before moving to the next location plus 0 1 ms for the time to move the pointing device Page 85 of 125 Ultima Manual ver 2 5 gt Set the Number of Pulses to 5 one pulse at the start of each new marked
97. o Press Snap in TriggerSync to collect a reference image on which to mark your points o Use any combination of discrete points lines of points or rectangular arrays of points Click on Acquisition and DAC Output Setup in 7riggerSync o Confirm or define stimulus control and acquisition parameters and close the dialog Enable the desired ADC input channels on main TriggerSync window Move to Different Points During a Single Acquisition NOTE The Point Number in the upper left corner of the main TriggerSync window does not update when collecting data in this mode Select Use Marked Points During Acquisition at the bottom left hand corner of the Mark Points dialog window in riggerSync o Move time is on the order of 0 10 0 20 ms o Setfirst point delay time o Verify the Hold and Move times Press to open the Line Scan dialog While the Linescan dialog is being started the operator will be prompted as to whether the Linescan acquisition should be synchronized with TriggerSync f the operator selects No then standard linescan acquisitions will be performed If the operator selects yes then a TriggerSync acquisition will be automatically performed when the linescan acquisition is started The riggerSync data will be automatically saved in the same directory using the same base name as the linescan data Position the line where desired Enter the number of lines you want to collect Press Beg
98. o be imaged which increases the scan rate up to hundreds of frames per second When this button is pressed the cursor is used to define a rectangular area in which to limit scanning To define ROIs after clicking the ROU button place the cursor at one corner of the area of interest Click and drag the mouse creating a rectangular ROI Click again to release S Ls gt ile bail 5 e e Be E 7 347 7 295 0000 2100 0000 0000 Figure 7 26 Define an ROI and ROI image X 347 7 295 0000 2100 0000 0000 The Image Window will automatically resize itself Press either Live Scan or Single Scan to refresh the display to reflect the ROI The laser beam is ONLY scanned over this portion of the specimen When LiveScan is stopped it is possible to right click on the ROP button to open a Save window to name and save ROls Page 67 of 125 Ultima Manual ver 2 5 7 4 6 Snap Tool The Snap button is used to save the current image displayed in an Image Window The image will be saved to the filename and path specified in the PrairieView Main Window lf any overlays are present on the image the exact displayed view including display zoom and overlays will be saved as an 8 bit TIFF file If there are no overlays present the displayed image will be saved as if it were a single image acquisition a 16 bit metadata file Page 68 of 125 Ultima Manual ver 2 5 7 4 7 PA
99. ol WARNING e may lead to serious bodily injury or death Neo Disregarding the instructions accompanying this warning symbol may lead to serious bodily injury or instrument damage Page 9 of 125 Ultima Manual ver 2 5 WARNING Use of this systems components or performance of procedures other than that specified in this manual may result in hazardous radiation exposure Intended use This system is designed as a Class 1 laser product intended for use in laser based imaging microscopy It is not intended for any other purpose WARNING VISIBLE and or INVISIBLE LASER RADIATION WHEN INTERLOCK DEFEATED AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION MAX AVERAGE OUTPUT POWER 5W EMITTED WAVELENGTHS 650 1100 nm PULSE DURATION 10 300fs CLASS 4 LASER PRODUCT Interlocked safety covers This system is enclosed within a light tight and laser safe covers These covers are designed to protect the user from exposure to Class IV laser radiation Therefore at no time should these covers be removed or modified The interlocks on these covers are designed to be defeated by authorized factory trained personnel during specific maintenance and service procedures During these procedures appropriate laser safe eyewear is required Page 10 of 125 Ultima Manual ver 2 5 WARNING VISIBLE and or INVISIBLE LASER DEFEATED AVOID EXPOSURE IR SCATTERED RADIATION MAX AVERAGE OUTPUT POWER 5W EMIT
100. on the red cursor in the image window to indicate the location of what would be the upper right hand corner of the grid Press the Mark Second Corner button If this second corner location is not to the right of the first corner then a message explaining this problem will be displayed and the operator must start the grid definition process over Position the red cursor in the image window to indicate the location of what would be the lower right hand corner of the grid gt Press the Mark Third Corner button VV Y Since the grid must be rectangular right angles at the corners the actual grid coordinates are determined using the following logic The defined locations of the first and second corner define one side of the grid The second side is defined by taking the distance between the second and third corners this defines the length of the second side and determining the new location of the third corner that will create a right angle between the first side and the second side The remaining sides are easily calculated now Page 104 of 125 Ultima Manual ver 2 5 At any time the operator may press the Clear Corners button to delete the previously defined corner locations and allow the operator to redefine the grid limits The defined grid of marked points can be either oriented along the image axes or at a random angle relative to the image axes To define a grid that is oriented along the image ax
