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Prairie Technologies User`s Manual

1. aiz Update Label Delete Selected Delete All Label Groups Label 1 Label 2 Label 3 Label 4 Add Label Update Label Delete Selected Delete All Label Group Group Values Label Group Groups Screen Shot Labels Tab The different values for each label created are displayed in the Labels chart Each label can have a customized Name for easy identification The status of the Channels in a label are shown as either True for on or False for off All PMT Gain values are shown for each channel The values are separated by commas Even if a Channel is not being collected False the PMT may have a voltage applied to it To ensure no photon collection for a channel set to False set the PMT high voltage to zero In addition to the PMT Gain values the DAQ Gains for each channel are listed as well The final setting for the label is the Dwell Time Per Pixel All images collected with the selected label will be at this dwell time Creating and Using a Label 1 Press Add Label A row named Label 001 with current settings will be created 2 Tochange the name of the label double click on the box with Label 001 in it and rename as desired 3 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 select Update Label Values 4 Selection of a specific label is done by clicki
2. C DANGER TET B Warning label for defeated interlocks on DEFLUTEO MOIO NTE FAKIN mamawa interlock defeat blocks AEPD RANA TAT 4301100 ren PULSE DURATE 10 AN CLASS 4 LASER PRODUCT Laser Warning IEC Logo 3 Introduction Introduction Alp 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 and describes not only hardware but also PrairieView software for image collection and scanner control and the TriggerSync software for synchronizing imaging with electrophysiological or uncaging events Conventions Used in This Manual When referring to a specific button icon or check box a bolded font is used Press Single Scan to collect an image Specific key strokes are denoted with bolded font within angle brackets Press lt Enter gt to continue Menu strings will be denoted with angle brackets to define each sub menu Go to File gt Preferences gt Z Series to edit these options When describing a specific action that the user is to take particularly as a part of a sequence of actions numbers are used to delineate the steps 1 Press ROI 2 Click and release the mouse once at one corner location of the ROI 3 Hyperlinks are present throughout the help file They are indicated with blue underlined text 4 Ultima Overview Ultima Overview La AlL gt T
3. HQ Bicubic 86 92 um Screen Shot Line Scan Overlay y Linescan Control Ed Number Of Lines Minimum Line Line Period Number Repetition Repetition 2 37 14 PM Linescan Acquiring 1 of 1 Define Period ms ms Lines to Skip Count Period s 2 37 14 PM Linescan Finished 7 2 37 14 PM Linescan Saved in C Documents and Sett L 2 880 2 880 b ee 0 000 2 37 14 PM Linescan ings prairie test Lin Acquisition Time ms __ 2 37 14 PM Li Scan 09272010 1048 0 57 600 _ Calculate Pixels Per Line 528 Total Acquisition Time s 10 058 2 37 14 PM Enasi 22 E Display Zoom mag D Sesi cona i Leave shutter open Chi vl in LinescanViewer A Update display lvl Open _ TriggerSyne short name Zoom Y Save Path C Documents and Settings prairie te Browse Append Linescants D Zoom XY Base Directory LineScan 09272010 1048 23 eal Start Linescan s Line O Circle Freehand 2mX o jes 4X 127 T 46 080ms 0011 0000 0000 Screen Shot Line Scan Dialog 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 Click on Start Linescan to begin the acquisition The acquired data will be displayed below Base Directory O
4. Screen Shot Image Window with BOT Regions Once a region is created there are some options for manipulating the position Right Click To offers three different manipulations The user can choose to Resize Rotate or Clone a region by selecting the desired change hovering over the desired region number and right clicking For Resize and Rotate the user must right click and hold down the mouse button until the region is the desired shape or orientation A single region can be moved only when Add New and Move All are off not highlighted The cursor will automatically turn into a hand cursor when hovered over a region number This indicates that particular region can be selected Click and hold the left mouse button to select the region and move it to the desired location When Move All is selected all of the regions will be shown in the selected highlight color of cyan The cursor can be placed over any region number in the image display window so the hand cursor is shown click and hold the left mouse button and move the mouse to move all of the regions Release the left mouse button to fix the regions in the desired locations As soon as the mouse is released Move All is turned off Brightness Over Time Regions Each region created on the Image is described in a table below the graph There are several columns of information specific to each region Display The user can choose which region s information to show on the graph by checking or unchec
5. Name New Action Action _ Rule g fa lt image ch gt TIF File Screen Shot Editing an Action Actions consist of a name filename and set of arguments Name Used to identify the action from others and give some indication of it s behavior Action will be referenced by their names throughout the application Filename The path to a program or script which will be executed For example notepad would launch a text editor Arguments A list of arguments passed to the program or script specified The arguments can contain tokens specified to the left of the text box for example in the screen shot above lt image ch1 gt would be replaced by the path and filename for the image containing the data for channel one Rules CO Name New Action Action Rule Scan Type Action to Perform te m x Screen Shot Editing a Rule Rules consist of a list of criteria with corresponding actions to perform if the criteria for a given line are met In the screen shot above actions can be performed based on the current scan type being acquired Note A rule can point to another rule but if a rule references itself directly or indirectly it will terminate rather than continue in an infinite loop Buttons to the right of the grid allow lines in the rule to be moved up or down as well as remove lines from the rule Types of Actions There are a number of different types of actions available in different places throughout the appl
6. 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 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 Marked Point Hold Time ms specifies how long the pointing device should stay at each marked point during this special acquisition mode Marked Point Move Time ms indicates 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 becomes too large it may be necessary to increase this value to 0 2 or 0 3ms Marked Point First Point Delay ms indicates an additional time delay at the start of the experiment in addition to 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 Synchronize Marked Points with Selected
7. 590 m Ethidium Bromide 595 I XRITC 595 E 6080 3 602 E Calcium Crimson 611 L Texas Red 615 L Propidium lodide 617 E SNAFRF BASE 630 E Fura Red 640 L Acridine Orange RNA 650 E 7 aminoactinomycinD 655 E TOTO 3 660 L Allophycocyanin 661 Screen Shot Channel Color Selection Dialog To select a display color for a channel click the color label to the right of the channel button and a dialog with color choices will appear Multiple channels can be displayed in the same image window with different colors Set Channel Color Freeze Channel Screen Shot Channel Button Context Menu Right clicking on a channel button will bring up a context menu which allows for color selection as described above as well as the ability to freeze a channel When a channel is frozen a snowflake icon appears on the channel button and the channel will no longer be updated in any image windows The frozen channel data will continue to be displayed To unfreeze a channel simply right click on the frozen channel and select Unfreeze Channel 10 3 3 Brightness Over Time Brightness Over Time 4a p BOT is used to measure image areas for pixel intensity over time This feature can be used on some active image collections and all collected data sets including W Series y Brightness Over Time Ea A WAA Jen n 1890 _ Best Fit e Fixed ba 26 0 1870 1860 N4 gt i 1850 Second
8. Emission Light Path for Quad Channel Detectors Sub Stage An optional source of signal detection is from sub stage detectors Placed below the specimen these detectors collect transmitted fluorescence and second harmonic generation signal To image with sub stage detectors move the two knobs in to the Image position see below Image Substage Detectors with mirror and shutter toggles labeled CAUTION Protecting the PMTs When the EPI light source is in use the two knobs for the sub stage detectors should be in the Close position see above to protect the PMT If the light from the illuminator is directed to the PMTs they will be damaged and need replacing This damage is not covered under warranty To achieve maximum intensity in sub stage detectors the condenser aperture should be in the open position and aligned for proper Kohler illumination Dodt Gradient Contrast Prairie s Dodt Gradient Contrast Imaging System is available as a secondary imaging system for the Ultima to help image unstained cells for electrophysiology experiments such as patch clamping It uses transmitted light from an IR laser scan to create a DIC like image To collect this information the Dodt tube and mirror are placed in line between the scope condenser and the lamphouse The mirror directs the signal to a PMT above the main housing To prevent accidental over load of the PMT an interlock is used to automatically shut off the lam
9. Manual Scale 0 10000 gh O Auto Scale C Log Scale Channel 2 Ch2 0 Lg l Manual Scale 0 i0000 edp O Auto Scale C Log Scale a jaen j v 1 0 0 1000 2000 3000 4095 1 01 0 A 10000 Wou Caangna 5000 0 l l l l 1000 2000 3000 4095 1 01 00 Manual Scale 0 10000 eaiiqh O Auto Scale C Log Scale lie jja j Screen Shot Look Up Table Dialog 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 10 3 6 Region of Interest Region of Interest Llall 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 to be imaged which increases the s
10. The labels in this control correspond to the labels on the BNC connectors on the BNC 2110 box Output Signal After selecting the desired output channel use the Output Signal control to turn this analog output on off when performing an acquisition Protocol allows the operator to select a pattern or Protocol for the selected analog output The operator may select from the following Protocols I V Ramp Pulse Train G F C and Custom Waveform These protocols are described in more detail below Graph Color 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 separate Ext Sensitivity value mV V for each analog output This 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 0 5 This scaling is applied to all voltages in all of the available protocols DAC Label allows the operator to specify a text string to display over the individual graph window along the right hand side of the dialog For example DAC 6 Out is o
11. by unchecking the Show Overlay box It is possible to manually run an action with the current data set by selecting one from the Action drop down and pressing Perform See Actions for more details The text box in the lower right corner of the playback controls will display an error message if the data set is corrupt due to missing samples at the time of the acquisition Due to hardware constraints while acquiring multiple channels with heavy processing it is not always possible to keep up with the data throughput Saving any heavy processing until after the data is acquired is a good practice in such cases Metadata Window The metadata window will appear by default once PrairieView enters playback mode There is a check box at the bottom of the window to disable this default behavior The metadata window can be brought up at any time in playback mode by pressing Info The metadata window displays information about system settings such as laser power and motor positions for each frame as well as providing controls to map data associated with a certain label and acquisition channel to a specific display channel There are only four display channels available so it is not always possible to view all data associated with a multi label acquisition in those four channels There are four combo boxes at the top of the window representing channels one through four left to right For each channel a label channel combination to be displayed is selected Somet
12. mirror is supplied as standard equipment in the Camera Port Mirror Dichroic position The knob on the right hand side of the scan head is used to move it in and out of position When it is in position the Ultima operates as a laser scanning system When it is retracted a normal CCD camera customer supplied may be used with standard trans illuminated or epi illuminated microscopy techniques This is not a user changeable mirror It is possible to mount a dichroic mirror in this position Contact Prairie for more details Note The Camera Port mirror is not user changeable If you think something is wrong with your Camera Port mirror please contact Prairie Technologies Primary Dichroic The Primary Dichroic is located in the epi fluorescence illuminator turret position one 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 PMT Dichroic The PMT Dichroic is the main user changeable dichroic It is based on a standard Olympus cube For a dual detector system this dichroic includes a 575nm dexr dichroic mirror and 607 45nm amp 525 70nm barrier filters in front of PMT1 and 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 1 For a quad detector system there are two PMT dichroics The standard cubes that come
13. this is now not used Most devices are no longer controlled through TriggerSync wvenenthirshedTriggersetup Liv m 5 ie rm be o E E E E l p s Screen Shot Movement Finished Trigger Setup dialog Set Amplifier This option is currently not updated Axopatch 200 B 1 Axopatch 200 B 0 Screen Shot Set Amplifier Sub Menu Leak Subtraction This option is to modify the signal from an outside amplifier Leak Subtraction is only applied to Input Channels ACHO ACHI ACH2 and ACH3 Leak Subtraction alll ON OFF Leak Subtraction of pre puses Screen Shot Leak Subtraction dialog Preferences This dialog controls many of the data output decisions for TriggerSync See TriggerSync Preferences for more information 11 6 7 ADC Configuration ADC Configuration Lal 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 Each of the eight inputs can be adjusted independently E ADC Configuration 1 1 vi ADC Configuration mmo O O OF O O nee SK eee se ven SANE tem a Se a ob ee soe Soe Ce re ma a OS SS a ee ee Be een PER See Pe SC a Pe a eS eS es SS Es 1 Ey Accept Cancel Screen Shot ADC Configuration The first three parameters Automatic Gain Sensing Y max and Y min are only used after da
14. 2 Tear Off Panels Tear Off Panels all As of Prairie View 4 0 0 0 certain panels may be torn off and placed on the user s desktop To tear off a tab click on the diagonal arrow button located on the right side of the tab label Prairie View will remember these selections and return the tear off panels to their last location when Prairie View is restarted To return a tear off panel to its original location click the X in the upper right corner ca BJ 0 J Zee 0 BIS J Zero Screen Shots Lasers tab with tear off option left Lasers tab tear off with revert location option right 10 2 3 Image Size Controls Image Size Controls iala p 525i Screen Shot Scan Resolution Tools Standard Image Size Image size is a definition of how the collected data is acquired and displayed In the case of 512 x 512 data is acquired at 512 bins in x by 512 bins in y with each bin sampled according to dwell time All images are collected at 72 pixels per inch Image Window Size There are four options under Image Window Size Fit is the default and the image is shown as 512 x 512 pixels If the image window is increased or decreased in size the display will be scaled to fit proportionally to fit in the new window 1 1 shows the image at its actual size as defined by the Image Size Smaller decreases the image by 10 The image can continue to be reduced by clicking Smaller repeatedly Larger increases the image size
15. Ch3 Ch4 The channel buttons indicate which input channel is being used to collect data for that image window See selecting an input channel Freeze Freezes current image contents in window Data for each channel continues to update but is not shown in frozen image windows BOT Used for measurement of average pixel intensity of user defined regions to be monitored over time See Brightness Over Time LS Used for definition and acquisition of a line scan See Line Scan LUT Used to adjust the display intensity scale for an image See Look Up Table ROI Used to define a region of interest to acquire a portion of an image faster See Region of Interest a fy Saves the current image displayed in the image window See Snap Tool lt lt RAYI Used to display intensity values over a line See Line Profile PA Used to defines masks to limit laser exposure to certain areas The button is only active when playing back a z stack See Photoactivation MIP Displays the maximum intensity profile for a stack of images The button is only active when playing back a z stack See Maximum Intensity Projection Mark Allows the user to perform a marked points experiment from PrairieView The button is only active when TriggerSync is open See Mark Points Note Button labels may be configured differently on different systems Along the bottom of the Image Window are descriptions of the image being shown At the left is the position of the cursor within the f
16. DAC Output automatically synchronizes 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 When Replicate Points and Synchronize Marked Points with Selected DAC Output are turned on TriggerSync will automatically replicate the currently defined mark points to match the number of pulses defined for the DAC Output being used for synchronization For example if the currently defined pulse train for the selected DAC Output has 12 pulses and there are currently five 5 marked points the software will see that only five points have been marked but there are 12 pulses that need to be synchronized with 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 11 7 2 Functional Mapping Functional Mapping 4a gt This feature of Mark Points contains controls to help the operato
17. Path Base Directory indicates the default path and base directory set up for saving data This is C Documents and Settings Prairie 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 to change this information When working with TriggerSync there are several options available triggering acquisitions and integrating TriggerSync experiments into the T Series With Trigger Cycle selected Prairie View will wait for an input trigger before processing the selected line as determined by the selection made in File Output Triggering Option By selecting Trigger Each Image Z Series Prairie View will wait for a trigger before acquiring each image or z series Adding a line using Add Insert TriggerSync Exp will cause TriggerSync to perform an Acquire operation as defined in the current TriggerSync experiment Collect a Basic T Series A Basic T series is one in which an image or Z series is collected at a desired interval N NONA OQ Select Add New A row with default settings will be created To select the number of images or Z series to acquire click on the Reps field type the desired number of images to collect and press lt Enter gt The default value is 1 so no changes are necessary if only one image or Z series is desired Click on the Period field type in the time required fo
18. a check box to allow the settings to be applied to all imaging lasers The user can choose the equation applied to the Pockels cell power by selecting Default or Custom The Default gradient is a slight parabola defined by Prairie View Custom allows the user to enter their own gradient line for the acquisition The user can enter a number in the field to the right of Custom or select graph If graph is selected a dialog will appear for viewing the custom gradient versus the default There may be additional lines for other imaging lasers A Custom Laser Gradient for Pockels 10 Laser Power Show All Lasers fio Cancel Accept Screen Shot Custom Laser Gradient Dialog The x axis horizontal of the graph represents the z position of the current Z Series while the y axis vertical of the graph indicates the laser power setting as a function of the z position As the Adjust Gradient slider is moved the graph will automatically update with the new relationship between the laser power and z position In addition the operator may type in a Gradient value in the text box located below the Adjust Gradient slider If more than one imaging laser is present then the operator may click on the Show All Lasers check box to display the graphs of all laser powers as a function of z position for the current Z Series definition Selecting Cancel will dismiss any changes made to the gradient with the graph and close the dial
19. and now movement commands from Prairie View will be executed To take advantage of the higher speed acquisition capabilities of the piezo controller selected by checking the Fastest Acquisition check box on the Z Series tab only available when the Z Series device is the E 665 piezo controller requires an input trigger to control the stepping of the piezo controller Typically this signal will be the End of Frame trigger The trigger signal is configured via the File menu option then select the Preferences option and then select the Output Trigger Type option This signal is found at the PFI8 connector on the BNC 2090A box connected to the PCI 6052E NI DAQ card The software configuration and necessary wiring information is provided in the trigger signals section This trigger signal is input to the E 665 on the I O Connector on the back of the controller via pin 9 this is a DB 9 connector 7 Ultima Quick Start Ultima Quick Start Alp Turn on the main power switch at the rack Following instructions from laser manufacturer put laser in on or ready state Check and or install the filters and dichroics into the upper PMT detection path Turn on the PC Note Disable anti virus software Start Prairie View Start TriggerSync if you plan to conduct electrical recording experiments oo et oe a Se SS Verify the communication link between Prairie View and TriggerSync
20. and or install the filters and dichroics into the upper PMT detection path Turn on the PC Note Disable anti virus software Start Prairie View Start TriggerSync if you plan to conduct electrical recording experiments ot et oe aS OS 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 oo Turn on the epi fluorescence mercury lamp to warm up 9 Push the trinoc plunger in to the B position 10 Position the epi dichroic wheel to an epi cube appropriate for the sample 11 Put sample under the objective 12 Open the turret s back shutter 13 Turn up mercury lamp power 14 Focus on specimen 15 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 epi fluorescence illuminator is NOT an adequate shutter as it leaks light from the lamp house that will flood the PMTs 16 Turn the epi dichroic wheel to position 1 17 Pull the trinoc plunger out to the LSM position 18 Close light box door 19 Turn off the room lights 20 Open the laser s hard shutter in PrairieView or the laser manufacturer s software 21 Press Live Scan This starts the scanning and opens the laser path hard shutter 22 In the Laser PMT DAQ tab bring the PMT high voltage up to mid range about 500 600 depending on the
21. as New Window in the Scanning Controls 10 4 4 Display Menu 10 4 4 1 Display Menu Options Display Menu Options Alp New Image Window Ghost Mode Interpolation Mode gt Scale Bar Options gt Fonts and Colors Screen Shot Display Menu New Image Window opens a new Image Window It functions the same as New Window in the Scanning Controls Ghost Mode Toggles ghost mode which causes all PrairieView windows to appear translucent It can be useful in minimizing the noise from external light sources in a darkened room Interpolation Mode Specifies a method to use to extrapolate interpolate intensity values when stretching shrinking the acquired image e Nearest Neighbor Bilinear Bicubic High Quality Bilinear High Quality Bicubic Screen Shot Interpolation Mode Menu Scale Bar Options Allows user to select position of scale bar in Image Window as well as units of scale bar Show Scale Bar Places a scale bar overlay on all image windows Upper Left Upper Right e Lower Left Location gt Lower Right Units gt nm Screen Shot Scale Bar Options Menu Fonts and Colors Brings up a dialog box to control the fonts and colors of image text and overlay properties See Fonts and Colors 10 4 4 2 Fonts and Colors Fonts and Colors Alp a Fonts and Colors x Font Comic Sans MS 11 25pt Regular Photo Activation Masks Next Mask Color R 0 G 0 B 128 Screen Shot Fonts and Colors Dialog O