101. one corner location of the ROI Hyperlinks are present throughout the manual Each entry in the Table of Contents is a link Internal references to other sections are also links and are indicated with blue text 3 3 Ultima Overview The Ultima is a unique laser scanning microscopy instrument that is capable of using one or two laser beams for multiphoton imaging and or uncaging experiments on tissue slices or very small animals With only one laser it is possible to perform traditional raster scanned laser imaging and optionally perform sequential imaging and uncaging experiments When two lasers are used it is possible to image and uncage simultaneously In either scenario the operator can choose to deliver electrical stimuli to the specimen as well as recording electrophysiological signals from the specimen through the use of trigger syncing software The Ultima IV is a version of the instrument designed specifically for in vivo studies of small to medium sized animals It is available in two versions one in Page 14 of 125 Ultima Manual ver 2 5 which the optics are fixed and the other which allows the optics to translate in the X amp Y directions Figure 3 1 Ultima left and Ultima IV in vivo system right without light boxes The Ultima uses externally mounted photomultiplier tubes PMTs to collect the fluorescence emitted from the specimen These detectors are located adjacent to the left of the epi fluorescence
102. one that has just been set up e Preferences Allows user to select options for post acquisition settings and t and Z series setting e Exit Stops PrairieView and closes the program Page 55 of 125 Ultima Manual ver 2 5 7 3 2 Tools Tools Window Display Hi e Calibrate Objective Lens See below Calibrate Objective Lens E E Er e Center Galvos Resets galvos to center Center Galvos position Manual L SE e Manual Lasers A special function which Notes F8 may or may not apply to your system Bran settings e Notes Open window for user notes Will Preamp Control be saved with all subsequent images e Scan Settings If this option is active on your system DO NOT change any settings except under the direction of Prairie personnel These settings are not intended for operator use Preamp Control Function not available at this time 7 3 2 1 Calibrate Objective Lens Each objective lens used on the Ultima must be calibrated The image size must be set to 512 x 512 and 1x zoom A calibration slide is needed for this process The calibration steps are as follows VN VY VV V Move the objective lens to be calibrated into position on the microscope Focus on a slide containing an object of known width a calibration slide is recommended for this purpose Under the tools menu select Calibrate Objective Lens The following window will appear i Objective Calibration Ei Calibration mustb
103. operator might enter a label such as mV in the Channel Units field for the third input 8 4 Point Calibration 8 4 1 Overview In system configurations where the image scanning galvanometers are not used as the pointing device for uncaging and so forth then it is necessary to calibrate the pointing device to the image The result of the point calibration process is a map that relates voltage values for galvanometer based pointing devices or motor positions for motor driven pointing devices to pixel locations on the image This map is then used within the Mark Points dialog for a fast method by which the operator can specify a location on the image that he she wishes the pointing device to be located It is important to note that since the calibration is dependent upon the pixel locations in the image any changes to the image generation parameters pan zoom rotation image resolution changing objective lenses and etc will necessitate a separate calibration At any time during the setup of the calibration procedure the operator may press the Done button at the bottom of the dialog to exit this dialog 8 4 2 Calibration Process To begin the point calibration process click on the Points menu option in the TriggerSync main window and then click on the Calibrate Points option If the Mark Points dialog is open when the Point Calibration dialog is opened the Mark Points dialog will automatically be cl
104. or the selected input channel Figure 8 4 View Channel Sensitivity 8 1 2 2 Change Scale and Auto Scale Along the left hand side of the graph area will also appear two button controls Change Scale and Auto Scale Change Auto Figure 8 5 Change Scale amp Auto Scale When the Change Scale button is pressed it will open the ADC Configuration dialog This dialog will allow the operator to specify whether each acquired data channel should be automatically scaled or whether the operator wishes to fix the minimum and maximum values that the data should be scaled against For a complete description see the ADC Configuration Section 8 3 2 later in this document Page 76 of 125 Ultima Manual ver 2 5 When the Auto Scale button is pressed each of the acquired data channels will be automatically scaled When automatically scaled the data in each channel is analyzed to determine the minimum and maximum values and these will be the values that the data in each channel will be scaled against 8 1 2 3 Sequential File Below the graph window and to the right of the Acquire F5 button is a box labeled Sequential When this box is checked a file selection dialog will be presented to the operator allowing him her to specify the directory and file name to be used for saving the current cycle data in a format readable by a spreadsheet program such as Microsoft Excel Sel
105. osed The Point Calibration dialog is used in the calibration of various pointing devices to various imaging devices For example the operator could calibrate a galvanometer driven pointing device to a galvanometer driven imaging device or operator could calibrate a galvanometer driven pointing device to a digital camera and so on This discussion will center on the calibration of a galvanometer driven pointing device to a galvanometer driven imaging device Page 89 of 125 Ultima Manual ver 2 5 e Calibration yvi a tf F L X l WT ne a d en tolysi Cer sten Finished Fd Figure 8 19 Point Calibration dialog Page 90 of 125 Ultima Manual ver 2 5 The calibration process is set out in a number of steps that the operator must proceed through in a specific order Due to various optical considerations it may not be possible to see the spot at the extremes of the image field left right top or bottom Because of this the area that will be calibrated is often not the entire visible image area Step 1 Device Selection The first step in the calibration procedure is to select the device for which the calibration will be performed For most systems the only available and hence default option will be Uncager This is the generic name given to any pointing device regardless if it is galvanometer driven or motor driven that is not the NeD Pinhole d