22. bar controls the selected frame while the horizontal scroll bar controls the selected sequence Sequences or datasets represent a cycle of a T Series a simple Z Series or a single image It is possible to navigate datasets by selecting one from the drop down to the right of the horizontal scroll bar or using the 00 buttons next to the drop down Next to each scroll bar is a set of five buttons to jump to the first or last frame sequence play through the images in either direction as well as stop any playback currently in progress There are also two check boxes associated with playback Loop which will continue playback when the last first image is reached and Wrap which will jump from the first to the last image or vice versa rather than reversing direction when the last first frame is reached When playing through a set of images the speed at which the images change is determined by the Delay ms text box By default this is set to 10 ms meaning that processing included the frame will not be displayed for any shorter than 10 ms To slow down playback increase this number When switching frames in playback it is possible to perform an action automatically by checking Perform Action This allows for some real time processing to occur as data is played back Other Playback Controls When in playback mode an overlay is displayed showing the time the frame was acquired as well as the motor positions at the time It is possible to hide this overlay
23. 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 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 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 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
24. blue cursor move and the values in the absolute and relative fields updated as well i o Make the necessary control changes to actually acquire the image of the pointing device the spot image 4 Acquire an image by pressing Snap 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 Intensity The operator would use this control to adjust the voltage of the uncaging Pockels If the selected pointing device is motor driven Optimize lt F6 gt will be activated for galvanometer based pointing devices this control is desensitized This control allows the operator to fine tune the calibration of the motor driven pointing device To use this feature Nudge the pointing device to the desired location Acquire an image of the pointing device W N gt Move the red cursor from its original position and center it on the spot from the pointing device 4 Press Optimize This process should eliminate small movement errors that sometimes occur with motor driven pointing devices 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
25. data Display and Analysis of Functional Maps For a description of the display and analysis options for Functional Map data see the TriggerSync documentation on Functional Mapping 14 8 Creating a Photoactivation Acquisition Photoactivation Acquisition All There are three major steps in creating a photoactivation acquisition define the mask test the mask and perform photoactivation Define the Mask for Photoactivation Collect a Z Series and open the file for playback Click PA to open the photoactivation menu on the right side of the Image Window Select a shape from the menu and define the region for photoactivation Apply the region to the desired frames by selecting Copy Ahead Back Le SP SS At this point the mask can be saved for future use 6 Exit playback mode Test the Mask 7 Under the T series tab click Clear All 8 Click Add New 9 On the new T Series cycle select Current Setting or a saved name for the Z Series and Photoactivation 10 Select Start T Series 11 Only the selected regions will shows up in the Image Window Note Mask regions are the regions that are scanned by the defined laser power Pockel Cell setting Regions that are not masked are still scanned but with minimum laser power Pockel Cell set as 0 If the defined Pockel Cell is 0 the selected regions and unselected regions will all display Only if the defined Pockel Cell is greater than 0 the selected regions are shown as translu
26. 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 Reset Relative will reset the relative position to 0 Marking points Basic Controls The button labeled Mark Point lt F9 gt is the basic control used for marking a location on the image for further study using the selected pointing device The actual location that will be marked when Mark Point is selected is dependent upon the setting of Mark Point Using Red Blue Cursor 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 press Mark Point 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 Nudge lt F7 gt to the desired location and then press Mark Point The list of marked points is maintained even after the TriggerSync program has quit running The next time TriggerSync is executed the last list of marked points will still be present in the program As locations are marked each point automatically added to the list of points shown in the Move To pull down menu The operator may u
27. front panel while the preamplifier controls can be accessed by selecting the green bar in the center of the tab In Prairie View 4 x this tab and others may be torn off and placed on the user s desktop kan Screen Shot Laser Controls The laser sliders are 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 If there is a laser launch associated with the system the laser line powers will also be accessed through this panel Which controls are visible for each laser depends on system configuration and calibration See calibrating laser power Check Interlaced scan pattern to enable interlaced scanning mode See interlaced scanning ai PLO 1 car BJ LO Zero Screen Shot PMT Controls The PMT HV controls the voltage which amplifies the photon electron signal to current pulse Increasing the PMT HV will increase the output current pulse and thus increase the brightness of image To reset all PMTs voltages to zero click Zero next to PMT Master To adjust individual PMT voltage slide the slider bar or click the arrows under that PMT HV The text field to the right displays the voltage that corresponds to the red LED displays on the Dual Preamplifi
28. gt This menu option will show a check mark in front of it when the Analyze Acquired Data mode is on When no check mark is visible then the Analyze Acquired Data mode is off TriggerSync 1 4 1 File Acquisition view Points FEQEIREEE Configuration Help Analyze Acquired Data Ctri 4 No online analysis Trace Averages Running Average Cumulative view Functional Mapping Full Width Half Maximum Screen Shot 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 check box labeled Disable Analysis When this button is checked enabled it will prevent the operator from turning the Analyze Acquired Data mode on amp NO B a Screen Shot TriggerSync Acquire This override control is useful for operators wishing to have full access to all of the input and or output controls as soon as the current acquisition is finished Analyze Acquired Data Off 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 As mentioned this is the default mode of operation when TriggerSync is first started If Disable Analysis is checked then this is the only mode of operation available until it is unchecked Analyze Acquired Data On This mode is intended for post acquisition evaluat
29. 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 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 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 us
30. is a two channel top mounted detector setup Most Ultimas can also be configured for four top mounted PMTs and or two sub stage PMTs Prairie offers two different types of PMTs multi alkali and GaAsP The multi alkali PMTs come standard and are side on Hamamatsu PMTs hand picked for their low dark current The GaAsP detectors are also Hamamatsu and are useful for applications requiring super high sensitivity in the collection of data The multi alkali PMTs have two cables attached One connects to the high voltage box to receive the voltage applied to it while the shorter cord sends the gathered signal to the preamp In addition to the high voltage cable and the signal cable the GaAsP detectors also require a power cable to power the cooling fan Top Mounted The standard signal collection for an Ultima system comes from either a dual or quad top mounted PMTs to the left of the epi turret The fluorescence from the sample is transmitted through the objective lens reflected off the primary dichroic through the IR blocking filter and through the various dichroics and barrier filters to the PMTs Barrier IR blocking Filter Sample Figure Emission Light Path for Dual Channel Detectors Laser Barrier Filters ao PNT 4 PNT Dichroic PNT 2 Barrier Filters Vee Ss a k o A S s s x PAT Dichroic Dichroic IR Blocking Filter Figure
31. 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 Pulse Train Screen Shot 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 should 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 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
32. laser and PMT type 23 Increase the Pockels cell percentage until an image is visible This should be about 2 5 9 Alignment amp Calibration 9 1 Beam Alignment Beam Alignment La AlL gt 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 The direction of the laser beam out of the laser cavity changes A mirror on the tabletop is adjusted A Pockels cell is added to or removed from the light path Something new is added to the light path that would cause the direction of the beam entering the Ultima to change WARNING VISIBLE and or pevisteLe LASER RADIATION WHEN INTERLOCK DEFEATED AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION MAX AVERAGE OUTPUT POWER SW EMITTED WAVELENGTHS 650 1 100 nm PULSE DURATION 1 300fs CLASS 4 LASER PRODUCT Never look into the laser beam path The lasers used with the Ultima system include Class 4 ultra fast infrared IR lasers These lasers do not have a beam visible to the naked eye The optics used in this system may cause back scattered or reflected laser light It is therefore critical to operate this system following all safety instructions and wearing appropriate laser safe eyewear 9 2 Scan Head Alignment 9 2 1 Imaging Beam Alignment Imaging Beam Alignment 4a
33. off mirror is placed in the path of the attenuated beam This mirror sends approximately 2 5 of the beam power to a power meter for monitoring purposes An interlocked electronic safety hard shutter is included in the beam path to prevent laser light from entering the scanhead when not imaging WARNING XPOSURE TO DIRECT OR SCATTERED RADIATION MAX AVERAGE OUTPUT POWER SW EMITTED WAVELENGTHS 650 1100 nm PULSE DURATION 1 300fs CLASS 4 LASER PRODUCT Never look into the laser beam path The lasers used with the Ultima system include Class 4 ultra fast infrared IR lasers These lasers do not have a beam visible to the naked eye The optics used in this system may cause back scattered or reflected laser light It is therefore critical to operate this system following all safety instructions and wearing appropriate laser safe eyewear 5 3 Pockels Cell Pockels Cell 4a P CAUTION The Pockels cell is a delicate consumable electro optical device The incorrect alignment of this component will cause it to fail Do not adjust unless instructed to by Prairie personnel The Pockels cell is a delicate consumable electro optical modulator used to attenuate the power of the laser to the scanhead The voltage applied to the Pockels cell is controlled through Prairie View The base voltage zero output is set by adjusting the knob on the front of the Conoptics box This number might change slightly depending on wavelength but s
34. outside of the range of the calibration file the operator will be warned that that is an illegal operation and the wavelength change will not take place If the system is equipped with the high speed optics option AOD it might not be necessary to perform the Pockels cell calibration This is due to the fact that the AOD system is set up and optimized for a single wavelength It might be better to simply take a couple of power measurements at the objective lens in Attenuation mode and then manually determine which setting in Attenuation mode will provide the desired sample power 10 4 2 4 Fluorescence Unmixing Fluorescence Unmixing All a Fluorescence Unmixing Channel to A E Gel i Bm E Dodt m Zeo lt li 2 isi Ch2 Zero lt i gt Dodt m Zero lt ij gt Screen Shot Fluorescence Unmixing When Fluorescence Unmixing is selected a selected percentage of a given channel is subtracted from another channel and added back to the original channel This is to correct for bleed through of signals from one channel to another By checking Enable the fluorescence unmixing will be applied The Source Channel is the channel to which the subtracted signal will be added to wile the Channel to Subtract From will be the channel the signal is removed from The Percentage slider allows the operator to control the amount of the Source Channel signal to be subtracted from the C
35. period is how many milliseconds it takes to create one frame This could also be arrived at by multiplying the scanline period by the number of lines per frame The frame rate is the number of frames scanned per second The second line displays the Field of View in pixels and microns and Pixel Size The field of view measurement and pixel size in microns are dependent upon the calibration of the objective lens selected The third line displays the X Y and Z motor positions The large window is used to display informational messages at start up and update status during an acquisition At the very bottom information is displayed by external program communicating with Prairie View such as TriggerSync 10 3 Image Window 10 3 1 Overview Overview KI a gt 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 Live Image or Single Scan is pressed 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 76 Y 0 0039 0000 0000 HQ Bilinear 160 12um Screen Shot Image Window Located on the left side of the Image Window are buttons for the following controls Ch1 Ch2
36. 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 time set to complete the defined active stimuli If the user attempts to set the time limit below the minimum required time an error will be shown and the time will automatically set to a safe length to complete the defined stimuli Acquisition Rate specifies the rate at which the inputs should be sampled during 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 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 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 specifies when the shutter should be opened and closed relative to the data acquisition There are three possible options Open Before Starting Data Collection Close After Data Collection Complete Start Stimulus Control with Data Collection the shutter will be opened first then t
37. the Acquisition and DAC Output Setup dialog in TriggerSync Confirm or define the stimulus control and acquisition parameters Press Accept Done to save changes and close the dialog Enable the desired ADC input channels on the main TriggerSync window Press Acquire in the main TriggerSync window The experiment will run as defined 14 3 Line Scans amp Electrical Recording Line Scans amp Electrical Recording oe NO a SS YS Start Prairie View Start TriggerSync Open the Acquisition and DAC Output Setup dialog in TriggerSync Confirm or define the stimulus control and acquisition parameters Press Accept Done to save changes and close the dialog Enable the desired ADC input channels on the main TriggerSync window In Prairie View use Live Scan or Single Scan to locate the area of interest Pan Rotate and Zoom to orient the target region Press to open the Linescan dialog Select Yes when prompted whether the Linescan acquisition should be synchronized with TriggerSync 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 Start Linescan s The TriggerSync experiment will be executed as it has been defined while line scan data is collected Start Linescan Viewer and load the created linescan file to see the electrophysiology data along with the fluorescence linescan profile s 14 4 Electrical Recording amp Uncaging Electrical Rec
38. which are used by Prairie during testing and configuration AOD An alternative laser scanning system for video rate imaging of specimens is available for the Ultima This system makes use of an acousto optic deflector to create the x raster scan of an image AOD systems will use the same Galvanometer Control Box as standard imaging systems In AOD scanning mode the X galvo is held still and used for panning the image while the AOD scans in X 6 12 Piezoelectric Z Device Piezoelectric Z Device KI a gt Piezo Controller This specially modified piezoelectric controller allows for up to 460um of high sensitivity travel for high speed Z series acquisitions There are two modes of operation for the piezo With the Servo switch set to on the piezo works in closed loop mode with 400 um of travel in closed loop mode When the Servo switch is set to off the piezo runs on open loop mode with a maximum travel of 460 um When used with PrairieView the current position offset of the piezo regardless of the state of the switch ANALOG DIGITAL will always be shown When this switch is in the ANALOG setting the position of the piezo is controlled via the DC OFFSET knob on the E 665 controller Any movement commands from Prairie View will be ignored When this switch is in the DIGITAL setting the VOLTS and MICRONS display on the controller will go blank and the DC OFFSET know will no longer affect the piezo position
39. with additional information about the proper syntax for the mathematical functions Some sample equations are as follows Let s say that instead of looking at the average intensity of a region over time standard graph of a region we want to see the relative change in the region intensity information over time Basically we want to look at the difference between the intensity values from one frame to the next If the region we were interested in was region 2 the equation would be 2 2 1 Many standard mathematical functions are available by using the following syntax Math Log Math Min and etc For example To compute the logarithm for the data in region 3 the syntax would be Math Log 3 To generate the absolute value of subtracting the value of region 1 from region 2 the syntax would be Math Abs 2 1 Sampling Options To update the graph when in playback mode create edit the regions and press Start The program will cycle through the current data set and generate the graph of data 10 3 4 Line Scan Line Scan KAD LS enables the line scan definition tool and brings up the Linescan Control dialog 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 Image Window 1
40. 1 e ee Ris C C 2 aL i KA aa a e l en LLLA D D Ze v P T D X 191 1 0000 0000 0000 0000 Nearest Neighbor 0 00um Screen Shot Automatically Generated Brightness Over Time Regions Freehand Linescan Path Generation These actions are run manually from the line scan dialog and are used to automatically generate freehand line scan paths based on the current image data There are two tokens available for use in the arguments field lt image ch gt where is replaced by a channel number 1 4 This token will be replaced by the path and filename of the image containing the current image data for the channel specified lt outfile gt which replaced by the path and filename of a temporary file created to pass line scan path data from the program or script specified into Prairie View The format of the output file is a comma delimited list of points that make up the path to line scan Each point is made up of an X and Y coordinate again separated by acomma that range from 0 0 0 0 upper left corner to 1 0 1 0 lower right corner Mark Points Point Generation These actions are run manually from the mark points dialog and are used to automatically generate a set of points to mark based on the current image data There are two tokens available for use in the arguments field lt image ch gt where is replaced by a channel number 1 4 This token will be replaced by the path and filename o
41. A message will appear at the bottom of the Prairie View window stating that the link has been established 8 Push the trinoc plunger in to the Bi position 9 Position the epi dichroic wheel to an open position Transmitted light will turn on automatically 10 Put sample on stage and focus through the binoculars 11 Make sure the epi fluorescence mercury lamp is completely shuttered or off if present on your system The shutter mechanism provided on the front of the epi fluorescence illuminator is NOT an adequate shutter as it leaks light from the lamp house that will flood the PMTs 12 Turn the epi dichroic wheel to position 1 This automatically turns off the transmitted light source and directs signal to the upper PMTs 13 Pull the trinoc plunger out to the LSM position 14 Close light box door 15 Turn off the room lights 16 Open the laser s hard shutter in PrairieView or the laser manufacturer s software 17 Press Live Scan This starts the scanning and opens the laser path hard shutter 18 In the Laser PMT DAQ tab bring the PMT high voltage up to mid range about 500 600 depending on the laser and PMT type 19 Increase the Pockels cell percentage until an image is visible This should be about 2 5 8 Ultima IV Quick Start Ultima V Quick Start all Turn on the main power switch at the rack Following instructions from laser manufacturer put laser in on or ready state Check
42. Before performing the actual calibration the operator must specify a few more control values 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 that 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 0 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
43. C 2090 PFI8 Ctrl C Start Acquisition Ctrl S Seal Test Screen Shot Start Experiment Sub Menu There are two different ways to start a TriggerSync acquisition The TTL Trigger Start lt Ctri T gt will start the acquisition after a TTL signal hs been received on PFI0 Trig1 This is usually a start of image or start of linescan signal TTL Trigger Count Start lt Ctri C gt will count the number of TTL pulses received on the PFI8 input and start the acquisition after the assigned number of pulses in the TTL Trigger Count field located on TriggerSync s main screen 0 34 Screen Shot TTL Trigger Count Selecting Start Acquisition will begin the defined TriggerSync experiment This is the same as selecting Acquire on the TriggerSync main screen Oscilloscope TriggerSync includes a basic oscilloscope for measuring inputs from outside sources or the NI 2110 outputs Up to four channels can be viewed on the oscilloscope These four channels correspond to the first four BNC inputs on the 2090A box labeled ACHO 3 Screen Shot Oscilloscope Seal Test The Seal Test dialog is a built in chart which calculates the resistivity between a DAC output and a 2090A input Screen Shot Seal Test dialog 11 6 3 View Menu Options View Menu Options 4a gt gt TriggerSync 1 4 1 Fie Acquisition WEWE Points Analysis Conl Overlay Traces Screen Shot View Menu Overlay Traces 11 6 4 Points Menu Options Poi
44. Image Screen Shot File Menu Open Experiment Previously acquired experiments can be opened in TriggerSync to be viewed again or analyzed Save Experiment The current experiment that has just been run can be saved to be analyzed later The experiment can be saved in two different file formats TriggerSync this includes all operating parameters as well as the data or Export this includes only the data which is readable by a spreadsheet program such as Microsoft Excel Load Parameters The parameters used and saved from a previous experiment can be loaded to run a new experiment Save Parameters The current parameters in TriggerSync can be saved to a file to be loaded at a later date Saved parameters include ACD channel DAC Output and playback configurations Save Image The currently displayed image from TriggerSync can be saved to be viewed at a later date Load Image A previously saved image from TriggerSync can be loaded for viewing Note Save and Load Image are associated with a now rare Ultima configuration 11 6 2 Acquisition Menu options Acquisition Menu Options KIAD Fe TriggerSy nc 1 4 1 File peu view Points Analysis Start Experiment gt Oscilloscope Screen Shot Acquisition Menu Start Experiment b gt TriggerSync 1 4 1 File Eitan view Points Analysis Configuration Help Start Experiment gt TTL Trigger Start Signal on PFIO TRIG1 Ctrl T Oscilloscope TTL Trigger Count Start Signal on BN