106. pecimen and the gain setting of the DAQ data acquisition board in the computer Laser PMT DA Z Series T Series 2P PC Oz AY Stage Misc 1 P Laser DAL gt CS Ca Mb 220 GER Hr Gi mmi crum xij Dr Ge Es m RF aj 2 x Cem Ak ze d Mp Lesser UI RH lo Z xj ge s o m metres D I Ph ze 20 Ak Ze e L Bib Gell Sit DI Ell en ERR 3p 3 Zee Vo DI Bib ze si SE De Figure 7 10 Laser PMT DAQ Controls 7 2 6 1 Laser The Laser slider is used to adjust the laser intensity that reaches the specimen In most Utima systems the imaging beam intensity is controlled via a Pockels cell that is tied to this control If your system has two lasers then a second slider will be activated to control a second Pockels cell for the uncaging laser intensity 7 2 6 2 PMT Gain voltage The sliders are used to set the PMT high voltage for each detector in the system The text field to the right displays the voltage that corresponds to the red LED displays on the Dual Preamplifier amp HV Control Unit Section 4 1 in the instrument rack The Zero button sets the voltage to 0 V Page 44 of 125 Ultima Manual ver 2 5 7 2 6 3 DAQ Gain The NI DAQ 6110 board in the computer has a gain setting that is applied to the PMT pre amp output voltage which is the signal from your detectors The drop down arrow is used to select the
107. point gt Finally the First Pulse Delay should be set to 0 When the Update button is pressed the composite graph should show the spikes that indicate when the system is moving to the next marked point and immediately after each spike there should be a pulse of 25ms duration indicating that the Pockels output is being executed 100 150 200 ZOU 300 EU 400 450 S00 350 Figure 8 16 DAC Out Composite graph 8 3 ADC Input Controls oection 8 2 outlines the process of setting up the various analog output signals and some of the parameters that affect the collection or acquisition of analog signals This section will cover the remaining controls and dialogs that are used for configuration of the analog inputs 8 3 1 Selecting Input Channels Figure 8 17 ADC inputs Across the top center of the main window of TriggerSync are a series of eight buttons with small color boxes just to the right and below these buttons These buttons are used for specifying which input electrical signals to record with Page 86 of 125 Ultima Manual ver 2 5 TriggerSync To perform a TriggerSync acquisition at least one of these buttons must be pressed When none of the buttons are pressed selected the Acquire button will be desensitized made inactive The eight buttons correspond in order from left to right to the first eight inputs on the BNC 2090 box labeled ACHO ACHT etc from left to right By left clicking the mouse o
108. r ODUONS estt ttr auia rae Vat d t rn c d n eren 55 T2321 mE NU TUTUP 55 9 2 MOONS m m 56 7 3 2 1 Calibrate Objective Lens 56 L SG SE A e Lee TEE of Page 4 of 125 Ultima Manual ver 2 5 1 3 4 DIS ec of 3 9 ACID EE 57 f image lee EE 58 1 4 1 Selecting an Input Channel Ch1 Ch2 Ch3 ChA 60 1 4 2 BOT Brightness Over Tume 61 7 4 3 LS SEINE SCAM NEE 62 TAA LUT LEo0k UD Ee EE 65 1 4 5 ROF Region of Interest E 67 7 4 6 SEET EE 68 1 4 1 PA NOLO ACU ALON airen a aiunt e a heat pato tih eran 69 1 4 8 um Line Profile Meaeurement 70 1 4 9 MIP Maximum Intensity Droiechon 71 7 4 10 MP Marked Homts 72 o JIOggeroypo SOMW EE 74 8 1 Man Operational Modes issoro aE 14 8 1 1 Analyze Acquired Data Off eenei 15 8 1 2 Analyze Acquired Data On 76 8 1 2 1 View Channel Sensitivity cccccccccecceeceeeeeeseeeseeseeeseeees 76 8 1 2 2 Change Scale and Auto Scale eeeseeeeeseese 16 8 1 2 3 EIERE ee T 8 1 2 4 Save Experiment cccceccsecceeceeeeeeceeeeseceeeeeeeseceeeeeeeeeeeeeeaes T 8 2 Acquisition and DAC Output Getup 78 8 2 1 ACOUISINON Se UDr a E Der E Vd dE Vi E Pis 19 8 2 2 SUUS ENEE 80 8 2 3 FrOlOCO M T Hx x 82 8 2 3 1 Idi gue VONAGE EE 82 8 2 3 2 FREIND EE 82 8 2 3 3 maii PL 83 8 2 3 4 G F C General Fluorescence Control
109. rSync and the program is in the Analyze Acquired Data mode The first parameter is labeled Automatic Gain Sensing By pressing this button and exiting the dialog the minimum and maximum data values for the Page 87 of 125 Ultima Manual ver 2 5 selected input will be determined from the data for that channel and that minimum and maximum value will be used as the minimum and maximum values for the vertical y axis of the graph In essence the Automatic Gain Sensing button means to automatically scale the graph to the data As data is acquired the graph is automatically scaled to the data which is why this button is only relevant after the data has been acquired and the program is in the Analyze Acquired Data mode To set the vertical y axis for a given input channel enter the desired values into the Y max and Y min fields for the selected channels After exiting the dialog the data will be redrawn on the graph with the data for the selected input channel scaled based upon the specified Y max and Y min values Newly acquired data is automatically scaled by default so that the operator will always be able to see the data as it is being acquired since it won t be impacted by the Y max or Y min values After the data is acquired however and TriggerSync is in the Analyze Acquired Data mode the Y max and Y min values can then be set for the desired input channels to the same values and d
110. reamplifier cccccccseccseceecsseceeeeeeceeeeeeeeeseeeeeeeeeeteeeaeess 25 4 2 Galvos amp Acousto Optic Deflector AOD esses 25 43 OPCS METTRE TUTTI TTE 2f DD MN Nep cm m 2f Z5 Jaca Beads uiti Medeae teilt e S nta un tat fin ura un RN 28 4 5 1 DICIWOICS Hm ME TR TT T T TT 20 4 5 2 MICTOSCOD G tdisudtesdessdnosdenidnsdidqu incus dieqa noc d stud mos DR n CADO DIU EDS IRE 29 4 5 3 Bc ee 29 4 5 3 1 Top Mounted detectors cece ccc cecsecceeeeeseeeeeseeeeeeeeeeeeeeees 30 4 5 3 2 oub 5tage GElECIOMIS EE 30 4 5 3 3 Dodt Gradient Contrast detechor 31 9 Uitinia startup ProCeGure EE 32 6 Alignment and Calibration Procedures cccccecccecceecceeeceeeseeeeeeeaeeeeeeeeeees 33 6 1 SE Well E 33 6 1 1 Imaging Beam Alignment in Scan Head 33 6 1 2 Uncaging Beam Alignment in Scan Head 34 6 2 Spot Detector Alnment 35 6 3 Calibrate e rn ll Le 36 6 4 Resetting the PMT High Voltage 00nanaanannanonnannnnanonnnnnannnnnnnnnnnnnnnnne 36 T FAITE VINW S ONW EE 37 7 1 Prairie view a ONE UN EA nie e UE 37 f 2 Mali VVindow GoRITOISescie er EERERENSEE DRIN E QU IAE ETC ITO 38 Page 3 of 125 Ultima Manual ver 2 5 7 2 1 Scan RESONON Ri ee UE 39 7 2 1 1 WMA QC SIZ CO NR UU 39 fld Pixels Per Line amp Lines Per krame 39 7 2 2 Zoom and Dwell time Tools eeeeeeeeeeee eee 40 7 2 2 1 Dwell Time Per Pixel
111. rner of the area to calibrate Manually move the spot location using the four blue buttons located to the left of the Acquire button The process is to move the spot left or right using the Blue buttons and then to use the Acquire button to capture a new spot image You will notice that during this step the Blue buttons that control movement of the spot left and right have been desensitized This forces the operator to only make up or down movement corrections when defining the lower right hand corner location relative to the upper right hand corner location Once the spot is in the desired location press the OK button to proceed to the next step or press the Back button to return to the previous step Step 8 Perform the Calibration Before performing the actual calibration the operator must specify a few more control values Page 95 of 125 Ultima Manual ver 2 5 gt The operator must specify the Number of Points in the x and y dimension to calibrate Depending upon the desired accuracy of the pointing device calibration typically a value of 2 in each of these fields will yield acceptable results At a value of 2 points in x and y the calibration is essentially being performed at the four corners that define the calibration area The four remaining controls Percentage of Points to place in the non linear region x and y and Percentage of outside edge of scan area t
112. rst point delay time o Verify the Hold and Move times In the TriggerSync main window select the menu option Acquisition then select the Start Experiment option and then select the TTL Trigger Start option this action will place a checkmark in front of this menu option indicating that it has been turned on Press the Acquire button in TriggerSync At this time the TriggerSync acquisition will begin but it will only proceed to the point of waiting for the trigger signal to arrive when the start of an image collection from PrairieView Press Single Scan in PrairieView When the image scan begins a trigger signal is sent to the 7riggerSync acquisition hardware causing Page 120 of 125 Ultima Manual ver 2 5 TriggerSync to collect the electrical data in synchronization with the image data gt The previous two steps must be repeated for each combined image electrical acquisition gt Inthe TriggerSync main window select the menu option Acquisition then select the Start Experiment option and then select the TTL Trigger Start option this action will remove the checkmark in front of this menu option indicating that it has been turned off Page 121 of 125 VV VVWV gt gt Ultima Manual ver 2 5 9 6 Functional Map Acquisition start PrairieView Start TriggerSync In PrairieView use Live Scan or Single Scan to locate the area of interest Use pan rotate and zoom to ori
113. s allows for the opportunity to manually indicate where the spot is located After placing the red cursor over the spot location if the operator presses the Accept button the dialog will disappear and the software will continue attempting to automatically perform the calibration At any location where the Page 97 of 125 Ultima Manual ver 2 5 software runs into a problem in finding the spot this manual override dialog will be displayed allowing for a means of continuing with the calibration If the Abort Calibration button is pressed then this dialog will be dismissed and the calibration procedure will terminate After the calibration has completed press the Done button to exit this dialog 8 5 Marking Points In the main window of TriggerSync select the Points menu option and then select the Mark Points option this will bring up the Mark Points dialog The Mark Points dialog is used to create a list of locations for performing uncaging experiments Figure 8 20 Mark Points window The image window occupies the largest part of this dialog There are two cursors displayed in the image window one red and one blue When two pointing devices are present on a system a third yellow cursor may also be present dependent upon the option selected in the Marked Points Device s control Since the blue cursor is used to indicate the current position of the selected pointing device i