45. Map Points 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 Overlay Color just below Show Map Boundary This allows the operator to visualize where exactly the marked points occur Screen Shot Show Map Points As soon as Generate Map Points 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 will be remembered so that experimenting with different Map Methods can be done by simply making the desired
46. No Online Analysis will prevent the cycles from coming up for analysis in the trace graph Trace Averages shows an average voltage for each active channel over all the cycles and Running Averages will show this same data as it is collected for each channel Cumulative View shows the actual trace for each cycle of each channel overlayed Functional Mapping Opens a dialog box where previously collected Function Mapping data can be analyzed More information can be found here Full Width Half Maximum This is an older FWHM calculator for use with images created in TriggerSync This calculation is now included in the PrairieView Line Profile tool 11 6 6 Configuration Menu Options Configuration Menu Options Screen Shot Full Width Half Maximum dialog The Configuration menu contains different dialogs for setting up the use of the controls of the main screen Use Shutter for Acquisition E gt TriggerSync 1 4 1 File Acquisition view Points Analysis Peay Meni Use Shutter for Acquisition Set Shutter gt ADC Configuration Ctrl C Continuous Pulse Setup Load Parameter Buttons Movement Finished Trigger Setup Set Amplifier gt Leak Subtraction Ctrl L Preferences Screen Shot Configuration Menu With this option checked TriggerSync will control the selected shutter during an acquisition Set Shutter There are various shutters that TriggerSync can be wired to control The stand
47. Prairie Technologies User s Manual 1 Preface Preface a gt Every effort has been made 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 2 Safety Precautions Safety Precautions KI a gt 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 Safety instructions in this manual are accompanied by the following symbols to highlight their significance Please pay attention to the instructions highlighted by these symbols Serious bodily injury or death may occur by disregarding the WARNING instructions accompanying this warning symbol Snia Serious bodily injury or instrument damage may occur by disregarding the instructions accompanying this warning symbol Arane Intended use This system is designed as a Class 1 laser p
48. Put reflective chrome slide on stage Start Prairie View Enable Channel 1 in the Image Window Start TriggerSync oe Sy SS 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 6 Close uncaging shutter in the TriggerSync Mark Points window 7 Image the slide with imaging beam 8 Activate mark points screen Make sure image in mark points screen is updating 9 Manually toggle shutter on uncaging beam to burn small spot on chrome slide 10 Put red TriggerSync cursor over mark on chrome slide Do not use Nudge 11 Turn off Channel 1 and turn on Channel 3 12 Close the hard shutter in the Ultima scanhead 13 Open uncaging shutter in TriggerSync 14 Activate focus 15 Move uncaging spot to be coincident to red TriggerSync cursor 16 Close uncaging shutter in TriggerSync 17 Open the hard shutter in the Ultima scanhead 18 Turn off Channel 3 and turn on Channel 1 9 4 Calibrate Point Image Calibrate Point Image KAD Important Each objective lens will require its own unique calibration file 1 Confirm imaging beam shutter is closed 2 Start PrairieView 3 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 Turn off Channels 1 amp 2 Turn on Channe
49. after that at address 26C is the number of data points in the sample stored as a 4 byte long The rest of the file starting at address 270 is a listing of all of the data values in the following order Channel 1 Value 1 Channel 1 Value 2 Channel 1 Value N Channel 2 Value 1 Channel 2 Value 2 Channel 2 Value N Channel M Value 1 Channel M Value 2 Channel M Value N Each of these values is stored in a 32 bit IEEE single precision floating point format All values are stored as big endian 11 5 Point Calibration Point Calibration La AlL gt 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 Note 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
50. alculation Equations The equations for determining the desired pixel size or z step size are as follows For a 1 P laser Desired x and y pixel size in nanometers 0 61 A NA OS Desired z step size in nanometers 0 61 A NA OS Tt NA For a 2 P laser Desired x and y pixel size in nanometers 0 61 A sqrt 2 NA OS Desired z step size in nanometers 0 61 A sqrt 2 NA OS 11 NA Where A is the specified laser wavelength in nanometers NA is the numerical aperture of the current objective lens OS is the desired over sample value and sqrt 2 is the square root of two 10 4 1 4 Trigger Signals Trigger Signals 4a p The Output Trigger Type option in the Preferences menu has a pull right menu with two options available to the operator Start of Frame Trigger and or End of Frame Trigger End of Frame Trigger Trigger Mode Generate No Trigger e Generate Start of Frame OR End of Frame Trigger OR Neither Generate Start of Frame AND OR End of Frame Trigger OR Neither Preferences Menu Output Trigger Type submenu e Start of Frame Trigger The output trigger is the signal that will be used to trigger the start of the TriggerSync experiment during a T series So it is possible to trigger the experiment at either the start or end of a frame Depending on the triggering type selected triggering a cycle while Max Speed is checked may or may not be possible T
51. an Mode to choose AOD If the AOD is not connected or functioning this box will be grayed out Differences between galvo mode and AOD mode The Acousto Optic Deflector AOD mode is capable of scanning at a much higher frame rate 25 frames per second FPS than galvo mode Its speed makes it ideal for capturing momentary phenomena that galvanometers cannot such as dendritic spikes AOD mode also has a faster minimum dwell time 1 compared to galvo mode s 4 which reduces photobleaching However the optical Zoom image size and dwell time are more restricted due to the current limitations of AOD technology Working with AOD mode Frame rate FPS is determined by the image size dwell time and optical zoom settings Default AOD settings e 512 x 512 pixels e 1 dwell time e 1 0 optical zoom e 25 FPS Note Changes in any of these settings will cause Prairie View to automatically compensate by changing the others This keeps the AOD calibrated correctly To select one parameter to keep constant click the checkbox next to it Prairie View will not change that setting unless you uncheck the box or restart Prairie View Frame Rate Frame rate is calculated automatically based on the image size dwell time and optical zoom settings For faster frame rates set those parameters lower Image Size The number of pixels in the scan area Set higher for cleaner images lower for faster frame rates Optical Zoom This setting is separat
52. and a series of toggles to control table components and motor movement The Coarse Fine and Normal Alt toggles affect the movement of the motors when controlled with the knobs Coarse Fine will switch the motor between a coarse movement of approximately 31um per knob turn versus a fine movement of 1 95 um per knob turn For all axes the amount of motion for a single motor step is an ideal estimate The actual amount of motion will vary due to internal factors within the motor drive electronics inaccuracies and friction within the mechanical hardware The Normal Alt toggle 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 keeping the stage stationary but possibly introducing noise into the image signal Axis Coarse Mode Fine Mode X 312 5 um 19 5 um Y 312 5 um 19 5 um Z 31 25 um 1 95 um Table Motor controller resolution 6 10 Preamplifier Preamplifier all The Prairie Preamplifier receives the signal from the PMTs and amplifies it before sending it to the input channels on the 2090B box It is powered by the High Voltage Box and controlled by the computer via a USB cable The preamplifier can receive input on
53. ard shutter signal is BNC 2110 DIO 1 Prairie personnel ADC Configuration inalysis Senini MEg Use Shutter for Acquisition Set Shutter Filterhousing Shutter Laser Shutter Ctrl C BNC 2110 DIO 1 OTF BNC 2110 DIO 2 BNC 2110 DIO 1 amp DIO 2 ADC Configuration Continuous Pulse Setup Load Parameter Buttons Movement Finished Trigger Setup Set Amplifier Leak Subtraction Ctrl b Preferences Screen Shot Set Shutter Sub Menu Sets the viewing parameters for the input channels See ADC Configuration for more information Lola Do not change this unless instructed to do so by Continuous Pulse Setup TriggerSync can be set up to send out a continuous pulse whenever an experiment is not running This is usually used to ping an external amplifier ContinuousPulseOutputSetup 1 0 i DAC 0 Out z a 7 Cir Yr AG E El E Screen Shot Continuous Pulse Setup dialog Load Parameter Buttons The Load Parameters Button Assignment dialog allows the user to define up to six commonly used Experiment Parameter files to be shown on the main screen Screen Shot Load Parameters Button Assignment dialog The files are then accessible by selecting Load Parameters on the front page Screen Shot Load Parameters Movement Finished Trigger Setup Once used to send signals after controlled motor devices were completed in their motion to trigger the next step in a cycle
54. ards 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 be used The value to use for steps is dependent upon the type of pointing device being used For a galvanometer based pointing device steps 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 steps 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 current X and Y positions are shown in four fields below the blue buttons 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 These values are shown in mV or motor steps the same as the steps field 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 position
55. area of interest y Line Profile Beket lalm i Best Fit 4000 C Fixed 3000 4000 0 E 2000 oo l __ Log Scale 1000 Full Width Half Max 0 0 Line Width 0 10 20 30 40 50 60 70 80 86 92 aR Legend Chl Microns um eu Z Mode Selected Channel Intensity 1344 0 1240 0 996 0 Chi por Microns um 12 749 78 230 65 482 Screen Shot Line Profile Dialog 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 Selected Channel and click Calculate When MIP is active See maximum intensity projection 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 10 3 9 Photoactivation Photoactivation Lalal The Photoactivation PA tool enhances the functionality of a Z series acquisition or single scans by allowing the user to apply masks to scan areas controlling where laser power is applied These masks can be on specific z slices applied to an entire stack or a single scan To use the PA tool for a stack of images a Z series must first be acquired see Z Series tab While still in the playback window after acquiring a Z series select PA This will open a toolbar on the right side of t
56. 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 40 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 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 nonlinear 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 10 of 512 After these values have been set press the Calibrate button to begin the calibration 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
57. bined image electrical acquisition 17 In the TriggerSync main window select Acquisition gt Start Experiment gt TTL Trigger Start this action will remove the check mark in front of this menu option indicating that it has been turned off 14 7 Functional Map Acquisition Functional Map Acquisition 4a gt Start Prairie View Start TriggerSync In Prairie View use Live Scan or Single Scan to locate the area of interest Pan Rotate and Zoom to orient the target region Load the correct point calibration file for your scan parameters Open the TriggerSync Mark Points dialog Press Snap in TriggerSync to collect a reference image on which to mark your points Load the appropriate calibration file in the Uncager Calibration File field oN ee oe eS PS Using the Mapping Setup section of the Mark Points dialog create a grid of marked points Note 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 10 Open the Acquisition and DAC Output Setup dialog 11 Confirm or define stimulus control and acquisition parameters then close the dialog 12 Enable the desired ADC input channels on main TriggerSync window 13 Turn on the Functional Map acquisition mode by clicking on the Functional Mapping check box along the right hand side of the main TriggerSync window 14 Press Acquire in TriggerSync 15 Save the Functional Map
58. by 10 The image can continue to be increased by clicking Larger repeatedly Pixels Per Line amp Lines Per Frame p4 U J Screen Shot Scan Resolution Tools Custom The Pixels Per Line and Lines Per Frame functions allow the user to define a rectangular scan pattern where the number of rows of x is not equal to the number of columns in y 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 Pixels Per Line will set the number of pixels in a single scan line x The minimum value allowed is displayed to the left of the highlighted field Lines Per Frame sets the number of lines y per image 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 10 2 4 Dwell Time Per Pixel Dwell Time Per Pixel Alp cal 08 40 Screen Shot Dwell Time Per Pixel Controls 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 The current dwell time is shown to the right of the minimum Increasing pixels per line Zooming or rotating may increase the minimum dwell time possible Averaging Summing During data acquisition the underlying sampling rate is 1 sample per 0 4 us for a galvo imaging sy
59. can 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 ROls select ROI and place the cursor at one corner of the area of interest Click and drag the mouse creating a rectangular ROI Image Window 1 ov oO NO ae a LUT ilele ESSESI EZE Window 4 X 396 Y 317 2340 0000 0000 HQ Bicubic Screen Shot Defining a Region of Interest 86 92 um The Image Window will automatically resize itself Press 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 To exit out of the ROI and begin scanning the entire frame click ROI again Enter ROI Edit New ROIs Edit Existing ROIs Load Delete All Import From File Export to File Save Reference Image 4uto Save Auto Edit Cancel Exit ROT Save gt Import From File 4uto Save Auto Edit Cancel Screen Shot ROI Context Menu ROI Not Active left ROI Active right It is possible to right click on the ROI button or click the drop down button to the right of it to bring up a context menu The context menu varies based on whether or not an ROI is currently active Enter Exit ROI functions the same as clicking ROI It allows a new ROI to be defined or closes the current ROI Load displays a list of saved ROIs which can be loaded by cli
60. cent green areas Photoactivation Photo Bleaching 12 Set the Pockels cell power high such as 70 13 Zero all PMT HV 14 Create a new label with these current settings named Burn 15 Under the Z series tab uncheck Adjust PMT and Laser 16 Under the T series tab select Clear All 17 Select the Burn label in the Label Select drop down list and click Add Label 18 Click Add New 19 Select Current Settings or a saved name for the Z Series and PA columns 20 Select Start T Series to start photobleaching the defined mask regions 21 To view the results collect a Z Series under normal PMT and Pockels cell settings 15 Contact Us Contact Us 4a Shipping Address Mailing Address 3030 Laura Lane Suite 140 P O Box 620677 Middleton WI 53562 0677 USA Middleton WI 53562 0677 USA Phone Fax 608 662 0022 phone 608 662 0023 fax Internet Support support Prairie Technologies com www Prairie Technologies com
61. change and pressing the Generate Map Points button without having to redefine the bounding corners Functional Map Display Select Analysis gt Functional Mapping A dialog will show the data as collected Functional Mapping Screen Shot Functional Mapping dialog 12 LinescanViewer 12 1 Overview niahi KI a gt C200 BOD Cet OF 20 OF ERD aoo iaa di fe 1 1a El AEN y i E E s b Screen Shot Linescan Viewer Image Windows It is possible to look at acquired line scan data by opening Linescan Viewer on the Ultima computer desktop or through the Applications menu in PrairieView Under File find and open the line scan data of interest Linescan Viewer can also load a line scan automatically after it completes by checking Open channel in linescan viewer in PrairieView Linescan Viewer must be running for the check box to become enabled In 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 If electrophysiological data 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 13 Swept Field Confocal Prairie View 13 1 Main Window Main Window not The SFC control screen in PrairieView has many of the same control features as the Ultima screen The scanning elements s
62. cking on the desired region Selecting Save saves the current ROI as a new ROI or overwrites an existing saved ROI Delete displays a list of saved ROIs which can be deleted by clicking on the desired region Delete All deletes all saved ROls Import from File imports a set of saved ROIs from a file Export to File exports the current saved ROls to a file to be imported and reused at a later time Save Reference Image saves the Image window contents with an overlay of the saved ROIs marked on it 10 3 7 Snap Tool Snap Tool Llai The a 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 Prairie View Main Window If 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 10 3 8 Line Profile Line Profile The line profile tool displays a plot of intensity along a user defined line on each of the active channels Image Window 1 fl mi Lisi 0313 0000 0000 282 V 0 HQ Bicubic Screen Shot Line Profile Overlay 86 92 um 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
63. cted laser and power to it using the button The same button can be used to update the power setting from a laser already added to the track To remove a laser from a track press the button Any lasers not included in a track will be turned off Channel Mapping During each track there are four possible channels to be acquired multiply that by the four tracks and there are sixteen possible data sources which need to be mapped to the four data channels To do this each display channel has a source channel and source track associated with it For example channel one could get its data from channel one of track one and channel two could get its data from channel one also but channel one of track two 10 2 12 Z Series Tab Z Series Tab La AlL gt The Z series tab contains the controls used to collect stacks of images called Z Series or Z Stacks This tab allows the user to define start and stop positions step size or number of steps and necessary laser power adjustments In Prairie View 4 x this tab and others may be torn off and placed on the user s desktop Z Series Slice Data z Pockels PMT1 Gain PMT2 Gain 270 61 0 271 11 0 Z Series Calculator Start Position um Step Size um Wv Adiust PMT amp Laser 70 10 beme D51 Coene X Leave shutter open C At all XY stage locations Set Number Of Slices Middle Position um 14 00 C Calculate K ef 273 15 Laser Power G
64. d 0 L Closed Jo 0 e000 0 Define Step Size nm Define Number Of Steps Calculate y Leave shutter open Save Path C Documents and Settings prairie i Browse Series 09272010 1048 C So Screen Shot 2 P Laser Tab If there are multiple lasers controlled through Prairie View each can be adjusted separately by choosing the desired laser from the Laser Selection pull down menu Depending on the laser manufacturer the Main Power can be controlled along with the laser cavity Shutter The wavelength is controlled with the Wavelength Adjust slider The status of up to four lasers is shown in a table along with their wavelength and power output The Wavelength Series Calculator offers controls to vary the wavelength of tunable lasers There are four variables that must be set three by the user and one to be calculated by the software This control can be combined with a T series to image samples as they change with different wavelengths The shutter can be left open during this process or can be closed At the bottom of the tab box the save path and base directory for the wavelength series is shown Once the variables have been entered the experiment can be started by pressing Start W Series 2 P Laser Status Eror Code 0 No Faults Key Switch ON Yeevelength Tuning Status Ready Diode 1 Current amps 46 59 Diode 1 Temperature Celcius 21 1 Diode 1 Set Tempe
65. d location on image by dragging center point Resize and rotate grid by dragging corners Click Configure to set Mark Points parameters Click Prepare Click Acquire to mark points Note Information on marking points in TriggerSync can be found here 10 4 Menu Options 10 4 1 File Menu 10 4 1 1 File Menu Options File Menu Options 86 92um Llall Load Image s Ctrl O Load Configuration Save Configuration Load T Series Settings Save T Series Settings Load XY Stage Locations Save XY Stage Locations Exit Ctrl X Screen Shot File Menu Load Image s Loads saved image folders Load Configuration Loads a system configuration file recalling control settings of the laser PMT scan rate scan rotation etc Save Configuration Saves a system configuration file using the current settings of the laser PMT scan rate scan rotation etc Load T Series Settings Loads previously saved T series settings Save T Series Settings Saves the current T series settings to a file for recalling at a later time Load XY Stage Locations Loads previously saved XY stage locations Save XY Stage Locations Saves the current XY stage location Preferences Allows user to select options for post acquisition settings and T and Z series settings See Preferences Menu Exit Stops PrairieView and closes the program 10 4 1 2 Preferences Menu Preferences Menu KIAD Adjust Live display rate for better co
66. ds This parameter is part of a Label definition This allows the operator to create labels that use either just one laser or potentially both lasers This also allows the operator to control the routing of the second laser for use in uncaging experiments with TriggerSync e g First image using both lasers then via a label change the routing of the second laser then uncage with TriggerSync and so on Actions to Perform Actions previously defined in the Actions dialog for after frame or after scan events can be selected in this section For information about defining Actions read here Custom Outputs This section specifies custom waveforms which will be synchronized with each line of an acquisition A Custom Output Waveform gi Percent of Scan Line JE 5 10 25 5 125 7 e Ix 5 i J J I J D g I z 0 5 10 15 2 25 30 3 40 45 50 5 60 65 70 75 80 85 90 95 100 Tane wa a Foroen oi Scan Line Fea Peco Sea ne 0000 toad Saveds Access Cancei Screen Shot Custom Waveform Editor Custom outputs available are defined in the configuration utility similar to any other analog outputs Up to eight custom outputs can be defined which can be further limited by hardware i e device only supports two analog outputs Once defined it is possible to specify a waveform for a custom output by selecting the output in the list and pressing Edit Selected Waveform The custom outputs are synchron
67. e controller is the main communication device between the separate components Galvonometer Control Each set of galvonometers has a separate control box on the electronics rack See Galvos amp AOD for more information BNC Breakout Boxes The three BNC Breakout boxes associated with each system provide in out triggers for imaging and synchronization See NI Boards and Boxes for more information PMT High Voltage Power Supply Depending on the number of PMTs present on the system there will be an HV box for each pair See Detectors for more information Pockels Cell Controller For each modulated laser beam entering the Ultima there is a corresponding pockels cell and control box See Pockels Cell for more information Main Rack Power Switch All of the rack components are connected to a rack mounted grounded power strip 6 5 Detectors Detectors KI a gt 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 The Ultima uses externally mounted non descanned side on photomultiplier tube PMT detectors to collect the fluorescence emitted from the specimen The standard configuration for PMTs