114. s more than one cycle If the increment is any value other than 0 then after the first cycle the voltage for the second cycle for this epoch will be incremented or decremented if a negative value is specified by this amount If a third cycle is to be acquired then the voltage for this epoch will be incremented decremented again and so on 8 2 3 2 Ramp When the Ramp protocol is selected the controls for the other protocols will be desensitized The Ramp protocol allows the operator to program a voltage ramp for output Epoch A is used to specify a voltage and the time leading up to the Page 82 of 125 Ultima Manual ver 2 5 start of the ramp Epoch B is used to define the actual ramp voltages starting and ending as well as for the length of time to spread the ramp over Epoch C is used to specify a voltage and the time from the end of the ramp to the end of the acquisition 8 2 3 3 Pulse Train Figure 8 13 Pulse Train When the Pulse Train protocol is selected the control for the other protocols will be desensitized The Pulse Train protocol allows the operator to define a single square voltage pulse or a train sequence of square voltage pulses or a sequence of multiple independently defined pulses or pulse trains Number of Pulse Trains defines the number of independently defined pulse trains to generate Number of Times to Repeat Pulse Train s defines the number of times that the defined pulse train s shoul
115. s the modulus function This function returns the remainder of the division of the first argument by the second argument The function int returns the integer value of its argument For example int 1 1 int 1 3 1 int 1 5 1 and int 1 99 1 For this algorithm to work the number of uncaging positions in the x dimension must be evenly divisible by 4 and the number of uncaging positions in the y dimension must be evenly divisible by 2 Page 106 of 125 Ultima Manual ver 2 5 The following table shows an example of the order of uncaging positions where Nx 8 Ny 4 and Px 0 4 2 6 1 5 3 7 X Pixel 15 16 1 2 3 4 5 6 T 8 9 10 11 12 13 14 Ce perpe es rs 2 2 9 00 2 22 s rea ao a1 sr ar ss ss ev 20 so uz so se oe 45 s EISE SES SSES EHS ESGSESESEE SSES SES ES ES ESGSES SES ra elle 7 za fos ar a 26 12 28 amp 26 vs s Fas sr as so 2o ss 47 69 35 52 24 oo 4o ss s e Ea OO EE e L Table 3 Non Neighbor order of uncaging example Y Pixel COIN OO P ow N Option C Random The order that the marked points will be generated for the grid will be random After selecting the Map Method the operator must press the Generate Map Points button It is only when this button is pressed that the actual marked points for the defined grid will be generated Once the Generate Map Points button has been pressed the Show Map Points b
116. sample at 2 5M samples sec while those with a 6115 board sample at 10M samples sec The pixel dwell time is a multiple of O 4us 6110 or 0 1us 6115 4 2 3 4 NI BNC 2090 amp BNC 2110 boxes There are two BNC 2090 boxes and one BNC 2110 box The 2090 boxes are referred to as 2090 A and 2090 B for the upper and lower rack mounted boxes respectively 2090 A is connected to the PCI 6052 card which is the acquisition card used by TriggerSync 2090 B is connected to the PCI 6110 or PCI 6115 card which is the scan control imaging card The BNC 2110 box is connected to the PCI 6713 card which is the output card used by TriggerSync Page 19 of 125 Ultima Manual ver 2 5 BNC 2090 A inputs ACHO ACH7 are the eight TJriggerSync input channels DACOOUT is unused DAC1OUT is used in photoactivation USER1 is the scanning shutter control signal and USER2 is the patch amplifier trigger input BNC 2090 B inputs ACHO ACH3 are the four high speed ADC inputs of the PCI 6110 or PCI 6115 card switches on each input must be set to the DIFF position DACOOUT and DAC1OUT control the X Y galvo in galvo systems or the AOD Y galvo in AOD systems USER is the Pockels cell blanking signal BNC 2110 outputs DACSOUT and DAC OUT control the X Y galvos for uncaging USER1 connects to the uncaging shutter DACOOUT DACAOUT and DACGOUT are available through TriggerSync for other experiments The PDIO TRIG 1 signal for all three breakout boxes should be connected toge
117. sted 3 A Pockels cell is added to or removed from the light path 4 Something new is added to the light path that would cause the direction of the beam entering the Ultima to change Nos Never look into the laser beam path The laser used in the Ultima system is a Class 4 infrared IR laser It does not have a beam visible to the naked eye The optics used in this system may also cause some back scattered or reflected laser light It is therefore critical to operate this system following all safety instructions and wearing appropriate laser safe eyewear 6 1 1 Imaging Beam Alignment in Scan Head Important Re aligning the imaging laser will introduce an offset into all of the spot calibration files Close Prairie View Close TriggerSync Remove sample Raise objective lens all the way up Install objective target in turret Open measurement and automation toolbox on windows desktop under o Devices and Interfaces o Traditional NI DAQ devices Select PCI 6110 Select Test Panels e Use analog output and DC voltage e Set channels 0 and 1 to 0 00 Volts Be sure to press update channel after selecting each channel VVVVVV Page 33 of 125 gt gt gt gt gt gt gt gt gt gt 6 Ultima Manual ver 2 5 Close shutter for uncaging beam Open shutter for imaging beam Verify that the image of the X axis galvo mirror is centered on objective target ring Use adjustments at top of tower to cent
118. sure optimal system performance Do not pull on or bend cables or fibers Do not handle filter cubes or dichroics except as recommended by the manufacturer Page 12 of 125 Ultima Manual ver 2 5 2 2 Warning labels used on the Ultima Multiphoton Microscopy System Warning label on beam cover and light box Warning label on interlocks components Warning label for defeated interlocks on interlock defeat bocks Warning label for defeated interlocks on interlock defeat bocks EMITTED WAVELENGTHS 650 1100 nm PULSE DURATION 10 200 CLASS 4 LASER PRODUCT Laser Warning IEC Logo Page 13 of 125 Ultima Manual ver 2 5 3 Introduction 3 1 Scope of this Manual Congratulations on your purchase of a Prairie Technologies Ultima Multiphoton Microscopy system This manual is intended to provide the information necessary to operate your Ultima system and describes not only the hardware but also the PrairieView software for image collection and scanner control and the TriggerSync software for synchronizing imaging with electrophysiological or uncaging events 3 2 Conventions Used in This Manual When referring to a specific button or icon single quotes are used Press the Single Scan button to collect an image When describing a specific action that the user is to take particularly as a part of a sequence of actions an arrow is used to highlight the action Press E Click and release the mouse once at