68. e from the zoom in out feature in the image window Zooming in out in the image window does not change scan parameters Setting optical zoom to 4 0 allows the greatest range of image sizes and dwell times To get the full field of view make sure the plunger position inside the AOD matches the optical Zoom level If you do not Know what the plunger position is restart Prairie View The software will automatically detect the correct optical zoom All other parameters will reset to default Dwell Time The amount of time spent imaging each individual pixel Decrease to reduce photobleaching The minimum possible setting in AOD mode is 1 Each 1 change in dwell time increases decreases optical zoom by 1 0 e g 2 0 to 3 0 Additional Tips Increase image resolution by increasing either image size total number of pixels or dwell time amount of time spent imaging each pixel Note that changing either of these settings will cause the optical zoom and frame rate to change accordingly Improve image quality with the Average Every N Frames feature Because AOD mode scans so quickly it can average frames much faster than galvo mode 10 2 8 Pan amp Scan Rotation Controls Pan amp Scan Rotation Controls al Pan Control Screen Shot Panning and Scan Rotation Controls Pan Control 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 yell
69. e 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 TriggerSync window For example the third input ACH2 might be connected to a patch amplifier so the operator might enter a label such as mV in the Channel Units field for the third input 11 6 8 TriggerSync Preferences TriggerSync Preferences nao EditPreferences 1 0 i Normal Acquire to Memory TriggerSync Format X Screen Shot TriggerSync Preferences Select Data Acquisition Mode The default acquisition mode is Acquire to Memory This saves the data as it is created With very long acquisitions where saving the data to disk might create a lag over time Stream to Disk is the
70. e laser setting displayed in the Laser Power s column will include mW or if the label was created when a calibration file or the Attenuation mode was in effect The selected laser mode and laser setting will be retained for the Interlaced scan pattern The selected laser mode will also be reflected in the laser sliders for the setup dialog for the Interlaced scan pattern The selected laser mode and laser setting will be retained for the Photo Activation masks and settings The selected laser mode will be reflected in the laser sliders for the Photo Activation palette edit dialog As mentioned previously all optical components in the light path between the Pockels cell and the light meter placed to measure the light output at the objective lens will have an impact on the power delivered to the sample Therefore it is necessary to perform a calibration for at least each objective lens used on the system to take proper advantage of this calibration process Since the maximum sample power for a given calibration is limited to the lowest measured power through the calibration wavelength range the operator might wish to make several calibrations for a given objective lens at various wavelength ranges If a laser control is configured to use a calibration file and there is a 2 P laser integrated into Prairie View and associated with the particular laser control if the operator attempts to tune the laser to a wavelength
71. e 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 Mark is selected a box appears on the image in the active Image Window and a second Mark Points window appears as shown below To select points click on the appropriate option in the Mark Points window and follow the steps described below Because this window is an interface between PrairieView and TriggerSync it is necessary to set the Mark Points parameters using a Mark Points Wizard which is accessed by clicking Configure Configure replaces many of the controls formerly accessed from the Mark Points dialog in TriggerSync To Ma 1 2 3 4 5 o Ma T 1 2 3 4 5 6 o Ma T o or a w Noa T oO 2 a ie ke 4 oto m wo 2 PS 4X 350 7 463 0029 0000 0000 HQ Bicubic Screen Shot Mark Points Dialog rk a Point Click Point Move point to desired location on image Click Configure to set Mark Points parameters Click Prepare Click Acquire to mark points rk a Line of Points Click Line Enter number of points in the X Point Density field Move line to desired location on image Click Configure to set Mark Points parameters Click Prepare Click Acquire to mark points rk a Grid of Points Click Grid Enter number of points in X and Y Point Density fields Move grid to desire
72. ea that you wish to perform the uncaging in is composed of 2Nx 2Ny pixels the position of the scanner X i Y i at the ith uncaging i 0 1 4 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 i 0 4 2 6 1 5 3 7 when Nx 8 In the statements listed above mod is 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 The following table shows an example of the order of uncaging positions where Nx 8 Ny 4 and Px i 0 4 2 6 1 5 3 7 X Pixel TPP EP PEP PPEPEPE ddd did didlo lad dled al lad PPE PEEP EE PEP EEEE rE ddd PPP PEPE PEP PIPE PF RPP PPP EE FEE PEPE PEPE Table Non Neighbor Order of Uncaging Example Option C Random The order that the marked points will be generated for the grid will be random After selecting Map Method the operator must select Generate Map Points It is only when this button is pressed that the actual marked points for the defined grid will be generated Once Generate Map Points is pressed Show
73. ed 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 Define 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 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 s
74. em measurements have been made and the laser power calibration procedure completed the operator will have the ability to specify the desired laser power to be delivered to the sample in mW units In addition once the laser power setting has been made if the operator changes the wavelength of the 2 P laser the drive voltage to the Pockels cell will be automatically adjusted to maintain a constant laser power to be delivered to the sample Warning The proper implementation of the Laser Power Calibration is dependent upon the initial Pockels cell installation as well as software during installation Any adjustments to the light path after the setup and calibration s have been performed would most likely result in the necessity of having the calibrations performed again a Laser Power Calibration To calbeate a laser select it in the drop Gown above and enter constants 10 the right if not siready filled in 1 C ck the New button below to create a new calibration 2 Choose a name which will identify when it should be used Maximum Power Setting 1 3 Speoty a range of wavelengths to cover as well as how many Gata points steps should be collected then press the Start button Wavelength 2 nm po G Enter power valses for each wavelength as prompted n f 5 i the rah tote arama E pa Maximum Power Setting 2 Selected Calibration Range fren Max Power mW Name EnterCalibration jon Namel Minimum Wavelength nm Ma
75. enu Options 4a gt TriggerSync 141 O O OOOO O O File Acquisition View Points Analysis Configuration MEJ Online Help kor TriggerSync 1 4 1 Screen Shot Help Menu Show Help This option opens a small text box which shows information about controls in TriggerSync when the user hovers over a control with the mouse Online Help for TriggerSync 1 4 1 Currently there is not an online help option for TriggerSync 11 7 Mark Points 11 7 1 Marking Points Marking Points al In the main window of TriggerSync select the menu Points gt Mark Points This will bring up the Mark Points dialog which is used to create a list of locations for performing uncaging experiments Many of these controls can now be found on the Image Window by selecting Mark if gt Mark Points Interface 4 Uncager amp amp p o00000 amp p 000000 4 Large Screen Shot 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 The blue cursor indicates the current position of the selected pointing device The placement of the blue cursor will change automatically in response to changes in the physical position of the selected pointing device Do not move the blue curso
76. er amp HV Control Unit in the instrument rack Clicking Zero at the end of a PMT HV slider sets that PMT HV to zero Preamplifier Q ai Chi Offset Ch2 Gain Filter lt i Screen Shot Preamp Controls To modify a preamplifier setting first use the Channel Selection pull down box to select the channel to change the control setting for Then use the Gain and Offset controls to modify the desired parameter The Filter Bank control affects collection rate of the channel Note When you change the Filter Bank value for one channel it will change the filter bank selection for all channels Screen Shot DAQ Controls DAQ is an abbreviation for data acquisition which samples the photo electric signal from the PMT to generate an image that can be manipulated by the data acquisition board the NI DAQ 6110 board The function of DAQ is to scale the amplified photo electric signal to match the acceptable input range of the data acquisition board for maximum image resolution The acceptable input range for the data acquisition board is fixed from 10v to 10v Because the dynamic photon signal from the preamplifier may be greater or less than the selected range adjusting the DAQs the dynamic photon signal will be scaled to the data acquisition board s range to achieve best image resolution DAQs values are preset in drop down list To change the DAQs click the drop down arrow and select the value from the dropdown list The higher t
77. er the current operating parameters would allow this feature to be used When this feature is not available will be displayed instead of a check box It will not be available in these conditions TriggerSync is not running The Output Trigger Type is set to Start of Frame Trigger and Max Speed is not checked A Z Series is selected TriggerSync Image is where the operator must specify the desired image number within the image sequence to start the TriggerSync experiment This value will be processed such that it can never be less than 1 and it can not be greater than the number of images in the image sequence Any attempt to enter a number outside of this range will result in the program automatically setting it to the nearest legal value when lt Enter gt is hit When TriggerSync Experiment is not checked or not allowed the number entered as the TriggerSync Image is not applicable 10 2 14 2 P Laser Tab 2 P Laser Tab nao The 2P Laser tab provides options for control of the laser s used by the Ultima system PrairieView can control up to four 2P lasers Laser PMT DAQ Z Series T Series 2P Laser bec Labels Misc ei tan j aa 1 0o H i 2 a90 oa 898 0 a Zl eo TER Series Calculator Start Wavelength nm Sa nm EJ Start EJ 900 0 0 Define Se Wavelength nm Set 5 ENJ ENJ Laser Selection Shaker n w Exciter Open 820 3027 Closed I0 0 Close
78. es 10 5 Multiple Users Multiple Users Alp By creating separate user profiles in Windows PrairieView can keep track of usage and settings for multiple users Monitoring Usage The file PrairieView log keeps track of several functions and actions taken while a user is in Prairie View Lines are added to the log when these certain events occur Starting and stopping Prairie View Execution of an acquisition Editing of the Scan Settings dialog The date time user name and in the case of an acquisition file location are shown for each line of action recorded in the log User Specific Configuration By default a number of settings a saved at the user level including labels Other settings will retain the same values as the last time Prairie View was run It is possible to customize which settings are user specific and which are not in the CustomUserKeys txt file found in the Configuration Files folder 11 TriggerSync Software 11 1 Overview Overview cl a D 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 TriggerSync 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 if TriggerSync 1 3 3 2 File Ac
79. es 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 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 ACH1 etc from left to right By left clicking the mouse on 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 11 4 Acquisition amp DAC Output Setup Acquisition amp DAC Output Setup noo The Acquisition and DAC Output Setup dialog controls the parameters for the available stimulus outputs on the Ultima system Depending on the configuration of a system there may be between two and eight outputs available for the user to define It is accessed through a button on the main TriggerSync window While this dialog is open the operator may not adjust any parameters in TriggerSync or any other TriggerSync dialog that is not part of this dialog The Mark Points dialog will automatically be closed when Acquisition and DAC Outpu
80. ess the Update Selected button and the group definition will be changed By clicking on the checkbox next to the group number the operator can alter what region graphs are displayed Located below the Region Groups is a set of controls for Regions Functions This section allows the operator to create mathematical graphs that are mathematical manipulations of the data from one or more defined region This is in addition to the predefined mathematical options presented by right clicking the mouse over a region definition as outlined above in section m In addition these functions will display as new graph data To create a function click on the Create Function button After you click on the Create Function button it will turn green showing that this mode is activated if you want to exit this mode without actually entering a formula or equation simply click on the Create Function button again This will activate a control just below the buttons that allows the operator to select from a list of pre defined mathematical functions e g f f0 Also activated is a control just below the list of functions that allows the operator to modify the pre defined equation or to enter an equation of their own choosing Located just below the control field where the equation is shown is a string shown in blue that reads Need help with function syntax If you click on this string a help dialog will appear
81. f the box is a dial This dial is only to be used if the control for that PMT is set to Manual All HV boxes are factory set to Computer Control to be used in conjunction with the Prairie View software The buttons located directly below the LED displays are only 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 re calibration is difficult and time consuming The PMT HV control unit has overload protection circuitry designed to prevent damage to the PMTs caused by exposure to an overly bright light source First is the photodiode that is located in the PMT HV box When the Sensor toggle is in the down position the PMTs will be set to 0 V if 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 The second protection is built in to the signal detection electronics The switch in the center of the unit labeled Protect Override is used to either enable this feature Protect mode or disable it Override mode In Protect mode when an excessively bright signal is detected by the PMT the high voltage is set to 0 V Note The LED display calibration buttons located directly below the LED displays should not be used These controls are only used to calibrate the LED displays They are factory set to display the c
82. f the image containing the current image data for the channel specified lt outfile gt which replaced by the path and filename of a temporary file created to pass point data from the program or script specified into Prairie View The format of the output file is a comma delimited list of points that make up the path to line scan Each point is made up of an X and Y coordinate again separated by acomma that range from 0 0 0 0 upper left corner to 1 0 1 0 lower right corner Photo Activation Mask Generation These actions are run manually from the image window when the photo activation mask editor is enabled and are used to automatically generate photo activation masks based on the current image data There are two tokens available for use in the arguments field lt image ch gt where is replaced by a channel number 1 4 This token will be replaced by the path and filename of the image containing the current image data for the channel specified lt outfile gt which replaced by the path and filename of a temporary file created to pass mask data from the program or script specified into Prairie View The format of the output file is a comma delimited list of vertices that make up polygon based regions Each vertex is made up of an X and Y coordinate again separated by a comma that range from 0 0 0 0 upper left corner to 1 0 1 0 lower right corner Each polygon is terminated by the palette number the polygon should use whe
83. f times the cycle is to be repeated The user can set the Period as the length of time to complete each cycle rep When max speed is checked the period is automatically set to the duration of the cycle When Max Speed is checked images are acquired continuously with no live update of the Image Window Max Speed is available for z series only when piezo control is available To save time in the processing of the images the image windows are not updated during a Max Speed acquisition Duration is the minimum amount of time necessary to acquire the image s in all of the repetitions as calculated by Prairie View By selecting a Z Series the user can choose a z series instead of a single image during a cycle To create a Z series see Z Series Tab The user can also add photoactivation masks or brightness over time measurement to pre defined slices or regions by selecting the Photoactivation or BOT respectively See Photoactivation and BOT The Cycle Completion function is not used at this time Add New places a new basic cycle at the end of the series and Insert New will insert a new basic cycle previous to the selected line Clear All will clear all T series cycles from the list while Remove Selected will only remove the selected line s from a T series The XY Stage ROI Label Select options can be selected and will apply to all subsequent relevant lines in the T series until changed See XY Stage Tab Region of Interest and Labels Tab The Save
84. ften 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 Protocols I V Current Voltage il i i i aieiaiee aa a a a a a i a a a a a i aLe a Le a Le a Le i bibl bili il i 3 a z F z r F E r E E z E F E r E F F F E rn F F E F z F F r a z il i laie Lat ae de a i a i i i i i i a e bili bill Lat ad ee had at i ad al a a L ili Screen Shot I V Setup When the I V 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 mV This field is used when running an acquisition that has more than one cycle If the increment is any value other than 0 then after the first cycle the voltage for the second and any subsequent cycle for this epoch will be incremented or decremented if a negative value is specified by this amount Ramp Screen Shot 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 start of the ramp Epoch B
85. g of data import and export See Actions Reset Dodt Controller If there is a Dodt detector present on the system this selection will reset the connection with Dodt controller in case of a connection failure Reset MAMC Device If there is a Multi Axis Motor Controller MAMC present on the system this option will reset the connection in the event of a failure Reset Preamplifier If the preamplifier is configured to run through Prairie View instead of its own Preamplifier Utility selecting Reset Preamplifier resets the connection with the preamplifier in case of a connection failure Scan Settings Brings up a panel of settings for the scan parameters of the system These settings should only be changed by Prairie personnel These settings are not intended for operator use 10 4 2 2 Calibrate Objective Lens Calibrate Objective Lens KAD To correctly use the measurement tools and have accurate field of view calculations in the metadata the objective lenses used need to be calibrated 7A Objective Calibration g OO a mst be peoe wih s 512x S12 image size ond We optical zoom seo T Objective Name Power NA alco edan X Microns Pixel Y Microns Pixel Calibrated 1 Olympus 40x Wate 40 08 107 186 101 186 0 19763 0 19763 True 2 Olympus 60x Wate 60 0 9 225 882 225 882 0 44118 0 44118 True Add New Objective Enter calibration distance here um FOO SA _ Remove Selected Obpective Calibrate Selected Objective Screen S
86. ge location based on current stage location It will also update all other stage locations with an offset equal to the difference between the first selected and shifted stage location Move to Selected Next Previous moves the stage to the indicated location in the saved locations list Goto Position is a drop down list allowing user to select a desired position to move the stage Load XY Stage Locations loads a previously saved stage location lists Save XY Stage Locations saves the current list of XY stage locations for later use 10 2 17 Misc Tab Misc Tab KAD The Misc tab contains controls for several different features of the PrairieView software Many of the features are used to control optional features on the Ultima This screen may contain fewer modules depending on the configuration of the Ultima Laser PMT DAQ Z Series T Series 2 P Laser XY Stage Labels Misc Photoactivation Mask None _ Single scan auto save C Documents and Settings prairie test Singlelmage 09272010 1048 439d Dodt Detector KE Background Image Miror out Record Background Image Filter 7 O Subtract 100 E of background image Cater Sean None 3 Screen Shot Misc Tab Single Scan Photoactivation Mask Specifies a photo activation mask to be used for all scans as defined in the photoactivation mask editor Single scan auto save When enabled the single scan file iteration is automatically incremented after a sing
87. ge on PFIO TRIG1 6 8 Device Control Box Device Control Box 4 l a gt The Device Control Box contains power supplies motor drivers and the logic for responding to commands from the RS 232 computer input or the DCRI Cables from the back of the Device Control Box go to many of the other boxes on the electronics rack 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 rightmost 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 6 9 Device Control Remote Interface DCRI Device Control Remote Interface DCRI 4a To provide easy access to x y and z motor control Prairie provides a Device Control Remote Interface DCRI box The motor system consists of the Prairie Device Control Box the DCRI an RS 232 cable the motorized stage and a z motor The DCRI box has a knob for each motor x y and z
88. grid will be created Screen Shot Show Map Boundary 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 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 ar
89. h Point Marked 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 will be the point number Save Cursor Locations A dat file will be created which will contain two columns of numbers The first column represents the x cursor position and the second column represents the y cursor position for each marked point The operator may select any combination of these options After selecting the desired options press Cancel to exit the dialog or press OK to save the desired data If OK is pressed the operator will be prompted to select a directory and base filename to use for saving the desired data 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 Screen Shot Line Definition To create a line of marked points place the red cursor in the image window where you wish to start the line and press Mark 1st EndPoint Next place the red cursor in the image window where you wish to end the line and press Mark 2nd EndPoint To specify the number of points you wish to create on a line including the two endpoints type or use the arrow controls to select the desired number in the Nu
90. hannel to Subtract From These controls will work either on a static image acquired with Single Scan or while in Live Scan mode This altered image cannot be saved For example there is bleed through of Channel 1 into Channel 2 The goal is to subtract out the Channel 1 signal from Channel 2 and add it back to Channel 1 Place a check in the box under the Enable column For the Source Channel select Channel 1 For the Channel to Subtract From select Channel 2 a Noe While either live scanning or after having acquired the images on Channels 1 and 2 with a Single Scan slowly move the Percentage control up The signal from Channel 1 that is present in Channel 2 should diminish At the same time the signal in Channel 1 should increase by the same amount as this data is recovered into Channel 1 One idea on how to do this visually so that not too much signal is subtracted from Channel 2 is to place Channel 2 into Range check color mode and then as the Percentage slider is increased some blue or zero intensity pixels will show in the area where Channel 1 was bleeding into Channel 2 This shows that the right amount of Channel 1 has been subtracted from Channel 2 10 4 2 5 Actions Actions KAD Actions are external programs or scripts used to automate repetitive tasks There are two types of actions actions and rules Actions simply perform a task rules determine another action to perform based on some criteria Actions