119. t should not be moved Using this dialog you will notice the Page 98 of 125 Ultima Manual ver 2 5 placement of the blue cursor changing automatically in response to changes in the physical position of the selected pointing device The red cursor is intended to be the primary method of selecting locations with the various marked points generation tools 8 5 1 Calibration Files and General Controls The section of the dialog along the upper right hand side contains the controls for selecting the calibration file for the selecting pointing device The Marked Point Device s control is used for selecting the desired pointing device Only valid options will be available for selection For the Ultima system the only available pointing device option is the Uncager option For an Ultima that contains only one set of galvanometers it is possible for a separate Prairie Photolysis Head to be installed This Photolysis Head would also be selected as the Uncager To the right of the Marked Points Device s control is the control NeD Marked Points Location This control has three options A Iris B Pinhole C Confocal The selection will determine which NeD Pinhole option will be used for generating the marked points The switch directly below the Marked Points Device s control is Mark Point using Red Blue Cursor Typically the operator will leave this control set to the Red cursor option In th
120. tart Position um Step Size um 2 00 de f Define Position um ln on C Home Position um 1 00 Define Step Size um Goto Start Pos 0 00 1 00 Define Goto Mid Pos mem Calculate Adjust PMT Save Path C Documents and Settings Prairie elt CG Base Directory 2 Series 12052006 1230 EES D Start Z Series 1 11 10 PM Linescan Acquiring 1 of 1 1 12 43 PM Linescan Acquiring 1 of 1 Scanline Period ms 3 120 Frame Period ms 1597 440 Framerate fps 0 626 x 0 Y 0 Z 0 FOV 512x512 1 0 1 0um Acquisition took 1 82 s Figure 7 12 Z series set up 7 2 8 1 Z Motor Control The Z Motor control allows the user to scan through the z axis of an image in user define step sizes Motor _ EE Position um Tom 0 m Figure 7 13 Z Motor control Use the and hd arrows to move the z motor by the number of um indicated in the Step Size field To change the step size click in the field replace the number with the desired step size and press Enter The step size value in this field is NOT used during acquisition of a Z series 7 2 8 2 Z series Calculator In order to collect a Z series stack the operator must define several parameters that define the range resolution and signal levels Four values are needed to define the range and resolution of a Z series Start Position Stop Position Step Size and Number of Slices or image plan
121. tern to indicate the direction the pointing device should move when the button is pressed For example if the top most blue button is pressed the pointing device should move up or towards the top of the current image by the amount specified in the steps field The selected pointing device will move an amount specified by steps The value to use for steps is dependent upon the type of pointing device being used For a galvanometer based pointing device the steps field should be specified in millivolts So a value of 200 in this field indicates a desired movement equal to 200 mV from the current location For a motor driven pointing device the steps field should be specified in motor steps So a value of 55 in this field indicates a desired movement equal to 55 motor steps from the current location When the Acquire button is pressed two image windows will appear One is labeled Original Image and the other is labeled Processed Image Within the Original Image window position the mouse over the location of the spot and click the left mouse button In the Processed Image window you should see a binary representation of the spot A binary image is an image where a pixel only has two values either black or white So the Processed Image window should show a primarily black image with a small white area that represents the spot Second adjust the values for the
122. ther This signal is the trigger signal that synchronized the start of an experiment acquisition among the three NI cards Normally these are also connected to the USER2 output of BNC 2090 B in which case the computer controls the start trigger Page 20 of 125 Ultima Manual ver 2 5 Ka Dt LEWg ug 1 BULO idid OL abba 11015 Uto ISP Odd 01 435 IMOLA XD AaS Ld 93 2343571 Sad 61 DOK 8 DOC NS W 060 2NG EE Ad HI peog OL LIN OL 8 d b If aug x open Bujuue s wog oen o puig Dreem aaepe Fi puig PURO AH Ld ZNI SCH AH Ld OL jeug DEE AH 1d LNI XCA AH Ld 01 hindu dwy uved INCOOWO OL LE OL anng anys 30201 UO Indu 2333s OY Jg 775 Buuu id acc uon2ajnad propaan wu 0 puy b Ao c ku Ld Buueyp o piding uod MN 0 wogsnoxd propaan z i 01 puy ay Nd Z bauen jo ehm au duj 123323 pods uonisog SY Ul S9u 1IMS 3SHN 3SH 108 uonisog 4410 U seu iMs 35 44IQ IV SHON gyae 10 spuump mdu g NI BNC 2090 boxes A top amp B bottom 3 Figure 4 Page 21 of 125 Ultima Manual ver 2 5 USER to FFIT DIOT to USERI USERT to CTRIOUT ur ETC Wir EFFE BRE rg d GEO SER irn Adr ah hide dag DIOO to PFI6 USER2 to FFIG Figure 4 4 Close up of NI BNC 2090 boxes Page 22 of 125 Ultima Manual ver 2 5 To NI 6713 Board in PC WIRE DIO 1 to USER 1 To Pointing Galvo Y input To Pointing Galvo X input Master Scan Start Trigger From USER 2 on BNC2090B