91. he DAQs the narrower the input range and the higher the image contrast Note Adjusting DAQs does NOT change the signal from the detector and preamplifier Setting the DAQs to an in compatible range may result in real signal being lost 10 2 11 Interlaced Scanning Interlaced Scanning 4a 79 interlaced Scan Pattern Setup n rterlaced scan pattem aquires data in up lo four racks each defining a subset of lasers and laser powers Each track is aquired one after another line at a time scanning this wey provides a meens to scan with mulbple laser ines eliminate cross tak and preserve temporal resolution STEP 1 Select a laser to include in one or more tracks along and adjust power as needed By default the power wt use the ms cuert power as defined by the sidar on the r3 Laos Power manfo To use a spec ic power uncheck LUAH Pockels 5 the check box and move the slider STEP 2 Enable one or more tracks and add x the previously selected laser using the add laser buon To remove an unwanted laser select it and press the remove laser button STEP 3 Select which data to display in each of the four channels The data can come from arty channel on acy track Accept the dalog to begin using the rtedaced scanning patem defined Screen Shot Interlaced Scanning Setup Dialog Interlaced scan patterns allow acquisition of frames using up to four different independent laser settings alternating each line fo
92. he Image Window By using the scroll buttons at the bottom of the window or cursor keys to change frames and the buttons on the right to edit the masks 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 below When all masks are defined disable the PA mask editing mode Click PA To use the PA tool for a single image click PA at any time except when in playback mode for a Z series Define a single slice PA mask as you would a set of masks for a Z series To use the mask it must be selected on the Misc tab Image Window 1 oul X 459 7 219 0149 0000 0000 HQ Bicubic 86 92 um Screen Shot Photo Activation Dialog To acquire a Z series using these masks it will be necessary to set up a T series being sure to select a set of masks in the PA column It is important to make sure Save images generated when Photo Activation PA is used is also checked in the Preferences menu Following acquisition the playback window will again open for review of the masked regions image data Enhanced Photoactivation Features For systems configured for interlaced scanning photoactivation masks are no longer simply on off states Laser powers can be set independently to different values for different masks within the same scan area When the photoactivation controls are active the
93. he Ultima is a unique laser scanning microscopy instrument that is capable of using one or two laser beams for simultaneous imaging or uncaging experiments on in vivo and in vitro specimens With one laser it is possible to perform traditional raster scanned laser imaging and 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 record electrophysiological signals from the specimen through the use of the TriggerSync software The Ultima scanhead can be integrated with your choice of OLM bases The base can be either an upright or inverted OLM This style of Ultima is best used for slice work or very small mice There are two different styles of Ultima Vs The standard is a Fixed Post IV but the Ultima V can also be installed as a Moving V In this case the regular In Vivo scope is placed on an x y motorized platform This is useful for times when the specimen is required to stay stationary ell pi Photo Ultima system with light box and beam cover 5 Table Components 5 1 Lasers Lasers KI D One of the major components of a multi photon microscopy system is the laser The laser used to create dual photon excitation has a range from 700 1000 nm or greater For Ultima Systems with the AOD option Prairie Technologies strongly recommends using a laser with pre co
94. he acquisition and stimulus control output signals will happen and after the acquisition and stimulus control has completed the shutter will be closed Open After Starting Data Collection Close After Open Shutter Time milliseconds Start Stimulus Control with Data Collection 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 Open After Starting Data Collection Close After Open Shutter Time milliseconds Start Stimulus Control after Shutter is Opened 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 specifies the amount of time in milliseconds that the shutter should be opened based upon the setting for the Open Close Shutter control Stimulus Control Depending upon the system configuration the operator has the ability to program 2 through 8 analog output signals The ability to edit these signals begins in the Stimulus Control box Screen Shot Stimulus Control DAC Out Select To program an analog output first select the desired output using the DAC Out Select control
95. he 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 At any time the operator may press Clear Corners 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 axes Place the red cursor in the image window at the location of the desired upper left hand corner Press Mark First Corner 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 Press Mark Second Corner a e OON 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 6 Press Mark Third Corner Once the three corners have been defined Show Map Boundary 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
96. he necessary wiring for this feature is as follows On BNC 2110 connected to PCI 6713 insert a wire jumper between PFI8 and USER in the push connector Add a wire jumper between PFI8 on BNC 2090A connected to PCI 6052 and CTR1OUT on BNC 2110 connected to PCI 6713 in the push connectors Add a BNC cable between USER1 BNC connection on BNC 2090B connected to PCI 611x and USER BNC on BNC 2110 connected to PCI 6713 10 4 1 5 T Series Preferences T Series Preferences KI a gt A T Series Preferences Jog 1 T Series Iterations 2 XY Stage Locations 3 Cycles TriagerSyne Execution Start next cycle immediately Screen Shot T Series Preferences The top of the dialog allows time format used on the T Series Tab to be customized from milliseconds to hours The format of the selected time periods will be shown above the selection T Series Execution Order Determines whether to do all iterations at a given XY stage location before proceeding to the next XY stage location 1 T Series Iterations 2 XY Stage Locations 3 Cycles or to perform each iteration at all XY stage locations before proceeding to the next iteration 1 XY Stage Location 2 T Series Iterations 3 Cycles TriggerSync Execution TriggerSync experiments can be executed two different ways either the software will wait for the entire experiment to finish before the next cycle starts or
97. hot Objective Calibration Objectives delivered with the system are calibrated upon installation Any other objectives purchased later will have to have their own calibration performed A marked calibration slide is necessary to complete this calibration The calibration steps are as follows 1 Set up the imaging parameters to 512x512 resolution and set the zoom to 1x 2 Move the objective lens to be calibrated into position on the microscope 3 Focus on a slide containing an object of known width a calibration slide is recommended for this purpose 4 Select Tools gt Calibrate Objective Lens 5 The dialog shown above will appear 6 Select Add New Objective 7 Highlight the new objective and fill in the objective name power and NA 8 Click and drag a straight horizontal line from the far left side of the screen to the far right Click to end line 9 Enter the distance indicated by the line drawn The longer the calibration distance the more accurate the calibration will be 10 Select Calibrate Selected Objective The FOV Width and Height field will now contain values and the Calibrated field should say True 11 Close this window The newly calibrated objective lens will appear in the Objective Lens pull down list on the main screen 10 4 2 3 Calibrate Laser Power Calibrate Laser Power cl a gt Laser Power Calibration extends the operating capabilities of the Prairie Technologies Ultima system Once the proper syst
98. hould be found in the same directory as PrairieView exe and add any number of MATLAB commands in double quotation marks in the arguments field For example to display channel one in MATLAB after each frame the arguments would be something like image imread lt image ch1 gt imagesc image colormap gray axis square axis off It is also possible to reference M files by changing to the directory where the M file is found For example if the command in the previous example were found in C Showlmage m then the arguments would be something like cd c Showlmage lt image ch1 gt Once run PrairieMLL exe continues to run until explicitly terminated This allows it to continue to use the same MATLAB command window thus saving the time required to load a new command window each time as well as preserving the state in the command window allowing variables to be saved and referenced again between actions To terminate PrairieMLL exe pass x as an argument Once the x argument is reached PrairieMLL exe will stop running any other commands passed after the x and will close the MATLAB command window it was using Calling PrairieMLL exe again afterwards will create a new MATLAB command window and execute commands normally 10 4 3 Window Menu 10 4 3 1 Window Menu Options Window Menu Options 4a gt New Image Window Screen Shot Window Menu New Image Window opens a new Image Window It functions the same
99. hould be within 30 The Pockels cell receives its signal from the Conoptics control box by two BNC cables while the Conoptics box receives commands from the Device Controller via a BNC connected to input J3 To check the baseline voltage of the Conoptics box Align a bright sample in the field of view and set up scope for 2 photon imaging Set Pockels cell power to 0 in Prairie View Begin Livescan 1 2 3 4 Set channel imaging color to Range Check 5 Bring up PMT voltage until a light image of the sample can be discerned 6 Adjust the black knob on the front of the Conoptics box to minimize the brightness of the sample It may be necessary to increase the PMT voltage again 7 Note the number displayed on the Conoptics box LED for the wavelength you are using If you find that you have to adjust the Conoptics box regularly or the base line number seems to be changing drastically please contact Prairie 6 Hardware and Electronics 6 1 Microscope Microscope 4a gt The Ultima Multiphoton Microscopy System is built around a modified upright microscope It can also be modified for use on an inverted microscope as well The manuals associated with this microscope have been provided to the user Please refer to these manuals for specific information about the microscope components The Ultima n Vivo System is built on a fixed or moveable pillar platform There is no standard transmitted light path or option for Dodt Gradien
100. ication After Frame Complete These actions are run automatically after a frame is acquired with the exception of max speed acquisitions In order for an after frame complete action to be performed it must first be specified on the Misc tab of the main window The only token available for use in the arguments field is lt image ch gt where is replaced by a channel number 1 4 This token will be replaced by the path and filename of the image containing the channel data for the frame that just completed C ee Newton Action Rule Scan Type Action to Perform p Single 7 Process Image y None T Screen Shot Editing an After Frame Complete Action The screen shot above is an example of a rule which will perform the Process Image action for a single scan but do nothing otherwise After Scan Complete Playback These actions are run automatically after a scan has completed In order for an after scan complete action to be performed it must first be specified on the Misc tab of the main window After scan complete actions are also available to be performed manually in playback mode from the image window Rule type actions are disallowed in this context The only token available for use in the arguments field is lt metadata gt This token will be replaced by the path and filename of the metadata file associated with the scan which has just completed or the scan which is currently open in playback mode The metadata file i
101. iew for the region of the image outside of any photo activation masks If the Custom option is selected then Edit is active and this will allow the operator to set the laser selection and power setting exactly how it is done for a Palette The difference being that this applies to the region of the image outside of any photo activation masks 10 3 10 Playback Playback Al After a multi frame acquisition completes or a set of images is loaded from the file menu PrairieView enters playback mode indicated by the playback controls visible on the first image window Playback Controls x aR B Dataset 1 ml lt i gt mi ganom as m Loop lv Show Overlay ivi O Wrap test Brightness Over Time 092720 10 1048 02 1 Brightness Over al 1 Elly Time 09272010 1048 021 xml v C Documents and Settings prairie A Screen Shot Playback Controls Note There is an option in the Preferences Menu to stop PrairieView from automatically entering playback mode after a multi frame acquisition When in playback mode many controls associated with scanning become disabled until the playback mode is disabled by selecting Exit Navigating Frames Depending on the sequence selected there are two scroll bars available one vertical for Z stack data and one horizontal for time lapse data If the number of frames is the same for all sequences in the open data set then both scrolls bars become active The vertical scroll
102. ified axis For the horizontal scale units the operator may select from Seconds or Frames The vertical axis is set to display intensity values Located below the horizontal and vertical scale controls Show Average For allows the operator to choose how many frames to average for each data point Save Changes will save the current regions definitions and data the original data set Save Changes As will create a new set of files with the current regions definitions data and a screenshot of the current image window in the same directory as the original file This includes creating a new xml file that is associated with the defined regions so different sets of regions definitions and data can be recalled for the same data set Region Options There are several different shapes that can be used to create BOT regions Add New allows the operator to draw a region in the selected shape onto the Image Using the mouse the operator can add up to 32 regions for analysis There are four different shapes available to create the regions Ellipse Rectangle Polygon and Brush The Brush option offers six different sizes for free hand region creation Tiny Small Medium Large Huge and Custom With Custom a user created shape can be imported to act as a brush shape If there is more than one channel active on the image window when a region is created an identical region is created for each active channel 1564 0000 0000 HQ Bicubic 86 92 um i
103. ill in the various fields in the Constants section as outlined above W N Press New 4 Enter a name for the calibration file in the Name field of the Selected Calibration Field The calibration name should reflect the objective lens being used as well as the range of wavelengths that will be used in the calibration Enter the Minimum Wavelength nm Maximum Wavelength nm and Step Size nm values to be used for the calibration Press Start If 2 P laser control is integrated for the selected laser the software will automatically tune the laser to the Minimum Wavelength value for the calibration If a 2 P laser control is not integrated for the selected laser the operator must manually tune the laser to the Minimum Wavelength value for the calibration 7 After the laser has finished tuning wait a couple of seconds for the reading on the power meter to stabilize and then enter the power meter reading in milliwatts in the Power field and hit Enter The entered value will now appear in the calibration table across from the current 2 P laser wavelength 8 After hitting Enter if 2 P laser control is integrated for the selected laser the software will automatically change the laser wavelength to the next value in the calibration table If a 2 P laser control is not integrated then the operator will need to manually tune the laser to the appropriate wavelength 9 Repeat last step until the calibration has been completed 10 Press Accept
104. imes for a given sequence of frames there is no data for certain label channel combinations so the image for that channel will remain unchanged Metadata CurrentS ettings 1 CurrentS ettings 1 CurrentS ettings 1 C Documents and Settings prairie test BrightnessO verT ime 0927201 0 1048 021 BrightnessO verT ime 09272010 1048 021 xml CurrentS ettings 1 Sequence 1 a Frame 1 CurrentS ettings Channel 1 Frame 2 Frame 3 Frame 4 Frame 5 Frame 6 Frame 7 Frame 8 Frame 9 Frame 10 Frame 11 l Frame 12 l Frame 13 Frame 14 Frame 15 Frame 16 Frame 17 Frame 18 x Sequence Absolute Start Time 1 5901 s Prairie View Yersion 4 0 0 50 Frame Relative Start Time 0 0000 s System Type Ultima Galvo Mode Pixels per Line 512 Lines per Frame 512 Objective Magnification 40 00 Binning Mode Averaging Frame averaging 0 Frame period 1441 792 ms Scanline period 2 816 ms Optical Zoom 1 00 x Microns per Pixel 0 5773 microns Y Microns per Pixel 0 5773 microns Field of View FOV 295 588 295 588 microns Scan Rotation 0 Position x 1546 24 y 3309 39 2 280 10 Laser 1 Wavelength 820 at 3061 0 milliwatts Label CurrentS ettings Ad Automatically show when entering playback mode V Screen Shot Metadata Window 10 3 11 Maximum Intensity P
105. ion of the data This includes analysis of the most recently acquired data as well as analysis of previously acquired and saved data When Analyze Acquired Data is On several menu options are disabled 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 wnen Analyze Acquired Data mode is On some additional controls will become visible in the main TriggerSync window 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 channel s 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 for the selected input channel Screen Shot View Channel Sensitivity 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 Scale Scale Screen Shot Change Scale and Auto Scale When Change Scale is selected 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
106. is faum ts applied to the sack see to mirate the range oF seag i the Tapat araa Saame maon fies ein aut fasa sadist doi BPE Arsin if Fy aes te Aces aes Step Ss Living the thus battens aber nove the Tapt bo Ue pir lets Darl carter ef ihe saioe Dy net hase the soot touch of atomi besoul the edgy of tha mage Step fn Lisirsg the Thay battens mbas mens the port te the sparar ott bond oarra of thee nizae Co yet aie the oe Paiki of pateri havurd the adge ef the mae Rep Fe Uber tas Whe MAG abaa hae thes Spt te Ut hese right heed os af the binaga Wo mot have the spot touch o evtend beyond tha migo of the image Bless th Sir fours the Taies Numba of Prats Pr temtage o Points te plasan the monie roghi Syrareaoy af maskis wee of sean ares that b roredhayr Th Done Esc Screen Shot Point Calibration Dialog 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 For most systems the only device 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 Pi
107. ized with each acquisition adapting to the current line period Waveforms are defined as a value for a certain percentage of the line period 100 of the line period is one line so such a waveform would occur once every line 50 would occur twice every line and 200 would occur once every two lines Limitations are put on the total percent such that it divides evenly into 100 or is an multiple of 100 such as 200 so that all custom outputs can share a common clock So it is not possible to define a waveform that occupies 75 of the line period but it is possible to define a waveform that occupies 300 of the line period and simply repeats the 75 waveform four times Custom outputs can also share the same physical output line as a laser In such cases when the custom output is enabled it overrides the laser control Disabling the custom output will return control to the laser slider 10 2 18 Scan Information Scan Information 4a gt At the bottom of the Prairie View Main Window status bars to show basic information about the scan being performed Scanline Period ms 2 696 Frame Period ms 1380 352 Framerate fps 0 724 FOV 512x 512 1 0 x 1 0um Pixel Size 0 002 x 0 002um X 000 Y 000 2 00 TriggerSync message received EXIT Screen Shot Scan Information The first line displays the Scanline Period Frame Period and Framerate The scanline period is the number of milliseconds required to scan across one line in the frame The frame
108. king the box to the left of the region number Each region number is shown with a color background representative of the graph line created for that region Channel Denotes which channel the region pertains to Position This number is representative of the top left hand corner of the region For ellipses polygons and brush shapes this is the top left location of the bounding box that would encompass the shape Width The x value of the rectangle or bounding box Height The y value of the rectangle or bounding box Area The complete area covered by the shape not the bounding box Rotation If a region has been rotated the degrees of rotation from the original position will be noted Shape Denotes the shape tool used to create the region In the right hand corner of the table the operator can choose to display the values for the regions in either Pixels or Microns When Microns is chosen the computations are based on the currently selected objective lens If the operator clicks the right mouse button over any of the defined regions in the region table a pop up menu will appear that will allow the operator to very quickly set up some basic region math to modify the analyzed shape This could be simply specifying that region 2 should be subtracted from region 1 or possibly subtract region 2 from all other regions When a mathematical relationship is defined via the pop up menu the region name will indicate the relationship If the relati
109. l 3 Set to 4ms line Open Uncaging laser shutter in TriggerSync o o oe Perform Point Calibration as described here 9 5 Resetting the PMT High Voltage Resetting the PMT High Voltage KZD 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 0 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 1 Correct the condition that caused the overload condition to occur i e turn off room lights or close turret shutter 2 Turn the Protect Override toggle on the front of the PMT high voltage power supply momentarily to Override 3 Turn the Protect Override toggle 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 Important When an image is not visible or suddenly disappears it may be due to this PMT HV safety feature 10 PrairieView Software 10 1 Overview Overview KI D PrairieView is the software that controls all of the scanning and image collection functions of the Ultima Image size scan rate pan zoo
110. le scan so that it will not be overwritten by the next single scan To manually save a single scan the snap tool button can be used on the image window Save Path Base Directory Specifies where acquisition data should be stored as well as the filename template for single scans Dodt Detector This section controls the Dodt gradient contrast detector if configured see detectors When the Dodt mirror is In the lamphouse will automatically turn off to protect the Dodt PMT from being saturated Filters can be placed before the mirror to filter data being sent to the PMT There are two filter positions Background Image A background image can be saved to use as a subtraction mask from the signal generated in a scan The intensities of the background image will be subtracted from the intensities of any new images when enabled A background image is stored by pressing the Record Background Image and will be used until replaced or the program terminates The percentage of the background image to subtract can also be specified Secondary Laser Beam Route This feature is a TTL level signal that is intended to drive a switch for systems that have two 2 P lasers where the second laser can be used as an imaging laser or as an uncaging laser This feature is enabled disabled and configured within PrairieConfigUtility exe at the bottom of the Misc tab This signal may be configured to use a digital output line from any of the National Instruments DAQ boar
111. lected laser light It is therefore critical to operate this system following all safety instructions and wearing appropriate laser safe eyewear 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 cords 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 discontinue use of the system turn off power and contact Prairie Technologies 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 assure optimal system performance Do not pull on or bend cables or fibers Do not handle filter cubes or dichroics except as recommended by Prairie Technologies Warning Labels Used on the Ultima Multiphoton Microscopy System Warning label on beam cover and light box Warning label on interlocked components Warning label for defeated interlocks on interlock defeat blocks
112. m 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 linescan brightness over time BOT region of interest ROI z series photoactivation marked points and T series 10 2 Main Window 10 2 1 Overview Overview KI a D Most of the controls of Prairie View are accessed through the Main Control Window Some sections have extra controls accessible through a green bar located to the left of the section such as the image size section Selected buttons are highlighted with a light orange outline on the button y Prairie View 4 0 0 50 File Preferences Tools Display Applications Help P Scanning O ey RT f Ehon window Dwell Time Per Pixel us Soft Shutter B 40 xY Step Size um User Def 10 00 Z Series T Series 2 P Laser xY Stage Labels Misc Lasers _ Zeo a el eS mila os gt Co Zeo Ch 2HV ra By 0 Zee Scanline Period ms 2 816 Frame Period ms 1441 792 Framerate fps 0 694 FOY 512 x 512 295 6 x 295 6um Pixel Size 0 577 x 0 57 um x 1546 24 Y 3309 39 zZ 280 10 2 08 22 PM T Series Finished 2 08 22 PM T Series Saved in C Documents and Settings prairie test T Serie 2 08 22 PM T Series 09272010 1048 035 Average iteration 16 16 Screen Shot PrairieView s main window 10 2
113. may press Back to return to the previous step or Done to exit this dialog 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 closed 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 I Pinhole Photolysis Calibration NeD Pinhole or Uncager Point Calibration Step i Select Device to Calibrate sUncager _ox Step 2 Salach Net Prije Spiton Bis Terni ov feet Shep 3 Taty Calls ten Fis Ret Accra Shutter F1 Yolsrance OSSA Lasor imensty pios J kand i Db teepreseiseneny COOMNTESS Gi PSR LG Lon Imege Channel to Display sichi Step 4 2 depan yn nang of the opat bo cuibrabs Lisia the menace itt cisien thes spat fete Ortginsl Diag Side ARA the Telus ariii honesty value to Teste 2 ane conker mane of the roe i the Srocssard image vende Th
114. mber of Points field The minimum number of points on a line is 3 There are two options for adding the line to the list of marked points Selecting Add to List will add the new points 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 Selecting Replace List will remove any previously defined marked points from the list The new list of marked points will only consist of those from the line definition Miscellaneous Controls Located below the lower right hand corner of the image window is a collection of miscellaneous controls Snap lt F3 gt will acquire a new image of the selected channel If TriggerSync is running in conjunction with a Confocal system then pressing Snap will cause the Confocal software to scan a new image using the scan parameters currently defined scan parameters in the Confocal software After the image has been acquired it will be automatically displayed in the image window in the Mark Points dialog Nudge lt F7 gt 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 Nudge you should see the
115. meter 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 Points 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 Created a list of five marked points in the Mark Points dialog In the Mark Points dialog the operator has set Use Mark Points During Acquisition to On Marked Point Hold Time to 100ms Marked Point Move Time to 0 1ms 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
116. mpensation WARNING VISIBLE and or pevisteLe LASER RADIATION WHEN INTERLOCK DEFEATED AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION MAX AVERAGE OUTPUT POWER SW EMITTED WAVELENGTHS 650 1100 nm PULSE DURATION 10 300fs CLASS 4 LASER PRODUCT Never look into the laser beam path The lasers used with the Ultima system include Class 4 ultra fast infrared IR lasers These lasers do not have a beam visible to the naked eye The optics used in this system may cause back scattered or reflected laser light It is therefore critical to operate this system following all safety instructions and wearing appropriate laser safe eyewear 5 2 Table Optics Table Optics nat Prairie Technologies provides the required table optics for the steering of an IR laser into the Ultima scanhead We have stringent quality guidelines to provide the customer with the best available products Our mirrors are specially coated to withstand high powered IR light Still IR light will damage these mirrors over time It is recommended to have your mirrors replaced and table re aligned every two years After exiting the laser cavity the beam is usually sent to a zero order half wave plate This used in conjunction with a beam splitting cube allows a selected amount of laser light to be sent to one of two paths For single scope setups the second beam path is sent into a beam dump After being sent through a Pockels cell modulator a pick
117. n added to the current mask prefixed by a minus sign For example an output file which would create a square region in the center using palette two and a triangle above it using palette one would look something like the following 3 5 7 5 7 8 3 8 2 2 5 5 2 8 5 1 Note There is only one palette available palette number one on systems not using national instruments cards to drive laser powers Which will look something like the following when loaded Al lasersusegourest A seega acs FEE Eee Aes Pookels Currers Add Rect ee Ecr F p a i A CC E AJ lasers off anai Ese UN arets off m Oo gr e A Toa lt et p lt lt lt X 201 Y 3 0000 0000 0000 0000 Nearest Neighbor 0 00 um Screen Shot Automatically Generated Photo Activation Mask Application Menu Extensions These actions are run manually from the Applications menu on the main window Actions defined here will added to the menu and add the ability to launch other applications directly from Prairie View Actions Using MATLAB Actions can make use of any number of third party tools one of which is MATLAB Unfortunately MATLAB does not provide command line support so an intermediary program has been provided Prairie MLL exe Prairie MATLAB Link To use this program in an action select PrairieMLL exe as the filename s
118. n the field and replace the number with the desired step size Use the home feature to store an XY location to come back to at a later time Selecting Set stores the current XY location as home and pressing the button will return to that position Select 0 to leave the motor where it is and set the current position to be zero Z Motor Control The z motor controls allow the user to scan through the z axis of an image in user define step sizes Use the a and iy arrows to move the z motor by the number of um indicated in the Z Step Size field To change the step size click in the field and replace the number with the desired step size Use the home feature to store a Z location to come back to at a later time Selecting Set stores the current Z location as home and pressing will return to that position Select 0 to leave the motor where it is and set the current position to be zero Z Devices If there is more than one z device available on the system such as a high precision piezo controller this control will be active to select which device to control More information about the piezo controller z device can be found here 10 2 10 Laser PMT DAQ Tab Laser PMT DAQ Tab Lal The Laser PMT DAQ tab is separated into three separate sections The Pockels cell throughput laser power at the sample is controlled on the left hand side while the PMTs and preamplifier settings are controlled on the right The PMT controls are on the
119. n will maintain a constant frame rate when enabling disabling Interlaced scan pattern under Laser PMT DAQ Tab 10 4 1 3 Nyquist Sampling Nyquist Sampling al Nyquist Sampling will automatically adjust the scan settings so that the pixel size will satisfy the Nyquist sampling theorem as well as the z step size for Z Series acquisitions The reasoning behind this is to set up the spatial acquisition parameters x y and z for optimal image data collection These system settings will help to eliminate unnecessary over sampling of the data and protect against under sampling of the data The Nyquist sampling algorithm is invoked by selecting Tools gt Nyquist Sampling or by pressing lt F9 gt A Nyquist Sampling Wavelength EJ Enter the wavelength to use for 0 SS Screen Shot Nyquist Sampling Dialog After invoking Nyquist sampling if the currently selected objective lens has not been calibrated a message box will display the necessary corrective action If the currently selected objective lens is calibrated and one or more of the following are true if the laser source is 1 P or the laser source is 2 P and not being controlled by Prairie View or always prompted is checked under preferences then a dialog will appear to collect the wavelength to use for the Nyquist sampling calculation The dialog will also allow the laser type 1 P or 2 P to be specified If Cancel is pressed then no changes will be made Once OK is pressed the sy
120. nce the line scan is complete this mode can be exited by again by pressing LS on the Image Window If TriggerSync is running bringing up the Linescan dialog will prompt to enable synchronization with the experiment that has been defined in TriggerSync When enabled a coordinated recording will be made that synchronizes the electrical recording from TriggerSync with the image data from the PMTs The Linescan Viewer displays both the line scan image and electrical data that has been collected in this manner for analysis The Linescan Viewer is discussed here 10 3 5 Look Up Table Look Up Table nao 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 0 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 y Lookup Tables X Channel 1 Ch1 Channel 3 Spot Bh le SOS lle Le les 10000 10000 5000 S s500 o 0 1 0 1000 2000 3000 4095
121. nd the image is saved as a temporary file If Average Every N Frames is set to a number more than one the selected number of frames will be acquired and averaged to create one image New Window Selecting 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 Running Frame Average The Running Frame Average affects the Live Scan imaging When this option is checked the software will average every chosen number of frames during Live Scan and update the Image Window accordingly This is useful for samples with little fluorescence or when using low laser power Average Every N Frames Average Every N Frames is designed to work with the Single Scan command When set to a number greater than one it will average the selected number of frames to create a Single Scan image Shutter Controls There are two different table shutters used when imaging The Hard Shutter is a physical shutter placed on the table The Soft Shutter controls the bias applied to the pockels cell The Hard Shutter automatically opens whenever an acquisition begins 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 by clicking Hard Shutter Open or Closed It is also po
122. ne Screen Shot SFC Settings Terminal tab The Terminal tab can be used to send line commands to the SFC control box directly 14 Acquisition Scenarios 14 1 Overview Overview KI D 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 Prairie View simultaneously with the data communication links between the two programs allows the operator to accomplish this IMPORTANT TriggerSync must be started AFTER Prairie View is started If both programs are running and you are unsure which was executed 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 Prairie View 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 It is assumed that the operator is already familiar with the individual commands as described elsewhere in this manual 14 2 TriggerSync Electrophysiology Acquisition TriggerSync Electrophysiology Acquisition KI a D oR ON Open
123. ng on a label listed under the Label Select drop down menu under Scanning Controls 10 2 16 XY Stage Tab XY Stage Tab KI a gt 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 DAG Z Series T Series 2 P Laser XY Stage Labels Misc Y 2309 39 Grid Definition Overlap J10 leach NoneSet Stop Location None Set 40 Y um Z um Dptical 1 166311 294320 29077 2 p 1585 30 3241 11 278 85 Add Current Location Remove Location Remove All Locations Goto Selected Location Goto Next Location Goto Previous Location Shift All Locations Shift Selected Location Grid Location Display J Record Optical Zoom Add to Grid Locations Goto Position XY 2 1585 30 3241 11 278 85 Replace Grid Locations Invert Xx Travel for Import Locations Export Locations a Mark Stage i Invert Travel for Mark Stage i Display Nothing Screen Shot XY Stage Tab None A Clear All removes any stage locations recorded in the table Remove Selected erases the currently selected location from the table Record Current Location adds the current stage location to the list of locations in the table Shift All Locations updates the selected sta
124. nhole device Using the Select Device to Calibrate control select the desired pointing device 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 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 Click on the Browse button and then use the pop up dialog to select the directory and enter the file name to use The cal extension will be automatically appended to the specified file name 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 Hints on Naming the Calibration File Since the calibration 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 operat
125. ns Electrical Recording amp Uncaging Line Scans Electrical Recording amp Uncaging 4a 1 Start Prairie View 2 Start TriggerSync 3 In PrairieView use Live Scan or Single Scan to locate the area of interest 4 Pan Rotate and Zoom to orient the target of the specimen 5 Load the correct point calibration file in Mark Points 6 Mark the locations to be uncaged 7 Open the Acquisition and DAC Output Setup dialog in TriggerSync 8 Confirm or define stimulus control and acquisition parameters and close the dialog 9 Enable the desired ADC input channels on main TriggerSync window 10 Select Use Marked Points During Acquisition 11 Set first point delay time 12 Verify the Hold and Move times 13 Press to open the Line Scan dialog 14 Select Yes when prompted whether the Linescan acquisition should be synchronized with TriggerSync 15 Position the line where desired 16 Enter the number of lines to be collected 17 Press Start Linescan s The TriggerSync experiment will be executed as it has been defined while line scan data is collected 18 Open Linescan Viewer and load the created linescan file to see the electrophysiology data along with the fluorescence linescan profile s 14 6 Non Line Scan Images Electrical Recording amp Uncaging Non Line Scan Images Electrical Recording amp Uncaging all gt Start Prairie View Start TriggerSync In Prairie View use Live Scan or Single Scan to locate
126. ntrol response 7 Automatically start playback after acquisition Automatically convert images to OME TIFF Leave soft shutter open Preserve Base Directory Names Nyquist Sampling Output Trigger Type Save images generated when Photoactivation PA is used T Series Z Series _tieracedScaming O C Preserve Framerate Screen Shot Preferences Menu Automatically start playback after acquisition When enabled this option will open the last acquisition in playback mode once it has completed Automatically convert images to OME TIFF When enabled this option converts TIFF images to conform to OME TIFF standard after each acquisition Nyquist Sampling Brings up a control panel for adjusting the scan setting to minimize over sampling and optimize spatial acquisition parameters See Nyquist Sampling Output Trigger Type Select when to trigger at the start of the frame or at the end of the previous frame See Trigger Signals Save images generated when Photo Activation PA is used When enabled images are saved normally when acquiring frames with photo activation masks By default these frames are discarded T Series Offers selections of how time data is displayed command execution order and how TriggerSync experiments are executed See T Series Preferences Z Series Offers different motor presets for z series execution See Z Series Preferences Interlaced Scanning Preserve Framerate When enabled this optio
127. nts Menu Options Al p gt TriggerSy nc 1 4 1 File Acquisition View Analysis Configurat Mark Points Show Points Calibrate Points Acquisition and DAC Output Setup Screen Shot Points Menu Mark Points Selecting this option will bring up the Mark Points dialog used to select points in the field of view to perform ablation experiments More information can be found here Show Points If there are points defined in the Mark Points dialog this option will be active When selected a scan of the active channel will be taken with the marked points shown in an overlay Calibrate Points When selected this option will bring up a dialog for the calibration of the uncaging galvonometers More information can be found here 11 6 5 Analysis Menu Options Analysis Menu Options 4a p gt TriggerSync 1 4 1 Fie Acquisition View Points EWEIREEE Configuration Help Analyze Acquired Data Ctril 4 No online analysis Trace Averages Running 4verage Cumulative View Acquisition and DAC Output Se Functional Mapping Full Width Half Maximum al Screen Shot Analysis Menu Analyze Acquired Data Toggles on and off the analyze data state for TriggerSync Trace Analysis The four options below require an acquisition to have more than one cycle The information collected from the active channel inputs are shown in the small trace graph below the main acquired data graph according to the type of analysis chosen
128. og If the operator chooses Accept the new gradient setting will be applied to the selected laser line s This will be shown by the updating of the value in the Custom field to reflect the changes made in the graph Triggered Z Series If No Input Trigger for Z Series is selected then the Z Series will start immediately when the Start Z Series button is pressed lf Start Z Series with Input Trigger is selected then after Start Z Series is pressed the z motor will move to the position of the first slice image the laser and or PMT settings will be set the hard shutter will open and the software will wait for the arrival of an external input trigger before acquiring the first slice image After that all subsequent slices images are acquired automatically If Use Input Trigger for each Image is selected then after Start Z Series is pressed the z motor will move to the position of the first slice image the laser and or PMT settings will be set the hard shutter will open and the software will wait for the arrival of an external input trigger before acquiring the first slice image This process is then repeated for each subsequent slice image in the Z Series These controls only effect Z Series executed as stand alone experiments They do not affect a Z Series executed as a cycle in a T Series Collecting a Z Series To collect a Z Series in which the system calculates the number of slices images to collect 1 Use a and x arrow
129. oint is not deleted Other actions besides pressing Erase All Points 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 To see where each Marked Point is located on an image select Show Points This 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 Show Points is Save Point Images When this is pressed it will display a dialog that presents the operator with four check boxes Save Open Image The current image will be saved with the specified file name Save Open Image with All Points Marked The current image will be saved and indicators for each Marked Point will be superimposed on the image The file will use the specified name with AllPoints appended to the name Save Individual Open Image with Eac
130. onship is defined as subtracting region 2 from region 1 the label in region 1 would change from 1 to 1 2 If Region Alone is selected in the pop up menu then any defined mathematical relationship for that region will be removed All Alone will remove all previously defined mathematical relationships Import Regions and Export Regions allow the operator to save a set of region definitions to a file for recalling later This could be useful if the operator wishes to use consistently sized regions over multiple data sets He she could save a set of regions using the Export Regions button and then when a new data set is loaded use the Import Regions button to load the regions The region locations could then be manually set where desired Located below the table of region definitions is a set of controls for Region Group To create a region Group first create the regions in the image window via the Add New button Next place a check in the box next to the region number in the Display column that you wish to be part of the region group Uncheck any regions you do not wish to have part of the group Now press the Create Group button and a group will be created that consists of the specified regions To change a group definition first check uncheck the desired regions in the region table then select the desired group left click on the group and it will be highlighted and then pr
131. or is using a 60x water lens at an image resolution of 512x512 and a zoom of 1 a useful calibration file name would be 60x w 512x512 zoom1 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 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 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 First acquire an image of the spot by pressing the Acquire button located
132. ording amp Uncaging Seno oe SY YS 10 _ _ Start Prairie View Start TriggerSync In Prairie View use Live Scan or Single Scan to locate the area of interest Pan Rotate and Zoom to orient the target region In TriggerSync open the Mark Points dialog Load the appropriate calibration file for the Uncager Calibration File Open the Acquisition and DAC Output Setup dialog in TriggerSync Confirm or define stimulus control and acquisition parameters Close the dialog by pressing Accept Done Enable the desired ADC input channels on the main TriggerSync window Mark the required points Lai Llib 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 1 2 3 4 Select Acquire Loop in the main TriggerSync window This will run the Cycle Number of acquisitions set in the Acquisition and DAC Output Setup dialog at each point of uncaging Set first point delay time Verify the Hold and Move times Press Acquire in TriggerSync 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 a 2 De Select Use Marked Points During Acquisition in the Mark Points dialog Set first point delay time Verify the Hold and Move times Press Acquire in TriggerSync 14 5 Line Sca
133. orrect high voltage values for each PMT but have no effect on the voltage itself Do not change these settings as re calibration is difficult and time consuming CAUTION PMTs are extremely sensitive to light Care should be taken to eliminate all sources of stray light that may reach 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 6 7 National Instruments Boards and Boxes National Instruments Boards and Boxes 4a gt There are three National Instruments PCI boards in the system computer the NI 6052E NI 6713 and the NI 6110 or 6115 These boards control the three external rack mounted National Instruments BNC breakout boxes two 2090 units and one 2110 The upper 2090 box is referred to 2090A and the lower is referred to as 2090B All of the NI boards and boxes are connected through the PFIO Trig1 channels to synchronize the experiment acquisition start triggers among the three NI boards NI 6110 6115 Board and BNC 2090B Box The 6110 or 6115 card is responsible for the creation of the imaging output waveforms and collecting the signals from the PMTs The 6110 card is used for regular galvo only imaging systems while the 6115 card is used for the high speed imaging AOD The 6110 board samples at 2 5 million