123. tmost LED indicates the status of the RS 232 send and receive lines Traffic through the RS 232 port causes very brief dim flashes on this LED This LED will flash continuously 10 times per second if an RS 232 transmission error causes the input command buffer of the controller to overflow In this state no further commands are recognized by the Device Control Box the motors will halt and the controller must be reset by either cycling power or pressing the reset button located on the front of the Device Control Box 4 2 6 Prairie Preamplifier The Prairie Preamplifier is a four channel fixed gain fixed bandwidth variable offset summing preamp in an electrically isolated housing The preamp requires supply voltages of 6 5VDC and a ground on a three conductor input connector Preamp offset voltage is controlled through a USB connection Each preamp channel has one BNC output and two BNC inputs If both inputs for one channel are connected to photomultiplier tube outputs PMTs the signals are averaged PMT gain is fixed at 10 A V for a single input with a 3 dB bandwidth of 2 0 MHz using a 2 pole filter 4 2 7 Galvos amp Acousto Optic Deflector AOD The Prairie Galvanometer Control Box controls two Cambridge galvos using two Cambridge MicroMax 671 Drivers Each galvo driver combination comes as a set carefully tuned for best performance Caution Switching cables so that the drivers control the wrong galvos can cause galvo damage
124. to have different laser intensity and or PMT voltage settings at each level For example more laser power may be needed to penetrate and provide sufficient signal levels at great depths in the specimen PrairieView provides such a mechanism In order to have a nonlinear PMT change applied to the laser intensity and or the PMT HV simply follow the steps outlined below Page 47 of 125 Ultima Manual ver 2 5 gt Use the al and el arrows to move the z motor to find the desired focal plane for the start and stop position of the Z series gt Turn on Adjust PMT in the Z series Calculator window VV VV NN Z Series Calculator ee Position Start Position um Set Start Limits Don Define eee Stop Position um Set Set Stop Limits Limits Mon o Define m ree 1 00 ES Define E Mid Pas Tu e orae OF Slices e Calculate Click on Go to Start Pos In the Laser PMT DAQ tab set desired laser ad PMT settings Click on Set Start Limits Click on Go to Stop Pos In the Laser PMT DAQ tab set desired laser ad PMT settings Click on Set Stop Limits Each line in the Z Slice Data Window will reflect the laser and PMT settings as calculated by PrairieView Page 48 of 125 Ultima Manual ver 2 5 7 2 9 T series The ability to generate a series of images and data using different labels region of interest ROI settings XY stage coordinates Z series photoactiv
125. trical recording amp stimulation gt gt gt start PrairieView Start TriggerSync Click on Acquisition and DAC Output Setup in riggerSync o Confirm or define stimulus control and acquisition parameters and close the dialog Enable the desired ADC input channels on the main TriggerSync window In PrairieView use Live Scan or Single Scan to locate the area of interest Use pan rotate and zoom to orient the target region with the feature of interest Press to open the LineScan dialog While the Linescan dialog is being started the operator will be prompted as to whether the Linescan acquisition should be synchronized with riggerSync f the operator selects No then standard linescan acquisitions will be performed If the operator selects yes then a TriggerSync acquisition will be automatically performed when the linescan acquisition is started The 7riggerSync data will be automatically saved in the same directory using the same base name as the linescan data Position the line where desired Enter the number of lines you want to collect or the amount of time to perform the linescan acquisition Press Begin Linescan TriggerSync will perform an experiment as you have defined it while line scan data is collected 9 2 2 Viewing Line Scan data Start the LinescanViewer application from the Desktop Load the line scan file created from the above example The graph displ
126. tter B scan dichroic j lens IS zi imaging galvanometers m b uncaging galvanometers laser camera 2 23 mode control imaging beam uncaging beam Figure 4 6 Ultima Scan Head light path 4 5 1 Dichroics The Ultima has multiple user changeable mirrors dichroics as shown in Figure 4 9 Primary Dichroic The primary dichroic is located in the Olympus epi fluorescence illuminator in position 1 Based on the specific system configuration selected this is a 660nm 700 nm or 720 nm LP dichroic Additionally there is a paired IR blocking filter located between the dichroic and the PMTs The cube is custom manufactured by Prairie PMT Dichroic A PMT dichroic is based on a standard Olympus cube For a dual detector this dichroic includes a 5 5nm dcxr dichroic mirror and 607 45nm amp 525 70nm barrier filters in front of PMTs No 1 and No 2 respectively This combination was chosen to optimize dual labeling using Alexa 594 and Alexa 488 If removed entirely all light is sent to PMT No 1 For a quad detector there are two PMT dichroics The standard cubes that come with a quad detector are selected at the time the system is ordered and are designed to maximize the available wavelengths To remove the PMT dichroics pull down on the magnetic cover on the bottom of the housing A 100 mirror is supplied as standard equipment in the Camera Port Mirror Dichroic position The knob on the right hand side of th
127. uired as described in Section 7 2 8 While still in the playback window click the MIP button This places a crosshairs cursor on the image and opens up a sidebar window to the right of the y z projection and below the image window the x z projection By moving the cursor to the x y coordinate of interest the user can observe changes in intensity in z As the cursor is moved along the x axis intensity changes in the y z plane are observed while as the cursor is moved along the y axis intensity changes in the x z plane are observed E Ki ave Fa ES Enk aj fi Dataset 1 nfo a wiet IV Loop hioo Delay ms lo Thickness W Show Overlay bs AE 125 0000 31 33 0000 0000 0 00 um Figure 7 29 MIP Maximum Intensity Projection window Page 71 of 125 Ultima Manual ver 2 5 7 4 10 MP Marked Points The Mark Points dialog is used to mark locations of points for photoactivation or uncaging experiments These points can be single or in a user defined line or grid Note Although the Mark Points function can be performed in a PrairieView window TriggerSync must be open and running in order to use this function When the Mark button is clicked a box appears on the image in the active Image Window and a second Mark Points window appears as shown in Figure 7 28 To select points click on the appropriate option in the Mark Points window and follow the steps descr