134. ose Prairie View Close TriggerSync Remove sample Raise objective lens all the way up Install objective target in turret So ee S Open National Instrument s Measurement and Automation Select Devices and Interfaces Select Traditional NI DAQ devices Select PCI 6713 Select Test Panels Use analog output and DC voltage Set channels 5 and 7 to 0 00 Volts Be sure to press update channel after selecting each channel 7 Open shutter for uncaging beam aorDoandeaoey 8 Verify that the image of the X axis galvo mirror is centered on objective target ring 9 Insert sidecar fluorescent target 10 Use adjustments at the sidecar turning mirror to center beam at the sidecar target 11 Remove sidecar fluorescent target 12 Use adjustments on the front sidecar mirror to center beam on objective target 13 Repeat the two sidecar adjustments until the beam is centered on both targets 14 Stop iris down only to the size of the ring on the objective target 15 Leave iris in this position to minimize back reflections into upper PMTs 16 Close measurement and automation 17 Close uncaging beam shutter 18 Restart system software 9 3 Spot Detector Alignment Spot Detector Alignment Lala Important This is normally a one time adjustment The only time this will need to be performed is if either galvo is replaced a mirror in the scan head is moved or the beam combining dichroic in the scan head is replaced
135. ow side is by definition the top side This box will change its size as a result of the Optical Zoom function described here Similar changes will be displayed when the image is panned or shifted laterally and when the scan is rotated Coarse Medium Fine 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 0 causes the pan to reset to the middle of the field of view 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 Zero will set the angle of rotation back to zero 10 2 9 Stage Controls Stage Controls noo The stage controls 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 to the left of the movement buttons and has a default of 10 um Saved locations can be modified on the XY Stage tab Stage Control ia a E 4 0 gt o MEME Y Home um Z Home um 20 00 0 00 Set 19 10 Screen Shot Stage Controls XY Stage Control The XY stage controls allow the user to scan through the x and y axes of an image in user define step sizes Use the arrow buttons to move the stage by the number of um indicated in the XY Step Size field To change the step size click i
136. p Important Re aligning the imaging laser will introduce an offset into all of the spot calibration files Close PrairieView Close TriggerSync Remove sample Raise objective lens all the way up Install objective target in turret DO CUL e w N gt Open National Instrument s Measurement and Automation Select Devices and Interfaces Select Traditional NI DAQ devices Select PCI 6110 Select Test Panels Use analog output and DC voltage Set channels 0 and 1 to 0 00 Volts Be sure to press update channel after selecting each channel 7 Open shutter for imaging beam ODQA0 0 8 Verify that the image of the X axis galvo mirror is centered on objective target 9 Insert sidecar fluorescent target 10 Use adjustments at the sidecar turning mirror to center beam at the sidecar target 11 Remove sidecar fluorescent target 12 Use adjustments on the front sidecar mirror to center beam on objective target 13 Repeat the two sidecar adjustments until the beam is centered on both targets 14 Stop iris down only to the size of the ring in the objective target window 15 Leave iris in this position to minimize back reflections into upper PMTs 16 Close measurement and automation 17 Close imaging beam shutter 18 Restart system software 9 2 2 Uncaging Beam Alignment Uncaging Beam Alignment ala Important Re aligning the uncaging laser will introduce an offset into all of the spot calibration files Cl
137. periments 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 DAC7OUT are set aside for driving the uncaging galvos DACOOUT DAC40OUT and DAC6OUT are available through TriggerSync for other experiments To NI 6773 Board in PC Lo FRE AO I toussi I To Pointing Galvo Y input f To Poirning Galvo X input ___ Master Scan Start Trigger Fram USER 2 on BMC 20808 A To uncaging shutter V Analog outputs yma Figure NI BNC 2110 of Channels 1 2 3 4 5 6 7 8 Max Input Samples Sec for NI 6052 333K 166K 111K 83K 67K 56K 48K 42K Max Output Samples Sec for NI 6713 1M 500K 333K 250K 200K 167K 143K 125K Table Maximum NI 6052 and NI 6713 sample rates Software Communication Through NI Hardware The software used for imaging Prairie View and the triggering software TriggerSync communicate by passing messages to each other using a TCP IP protocol In order for Prairie View to recognize that TriggerSync is running TriggerSync must be started or restarted after Prairie View 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 Prairie View and TriggerSync are triggered by a falling ed
138. phouse when the Dodt mirror is in the path CAUTION Protecting the PMTs When the EPI light source is in use the Dodt mirror should be in the out off position under misc tab in PrairieView software to protect the PMT If the light from the illuminator is directed to the PMT it will be damaged and need replacing This damage is not covered under warranty The Dodt aperture motor is controlled by an external control box which includes a remote for the user to control the portion of light sent to the PMT The Dodt mirror and filter tray are controlled by the computer via a USB cable 6 6 Dual Preamplifier amp HV Control Unit Dual Preamplifier amp HV Control Unit Alp o PMT High Voltage 1 gt PMT High Voltage 2 a 7h Ban E me et g Wz Ca any ET ote Po an pA S C Sansor a i E m S charge o _ Override Mee 5 o S PECHNDLOGHES DUAL PREAMPLIRER amp FY CONTROL 9 Figure Dual Preamplifier amp HV control unit The PMT High Voltage HV is controlled via the PMT control unit mounted in the electronics rack Each unit controls up to two PMTs The commands for the voltage are received by the HV box via the C C Inputs on the rear of the box These commands come from the Device Control box The signal is sent to the PMTs by the BNCs attached to the HV Outputs on the rear of the box There are several controls on the front of the HV box Included on the LED panel showing the HV output o
139. preferred option As noted the data will not be displayed at the end of the acquisition The only format available for this type of acquisition is the Spreadsheet Format Save File Type TriggerSync acquisition data can be saved in three different file formats The default is TriggerSync Format This can then be re opened in TriggerSync for later analysis The other two formats Igor and Spreadsheet Format are designed to export the data for analysis in other programs These cannot be opened in TriggerSync Spreadsheet format can be opened in programs such as Microsoft Excel Spreadsheet Delimeter Defines the keystroke used to separate datapoints in the program being exported to The default is Tab Scale Exported Data Defines whether data saved in the Igor or Spreadsheet Format should be Scaled or left Raw Save File Structure When Sequential Auto Save or Acquire Loop acquisitions are selected there is the option of saving All Data in One File or saving Separate Points in Separate Files Reset Acquisition Mode The user can determine whether when previous points are deleted and new points are defined the acquisition mode should reset Save Mark Points Image s The user can decide to have an image saved for the Mark Points portion of an acquisition or not Point Calibration Pause Defines the number of seconds to delay after a move is complete before acquiring an image during the Point Calibration procedure 11 6 9 Help Menu Options Help M
140. quisition View Points Analysis Configuration Help i Acquisition and DAC Output Setup E Acquired Data joo 0 000 0 000 0 000 0 000 PointO n421 T T Lj f T 1 lj T T lj T T T T T T T T T 1 0 0000 0 0500 0 1000 0 1500 0 2000 0 2500 0 3000 0 3500 0 4000 0 4500 0 5000 0 5500 0 6000 0 6500 0 7000 0 7500 O8000 0 8500 0 9000 0 9500 0 9990 Sec ie s oad Parameters SS ao Comme Mma amp amp am Load Parameters 2 A ccoseo M _ ShowTools i Load Parameters 3 44 a ee Load Parameters 4 Cert ad Screen Shot TriggerSync Main Window 11 2 Main Operational Modes Main Operational Modes Alp TriggerSync 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 enters 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 acquisition is completed TriggerSync will go into the Analyze Acquired Data On state The operator may manually switch between these two modes at any time by selecting Analysis gt Analyze Acquired Data lt Ctrl A
141. r The red cursor is intended to be the primary method of selecting locations with the various marked points generation tools Calibration Files and General Controls The section of the dialog along the upper right hand side contains the controls for selecting the calibration file and desired pointing device The Marked Points 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 If a NeD Pinhole device is selected in the Device field the control NeD Marked Points Location will be active This control has three options Iris Pinhole Confocal The selection will determine which NeD Pinhole option will be used for generating the marked points Point 0 v Screen Shot Marked Points Device s Mark Point using Red Blue Cursor is usually set to the Red cursor option In this 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 With this option selected the selected pointing device must have its ph
142. r create a grid of marked points Currently this option only works with the Mark Point Using Red Blue Cursor control set to Red A Large Screen Shot Mapping Setup While this grid of marked points may be used in any experimental way that any of the other marked points may be used within TriggerSync a grid of marked points provides a new data acquisition and analysis option This option is referred to as Functional Mapping To create a grid of marked points 1 Position the red cursor in the image window to indicate the location of what would be the upper left hand corner of the grid 2 Press Mark First Corner 3 Position the red cursor in the image window to indicate the location of what would be the upper right hand corner of the grid 4 Press Mark Second Corner Note 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 5 Position the red cursor in the image window to indicate the location of what would be the lower right hand corner of the grid 6 Press Mark Third Corner 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 t
143. r higher temporal resolution than is possible with using labels to alternate settings each frame One line of each track is scanned to acquire one line of the final image Note While this feature is available for systems with only a single laser its usefulness with a single laser setup is limited at best This feature is primarily useful when a system has more than one laser either 1 P or 2 P For example if a system has two imaging lasers one tuned to 800 nm and one tuned to 900 nm this feature allows the operator to scan a 512x512 image alternating wavelength with each line The data for track one will be saved as the Channel One data and the track two data will be saved as channel two Whenever the Interlaced scan pattern check box is checked the defined interlaced settings will be used This applies to Live Scan Single Scan T Series Z Series etc Laser Selection Specifies one of the lasers currently configured on the system as well as a power setting The laser and power here can be added directly to an active track By default the current power check box is checked which indicates that the current power setting should be used even as the current value changes When unchecked a static power value can be specified Tracks A Track is a sequence of laser lines and powers to control Up to four different Tracks may be defined There is a check box on the left of each track to enable it Once enabled it is possible to add the sele
144. r this cycle to take Press lt Enter gt Click the Max Speed check box if this is desired Note This will override the entered Period time Select a Z series from the drop down This will cause the Z series to be collected as defined on the Z Series Tab Verify the Save Path Press Start T Series 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 It is possible to collect a Z series at a set interval or to collect individual images and Z series alternately This example shows a 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 Select Add New A row with default settings will be created To select the number of images or acquire click on the Reps field type 10 and press lt Enter gt Select Add Wait A second line appears Click on the Period field type 30 the total time for the system to wait between the preceding and following acquisitions Press lt Enter gt Select Add New A third line appears Select a Z Series from the drop down menu This will cause the Z series to be collected as defined on the Z Series Tab Verify the Save Path Press Start T Series Ro eS oS Se TriggerSync Experiments Within a T Series The column labeled TriggerSync Experiment will display a check box whenev
145. r to be adjusted when setting the Nyquist sampling values Depending on the current system configuration both the number of pixels and the optical zoom may need to be adjusted For example if optical zoom was specified but the computed optical zoom would be less than 1 0 the system minimum then the number of image pixels would also be changed to achieve the desired pixel size Laser Type specifies the laser source to be used for the Nyquist calculations There is a difference in the equations if the laser source is a 2 P two photon laser versus a 1 P single photon laser Over Sample Constant specifies the over sampling constant to use Always Prompt for Laser Type and Wavelength specifies whether or not a dialog should appear to collect the desired wavelength value and laser type 1 P or 2 P when the Nyquist option is invoked This option is particularly useful with system configurations that include both 1 P and 2 P lasers Since when the Nyquist option is invoked the software won t know in advance what wavelength to use The type specified under Laser Type will be the default laser type shown in the dialog mentioned above If More Than One 2 P Laser is Present use the Wavelength of Laser is only active if the system has more than one 2 P laser being controlled by Prairie View If more than one laser is being controlled by Prairie View then this will specify which laser to use for determining the wavelength for the Nyquist sampling c
146. radient _ _ Acquire until 20 drop Stop Position um Pockels v in image intensity detected 276 20 Define y Applies to all lasers No input trigger for 2 series Set fe Default Saved Z Series Custom 00 fez O Manual z x C Documents and Settings prairie tes Base Directory ZSeries 09272010 1048 L e Stat Z Series Screen Shot Z Series Tab 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 planes By entering values for any three of these values the fourth value will be calculated automatically The Define Calculate radio buttons indicate the three values that the operator must define as well as the one that is calculated by the system Laser Power Gradient The Laser Power Gradient control affects the way Pockels cell power is controlled throughout a Z Series When Adjust Laser amp PMT is checked the Laser power Gradient control is active This pull down menu allows the user to specify the laser control that the gradient control operation will be applied to This is only active if there is more than one imaging laser The settings are retained for each Z Series definition and each saved z series can have a different gradient setting There is also
147. rame Next if a dataset is being shown the time of the frame shown will be displayed To the right of that is a description of the interpolation method used On the far right is a readout of the z position 10 3 2 Selecting an Input Channel Selecting an Input Channel KAD The top four buttons on the left hand side of the Image Window indicate which input channels are to be displayed in 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 Ch 1 represents PMT 1 on the PrairieView Main Window Ch 2 represents PMT 2 etc To start collecting data from a certain PMT simply click on the corresponding channel button The background of the button will change from grey to green Channel Colors ES haa Grayscale 000 E Pseudocolor 001 Range Check 002 Cama Temperature 003 E INDO 1 Ca 405 E Cascade Blue 423 B AMCA bound 445 E Hoechst 460 J INDO 1 490 E bio 601 GFP 509 E voyo 509 E Bodipy FL 512 E Acridine Orange DNA 520 E FITC 520 E Fluo 3 526 Ec 629 Sy BCECF 530 Calcium Green 533 Lucifer Yellow 533 m Rhodamine 123 533 E TOTO 1 533 E GFP 540 __ Cv3 565 Dil 565 TRITC 576 E R phycoerythrin 578 SNAFF ACID 580 Chromomycin 43 590 JC 1 High Con 590 E Lissamine Rhodamine
148. rature Celcius Z Diode 1 Heat Sink Temperature Celci Diode 1 Temperalure Servo Status L Diode 1 Hours 1909 Diode 1 Voltage fvolts 0 Diode 1 Servo Drive Setting 1971 Diode 2 Qurrent amps 46 19 Diode 2 Temperalure Cedcius 20 64 Diode 2 Sat Temperature Calciuz 21 Diode 2 Heat Sink Temperature Celci Diode 2 Termperalure Servo Status L Diode 2 Hours 1900 Y b Diode 2 Voltage folts 0 Update Write Laser Status to File Screen Shot Laser Status By clicking on the green arrowed bar in the center of the tab the right hand side of the tab will be replaced with the 2 P Laser Status This is a text readout of the commands and information received from the laser itself It is useful for diagnosing errors and checking the status Update will query the laser for the most recent information The information displayed in the window can be sent to a text file for printing by clicking Write Laser Status to File 10 2 15 Labels Tab Labels Tab KIAD The Labels tab 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 Labels Name Channel s PMT Gainfs 1 gt 2 Label 001 False True 0 650 0 0 10 10 10 1 3 Label 002 False True 0 0 0 0 10 10 10 1 PMT Sub Stage Gairf s DAA Gain s WEN
149. re will be an additional column of controls labeled Laser Palette There are eight available user configurable palettes The first palette by default has all lasers at their current settings This palette is represented by a solid color when the operator is drawing the mask s Like all of the palettes the operator may select Edit to change the laser selection and or power values for a custom setting When Edit is selected a new dialog will appear that allows the operator to define the laser selection if more than one laser line is available and the desired laser power to be used when that particular photo activation palette is selected After the dialog is closed the specified laser settings will be shown in the text area to the right of the palette symbol To use a desired palette simply click on the palette symbol pattern icon and then draw the desired photo activation masks The display of the photo activation mask will be the same as the selected palette symbol pattern Below the Palette selections there is another set of controls labeled Background Power These controls are used to define what laser s if any and the laser power setting for the selected laser to use for the region of the image outside of any photo activation masks If All Off is selected then all lasers will be off outside of the photo activation mask areas If All Current is selected then the laser s will be at the current setting of the laser slider controls in Prairie V
150. red 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 This 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 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 11 6 Menus 11 6 1 File Menu Options File Menu Options 4a gt gt TriggerSync 1 4 1 Acquisition View Points Open Experiment Save Experiment Load Parameters Save Parameters Save Image Load
151. roduct intended for use in laser based imaging microscopy It is not intended for any other purpose Use of this system its components or performance of procedures other than those specified in this manual may result in hazardous radiation exposure VISIBLE and or pevisteLe LASER RADIATION WHEN INTERLOCK DEFEATED AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION MAX AVERAGE OUTPUT POWER SW EMITTED WAVELENGTHS 650 1100 nm PULSE DURATION 10 300fs CLASS 4 LASER PRODUCT Interlocked safety covers This system is enclosed within light tight and laser safe covers These covers are designed to protect the user from exposure to Class 4 laser radiation Therefore at no time should these covers be removed or modified The interlocks on these covers are only to be defeated by authorized factory trained personnel during specific maintenance and service procedures During these procedures appropriate laser safe eyewear is required WARNING VISIBLE and or INVISIBLE LASER RADIATION WHEN INTERLOCK DEFEATED AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION MAX AVERAGE OUTPUT POWER SW EMITTED WAY GTHS 650 1100 nm PULSE DURATION 10 300fs CLASS 4 LASER PRODUCT Never look into the laser beam path The lasers used with the Ultima system include Class 4 ultra fast infrared IR lasers These lasers do not have a beam visible to the naked eye The optics used in this system may cause back scattered or ref
152. rojection Maximum Intensity Projection Alp While in playback mode the active dataset can be shown as a Maximum Intensity Projection MIP This control displays the maximum intensity profile for a position in the z plane at a user selected x y coordinate point image Window ij Exit a E i Detaser maae i a7 S008 Galen elem m iA Loop i Show Overtay z I pne E ee 2 227 98 0028 M85 0000000 Z 121 113 000 000 Nearest Neighbor 0 00um Screen Shot Maximum Intensity Projection To use MIP a Z series must first be acquired see Z Series tab While still in the playback window click MIP This places a cross hairs 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 Options for display can be found by selecting the arrows to the right of MIP These options include an average intensity projection to show the dataset at the average for each pixel instead of maximum 10 3 12 Mark Points Mark Points KAD The Mark Points dialog is used to mark locations of points for photo activation or uncaging experiments These points can be singl
153. roller Reset Preamplifier Scan Settings Screen Shot Tools Menu 1 P Pinhole If a 1 P Confocal pinhole detector is present on the system selecting 1 P Pinhole will bring up a control dialog for the pinhole motors Calibrate Objective Lens Brings up an instructive dalog box for calibrating the field of view for objectives See Calibrate Objective Lens Calibrate Laser Power Brings up a dialog box for calibrating the power output from the pockels cell to the power output from the laser cavity for mW control of laser power at the sample See Calibrate Laser Power Center Galvos When selected Center Galvos will apply a zero voltage to the imaging galvos for the purpose of checking alignment While active some scanning controls will be deactivated This state is exited by clicking Center Galvos a second time Fluorescence Unmixing Brings up a control panel for quick adjustment of signal data between the channels See Fluorescence Unmixing Notes Brings up an empty window for users to enter notes These notes will be saved with each subsequent saved image Nyquist Sampling Brings up a control panel for adjusting the scan setting to minimize over sampling and optimize spatial acquisition parameters See Nyquist Sampling Oz If the system is configured as a PC Oz system this selection will bring up the controls for the PC Oz system Actions Brings up access to creating sets of external commands to use within Prairie View for the controllin
154. s JF gt 1840 Frames Vertical Scale 1830 Best Fit C Fixed va x 1890 0 1910 X z s 1780 0 d 1800 1790 Show Average For L 1E Frames 1780 10 12 14 16 18 20 22 24 26 Seconds Brightness Over Time Regions Units pixels C um Display Channel Position Width Height Area Rotation Shape 1 Chi 38 69 148 149 17320 0 Ellipse Ph 158 234 157 157 19359 0 Ellipse Add New Ellipses Rectangles Polygons J Brush Medium Mj sooch Import Regions Export Regions Clear Regions Move All Region Groups Right Click To Create Group O Resize _ Rotate Update Group Clear Groups icone Region Functions Create Function Edit Function Clear Functions File Options Sampling Options C Documents and Settings prairie Browse BrightnessOverTime 09272010 104 22 v Update Display __ Start with Input Trigger Screen Shot Brightness Over Time Dialog Graph Options Along the right hand side of the graph window are controls for the graph display There are separate controls for both the horizontal x and vertical y axes Both axes can either be scaled as Best Fit an auto scale or Fixed Best Fit will adjust the visible values as necessary according to the data set while Fixed will be limited to the value entered in the Range field Also when Fixed is chosen the Offset allows the operator to specify the bottom left limit or first value for the spec