128. uisition is completed the TriggerSync application will go into the Analyze Acquired Data On state The operator may manually switch between these two modes at any time by clicking on the Analysis menu option and then clicking on the Analyze Acquired Data CtrI A option This menu option will show a checkmark in front of it when the Analyze Acquired Data mode is on When no checkmark is visible then the Analyze Acquired Data mode is off Points deny Configuration Help Analvz amp Acquired Data Ctri 4 vd Automatically Scale Data Wo online analysis Trace Averages Running Average DM Amplitude analysis Arniplitude Analysis Using cursors Exponential Fits Fluorescence Quantification Functional Mapping Figure 8 2 Analyze Acquired Data mode There is an additional control that overrides the Analyze Acquired Mode menu option To the immediate right of the Acquire button is a checkbox control labeled Disable Analysis When this button is checked enabled it will prevent the operator from turning the Analyze Acquired Data mode on Figure 8 3 TriggerSync Acquire This override control is present because some operators wish to have full access to all of the input and or output controls as soon as the current acquisition is finished without having to first turn the Analyze Acquired Data mode off 8 1 1 Analyze Acquired Data Off As mentioned this is the default mode of operation wh
129. utput is turned on will automatically replicate the currently defined mark points to match the number of pulses defined for the DAC Output that is being used for synchronization For example let s say that the currently defined pulse train for the selected DAC Output has 12 pulses Now let s say that there are currently five 5 marked points If the Replicate Points control is on then when the Acquire F5 button is pressed or the JriggerSync acquisition is started the software will see that only five points have been marked but there are 12 pulses that need to be synchronized with So the software will automatically temporarily just for the acquisition loop through the five marked points again which will bring the new total of marked points to 10 which is still two shy of what is needed so the software will add the first two points again Now the sequence of points during the acquisition will be 1 2 3 4 5 1 2 3 4 5 1 2 Page 113 of 125 Ultima Manual ver 2 5 9 Acquisition Scenarios One of the most powerful aspects of the ULTIMA system is the ability to collect fluorescence data in synchrony with Uncaging and or electrophysiology recording Running TriggerSync and PrairieView simultaneously with the data communication links between the two programs allows the operator to accomplish this IMPORTANT TriggerSync must be started AFTER PrairieView is started If both programs are running and you are unsure which was executed
130. utton A row with default settings will be created To select the number of images or acquire click on the Images Stacks field type the desired number of images to collect and press lt Enter gt Click on the Period field type in the time required for this line of data acquisition to take Press Enter Click the Max Speed check box if this is desired Press Add Wait A second line appears Click on the Period field type in the total time for the system to wait between the preceding and following acquisitions Press lt Enter gt Press Add New A third line appears Click the Z series check box This will cause a single Z series to be collected as currently defined in the Z series dialog Verify the Save Path Press Start T series The active T series line is the one marked by an arrow in the leftmost column of the T series cycle data list Page 51 of 125 Ultima Manual ver 2 5 7 2 10 XY Stage The XY Stage tab allows the user to select and save specific x y and z coordinates This makes it possible to return to locations of interest in the sample and is especially useful in setting up complex experiments LaserPMT DAQ Z Series T Series 2PLaser PC O2 XY Stage Misc hal viel Zum P3 Al 1 J 714633 129 06 94 90 216 60 48 48 98 40 p 32 89 100 43 89 40 vi A um 22 93 Clear All Move to Selected 100 43 Remove Selected Step Size um 5
131. utton becomes active If the operator presses this button a separate image window will appear with a each of the marked points within the grid area shown The color of the marked points is determined by the color specified in the Overlay Color control just below the Show Map Boundary button This allows the operator to visualize where exactly the marked points occur Page 107 of 125 Ultima Manual ver 2 5 Figure 8 22 Show map points As soon as the Generate Map Points button is pressed any previously defined marked points will be deleted Directly below the Map Method there is a field labeled Functional Mapping that indicates whether the current marked points list will allow a Functional Mapping acquisition Enabled or will not allow a Functional Mapping acquisition Disabled If a grid of marked points is to be used with either random marked points and or a line of marked points the Functional Mapping acquisition will be turned off To use a grid of marked points along with either random marked points and or a line of marked points first define the grid marked points and then add the other desired marked point locations As soon as one or more marked points are added to the grid definition or one or more marked points are deleted from those created in the grid definition the field labeled Functional Mapping will indicate Disabled The most recently defined grid corners wil be remembere

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