155. s Terminal Tso so PPV Sun pinhole Si 483560 z 3300 ef Fas Sep S Gen Ww Face roe C Ee er Be BS CO iipaimen 5e amnen omms Cosmea 297C 1OMHzMutpicaionGan Stopped Stopped 27 CycesFrane Screen Shot SFC Settings General tab The General tab includes the basic settings for creating an SFC scan The desired aperture and filter is set here along with Fine control of the piezo Y scan peak to peak voltage The Camera Settings will control the image collection properties of the camera itself The Gain and Mult Factor both amplify the signal after it is acquired by the camera The Port selection tells what speed the camera transmits data at Mode determines whether the camera creates the scan trigger or it waits to receive one from the software Preset Scan Configurations offer a quick pick for most used scan settings 13 4 Triggers Tab Triggers Tab all amp SPC Settings I eal Gere Taer Motors Scan Setting Termina stan olf id Got jae JL chs JL ENEFA EEE TE 298 10HHz Mulipicstion Gan Stopped Stopped 27 Cycles Frane Screen Shot SFC Settings Triggers tab The Triggers tab can be used to set the trigger for each channel independently 13 5 Motors Tab Motors Tab all gt uresen E Corn e C caaea _ OoOO 297 C 10MHz Multiplication Gan Stopped Stopped 27 Cycles Frane Screen Shot SFC Settings Motors tab The Motors tab can be u
156. s in an XML format and contains all information about the scan The program or script specified must be able to parse the metadata file in order to retrieve scan data Brightness Over Time Region Generation These actions are run manually from the brightness over time dialog and are used to automatically generate brightness over time regions based on the current image data There are two tokens available for use in the arguments field lt image ch gt where is replaced by a channel number 1 4 This token will be replaced by the path and filename of the image containing the current image data for the channel specified lt outfile gt which replaced by the path and filename of a temporary file created to pass region data from the program or script specified into Prairie View The format of the output file is a comma delimited list of vertices that make up polygon based regions Each vertex is made up of an X and Y coordinate again separated by a comma that range from 0 0 0 0 upper left corner to 1 0 1 0 lower right corner Each polygon is terminated by the channel number the region should be created for prefixed by a minus sign For example an output file which would create a square region in the center for channel two and a triangle above it for channel one would look something like the following 3 5 7 5 7 8 3 8 2 2 5 5 2 8 5 1 Which will look something like the following when loaded Image Window
157. s in the stage controls to move the z motor to the correct focal plane for the starting position of the Z series 2 Select Set or in the stage controls to enter the start position 3 Use a and M arrows to move the z motor to the desired focal plane for the ending position of the Z series Select Set or the in the stage controls to enter the end postion Enter the desired Step Size if different from the distance displayed and press lt Enter gt Confirm that the Save Path and Base Name are correct oS 0 e 7 Press Start Z series Acquisition of the Z series can be monitored in the Scan Information Window Collecting a Z Series Over a Large Depth When collecting a Z series over a large depth it is sometimes desirable 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 Prairie View provides such a mechanism In order to have a nonlinear laser power change applied simply follow the steps outlined below Use a and M arrows to move the z motor to find the desired focal plane for the start and stop position of the Z series Check Adjust PMT amp Laser Click Goto under start position In the Laser PMT DAQ tab set desired laser and PMT settings Click Set under Start Position in the Z Series tab Click Goto under stop position In the Laser PMT DAQ tab set desired laser and PMT set
158. s 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 corner 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
159. samples per second and allows dwell times in multiples of 4us The 6115 board samples at 10 million samples per second and allows dwell times in multiples of 1ys The output waveforms for the imaging galvos are output on the 2090B s DAC 0 Out and DAC 1 Out The 2090B box also provides the input for the PMT data on BNCs ACH 0 3 These four channels correspond to four high speed ADC 12 bit input channels on the 6110 or 6115 board NI 6052E Board and BNC 2090A Box The NI 6052E board collects all the input signals generated while running an experiment These input signals are collected by the 2090A box at BNCs ACHO 7 They are represented in the software as the eight TriggerSync acquisition channels The maximum sampling rate for this board can be as high as 333K samples sec For each additional channel enabled the maximum sampling rate is proportionally decreased Gerty Perera han beget reon ham Dm PIRON Teh a ECA TIN ID w n gt 5 mpa jawan A salen NS Va MAT wv Be PR vy Camran aged Figure NI BNC 2090 boxes A top amp B bottom Bigeye wae AARETE che phat pte pty aP PEE y Regi its a tpt ae USER to PFI USERI to CTRIOUT vee ee DIDO to PRG USER to PFI6 Figure Close up of NI BNC 2090 boxes NI 6713 Board and BNC 2110 Box The NI 6713 board generates the output waveforms for the uncaging galvos the Pockels cell and the stimulus signals available in TriggerSync for use in ex
160. scaled against For a complete description see ADC input controls When Auto Scale is selected 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 Sequential File Below the graph window and to the right of Acquire 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 Select Sequentia File vi Select the baca flename Select the First value to bo be used inthe sequence be placed alter the filename First File 001 dak Seet Directory to save sequertia Files Screen Shot Sequential File 11 3 ADC Input Controls ADC Input Controls KI a gt The previous section Acquisition DAQ Output Setup 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 Selecting Input Channels Screen Shot ADC Inputs Across the top center of the main window of TriggerSync are a seri
161. se 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 and the values for the absolute and relative position will be updated Selecting Move to Next Point lt F11 gt will automatically move to the next point in the marked points list from the current point For example if Point 4 is the current value for the Move to control and the operator selects Move to Next Point the Move to control will increment to Point 5 and the blue cursor in the image window will move to the new point location just selected When the end of the marked point list is reached it will automatically wrap around and start over with Point 0 There are several ways to erase previously defined points Erase All Points will remove all points from the Marked Points list To remove a single point in a list of points select Erase Point lt F10 gt A dialog will appear asking the operator to select the marked point to delete Select the desired point and press OK If the operator does not wish to delete a point press Cancel If OK has been pressed 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 p
162. sed to tweak the positioning of both the aperture and emission filter motors Motor Configuration t wean G oc i 88 660 88 553 540 ae lies ilo lt lela Let i Screen Shot Open Configuration dialog Selecting Open Configuration will bring up a second dialog box where one can edit the filter names and move a selected aperture To Top for easy removal If there seems to be an issue with the motor movement selecting Reinitialize will reset the motor and its position 13 6 Scan Settings Tab Scan Settings Tab KI a gt Geresal Taggers Motors Scan Settings Teina Frese Scan saing nee Oat J Gohet Time Reit Tine pono 2 00004 aso Speedle Speed Lina Bo n Camera Piezo Lag Tine ip 00000 4 Update Scar Setiegs Abn Gao 296 1OMHzMulpicationGen Stopped Stopped 27 Cycles Frane Screen Shot SFC Settings Scan Settings tab The Scan Settings tab allows the user much finer control over the movements of the galvanometer and the piezos in the system After changes have been made the user must select Update Scan Settings for the changes to take effect Selecting Align Galvo will set the galvanometer to OV centering the beam in its travel path 13 7 Terminal Tab Terminal Tab nao aas Gerasa Triggers Motors Scan Settings Terminal ey Comaands ATC 1OMHzMuipicationGan Stopped Stopped 27 Cycles Fra
163. separate each pulse G F C General Fluorescence Control Screen Shot G F C 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 Custom Waveform When the Custom Waveform protocol is selected the controls for the other protocols will be desensitized This protocol allows 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 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 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 Number of Elements in File indicates the number of elements that were actually in the file 0 00 osne O SSS O Screen Shot Custom Waveform To load a user defined file press Browse and select the appropriate file in the pop up dialog If the file contains too few elemen
164. 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 Set the Duration to 25 ms 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 Set the Number of Pulses to 5 one pulse at the start of each new marked point Finally the First Pulse Delay should be set to 0 When Update 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 Out 50 0 45 0 4 0 450 30 0 25 0 20 0 15 0 10 0 50 00 l 0 50 100 150 200 250 300 360 400 Screen Shot DAC Out Composite Graph Acquisition Binary File Format The format for the binary file that is used within TriggerSync for saving experiment data is as follows The file begins with 616 bytes of header starting with DTLG which appears is irrelevant for the purposes of extracting data but a good check to validate the file format The next byte first important value at a hex address of 268 is the number of channels sets of data stored as a 4 byte long The value
165. ssible 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 experiments This can be controlled by clicking Soft Shutter Open or Closed Scan Mode Galvo or AOD AOD is Acoustic Optic Deflection which can provide up to 25 frames per second for a 512by 512 image It is option and not available on all systems The Scan Mode button should be enabled if the system has AOD option To switch between the standard galvo driven imaging mode and the AOD driven imaging mode click the Scan Mode button See also AOD Mode Label Selection Label Select allows the user to select previously defined and saved experimental protocols from a pull down menu These protocols include saved settings for active channels PMT selection and DAQ gain laser power settings Setting up labels is covered here 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 here 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 10 2 7 AOD Mode AOD Mode A gt Switching between galvo and AOD modes Scanning Controls with AOD selected In the Prairie View Scanning menu click on Sc
166. stem This means that for a 4 us dwell time the system is either averaging or summing 4 us 0 4 us 10 samples per bin 10 2 5 Optical Zoom Optical Zoom mag KAD Screen Shot Optical Zoom Controls Controls the size of the area of the specimen that is being scanned An optical zoom of 2 will cause the microscope to scan an area that is 1 2 the width and 1 2 the height of a scan at a zoom of 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 Selecting Reset will cause the zoom to return to the default value of 1 The minimum value allowed is displayed below and to the left of the slider The current selected zoom is shown to the right of the minimum Note It is not possible to set the optical zoom to less than 1 10 2 6 Scanning Controls Scanning Controls La AlL gt Average Every N Frames 1 CurrentS ettings Screen Shot Scanning Controls Live Scan To start the Ultima scanning click on Live Scan During a live scan this button changes to read Stop Scan Clicking it a second time causes the scan to stop and the laser beam to be shuttered so that it no longer scans across the specimen Only the last image of a Live Scan can be saved Single Scan When Single Scan is selected one image is acquired with the result displayed in the image window s The laser is shuttered following collection of the image a
167. stem settings will change as necessary If the currently selected objective lens is calibrated and the laser source is 2 P and the laser is being controlled by PrairieView then the system settings will change as necessary without any further interaction Note In addition to adjusting the number of pixels in the x and y dimension and or the optical zoom the Nyquist sampling logic will also set the proper Z series step size This action will only happen if on the Z Series tab the radio box next to the Step Size IS NOT set to Calculate Nyquist Sampling Configuration 7A Nyquist Sampling Preferences J Parameter Adjustment Selection Adjust Number of Image Pixels X and Y Ki Laser Type Perform Calculations for 2 P Laser Source Over Sample Constant 22 a Always Prompt for Laser Type and Wavelength lf More Than One 2 P Laser is Present use the Wavelength of Laser v i K Screen Shot Nyquist Sampling Preferences There are several settings to be configured in the File gt Preferences gt Nyquist Sampling dialog Parameter Adjustment Selection allows the operator to choose between having the number of pixels x and y adjusted or by having the optical zoom adjusted Basically there are two methods for getting the desired pixel size one is to increase decrease the number of pixels and the other is to increase decrease the optical zoom The value specified determines the first paramete
168. t Contrast System 6 2 Laser Light Path Laser Light Path 4a After passing through the standard table optics and hard shutter the beam passes into an enclosed beam steering periscope assembly which steers the aligned beam into the sidecar As it enters the sidecar the beam passes through an alignment iris and into the scanhead 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 raster scanning a microscope image when coupled to an appropriate objective lens The beam exits the objective and is scanned across the sample as a means of fluorescent excitation Ultima Scan Head Light Path Top View comers shutter E Seih n scan ne Tt P _ fens 7 imaging pioni i t galvanometers laser camera Que 6 mode control ie Figure Ultima Scan Head Light Path 6 3 Dichroics Dichroics KI a gt The Ultima has multiple mirrors dichroics as shown in the photo above Some of these dichroics are fixed and others are user changeable Combis pae 9 tSS Dichroic Camera Port Mirror Dichroic Image Ultima Scan Head Dichroic Locations Beam Combining Dichroic The Beam Combining Dichroic is a 760nm dichroic used to combine the two 2 photon laser beams so one may be used to image and the other to uncage In single galvo systems this dichroic is not necessary Camera Port Mirror Dichroic A 100
169. t Setup is opened Acquisition and DAC Output Setup Screen Shot Main page access to Acquisition and DAC Output Setup E gt Acquisition and DAC Output Setup I 1000 00 Open Before Starting Data Collection Close After Data Collection Complete Start Stimulus Control with Data Collection 1 1 1 1 0 000 25 000 50 000 75 000 100 000 1 1 0 000 500 000 1000 00 DAC 0 1 2 3 Out 0 00 0 000 Ue 28 Ig ssf gh pt Screen Shot Acquisition and DAC Output Setup The current waveforms for each output are shown graphically on the right hand side of the dialog In the bottom window all active outputs are shown overlaid with each other Each output graph is controlled by the settings entered on the left hand side of the screen To save changes and exit this dialog there are three buttons in the top right corner Update will change the graphs on the right to show any changes made to the outputs Accept Done will save any changes made to the outputs and close the dialog while selecting Cancel will close the dialog without saving changes Acquisition Setup The Acquisition Setup box contains the settings for the overall acquisition Open Before Starting Data Collection Close After Data Collection Complete Start Stimulus Control with Data Collection Screen Shot Acquisition Setup Number of Cycles indicates the number of acquisitions to perform when the Acquire is pressed Cycle Period
170. ta has been collected within TriggerSync and the program is in the Analyze Acquired Data mode is On The first parameter Automatic Gain Sensing takes the minimum and maximum data values for the 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 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 differences in data recorded on those input channels can be more easily compared Manual Sensitivity mV unit is a scale factor applied to the input voltages just as Ext Sensitivity is a scale factor for the output 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 th
171. teps 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 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 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 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 button
172. the area of interest Pan Rotate and Zoom to orient the target region Open the TriggerSync Mark Points dialog Load the appropriate calibration file for the Uncager Calibration File field Mark the locations to be uncaged Open the Acquisition and DAC Output Setup dialog o NDN AON gt Confirm or define stimulus control and acquisition parameters and close the dialog 10 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 11 Select Use Marked Points During Acquisition at the bottom of the TriggerSync Mark Points dialog 12 Verify the Hold and Move times 13 In the TriggerSync main window select Acquisition gt Start Experiment gt TTL Trigger Start this action will place a check mark in front of this menu option indicating that it has been turned on 14 Press Acquire in TriggerSync At this time the TriggerSync acquisition will begin but it will only proceed to the point of waiting for the Trigger to arrive when the start of an image collection from Prairie View 15 Press Single Scan in Prairie View When the image scan begins a Trigger is sent to the TriggerSync acquisition hardware causing TriggerSync to collect the electrical data in synchronization with the image data 16 The previous two steps must be repeated for each com
173. the next cycle can be started directly after the TriggerSync experiment is started Start next cycle immediately 10 4 1 6 Z Series Preferences Z Series Preferences KI a gt 7 T Series Preferences x Z Motor Position After Z Series Default Z Motor returns to position before Z Series started Sal Y Align Z Series center to stage location Align Z Series start to stage location Screen Shot Z Series Preferences Z Motor Position After Z Series Specifies where to move the Z Motor after a Z series has completed There are four options Default will return the z motor to the location it was at before start of the Z series Start will return the z motor to the start position of the Z Series Middle will return the z motor to the middle position of the Z Series Stop will keep the z motor at the stop position of the Z Series These setting will also apply to the z motor placement when executing Z Series from within a T Series protocol Align Z Series center start to stage location When performing Z series at multiple XY stage locations this determines where the Z series will start end By default it will center the Z series on the XY stage location 10 4 2 Tools Menu 10 4 2 1 Tools Menu Options Tools Menu Options 4a gt 1 P Pinhole Calibrate Objective Lens Calibrate Laser Power Center Galvos Notes F8 Scripts b Nyquist Sampling F9 Actions Reset Muti Axis Motor Cont
174. third field indicates the elapsed time since the Calibrate button was pressed 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 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 of the image window to zoom the image window to provide for greater accuracy in placement of the red cursor 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 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 desi
175. tings 8 Click Set under Stop Position in the Z Series tab Each line in the Z Series Slice Data grid will reflect the laser and PMT settings as calculated by Prairie View NODON AOUN gt 10 2 13 T Series Tab T Series Tab all The ability to generate a series of images and data using different labels regions of interest ROIs XY stage locations Z Series Photoactivation masks and Triggers are possible in a T Series In Prairie View 4 x this tab and others may be torn off and placed on the user s desktop T Series Cycle Data Period Max Duration Resource Seri Fhoto Reps s ms Speed s ms Selection SSS activation gt 14814 282907 None None 7 Ei a Add Insert Cycle Remove Selected Add C Insert Clear All a Total Duration s msj ESimated Time Le ThagerSyne otal Duration s ms sms Acquisition Time s Total Images 282 907 289 846 1 Update 20 At all XY stage location Bidirectional Z Series Leave shutter open At all Mark Point location Start with input trigger Save Path C D ocuments and Settings prairie tes BaseDiectoy TSeies0327201071048 O E Star T Series _ Screen Shot T Series Tab The T Series tab allows the user to optimize a complex set of experimental parameters to the acquisition of key data The selected T Series cycle is denoted by a triangle on that line to the left of the Reps column Reps shows the number o
176. to utilize the calibration file After at least one calibration file has been generated then the Mode control associated with the calibrated laser line will include the names of the calibration file s in addition to Default and Attenuation as outlined above When one of the calibration files is selected the laser control label will include the symbol mW after the laser line label When one of the calibration files is selected then the laser slider is internally rescaled such that based on the current operating wavelength for the associated 2 P laser the drive voltage for the Pockels cell will go between the minimum achievable output power not necessarily OmW and the maximum output power that can be attained at all wavelengths within the calibration range For example if the calibration wavelength range was 780nm to 950nm and the lowest maximum power measured across that range was 50mW then when this calibration file is used the maximum output power at any of the calibrated wavelengths will be 50mW If the association between the 2 P laser and the laser control was set in PrairieConfig Utility exe then when the operating wavelength is changed the laser setting will be automatically adjusted to maintain the desired sample power Additional Information The selected laser mode Default Attenuation or Power Calibration file and laser setting will be retained when a label is created used in Prairie View Th
177. ts 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 Use Marked Points During Acquisition The lower left hand corner of the dialog is labeled Use Marked Points During Acquisition Screen Shot Use Marked Points During Acquisition If a list of Marked Points has been created and Use Marked Points During Acquisition in the Mark Points dialog is turned on then these controls are activated If 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 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 msy para
178. uch as Live and Single Scan are the same along with averaging options shutter control and label selections The stage control is also the same with positioning home selection and limit locators for z series The left hand column has several differences Because the camera is a fixed 512x512 image resolution there are no image size options The Image Window Size can be altered but that only magnifies the collected image it does increase or decrease the field of view Also there is no Optical Zoom or Pan Control Dwell Time Per Pixel is also disabled This will be set by the imaging parameters chosen in the SFC Settings dialog Many of the Tabs are the same for SFC and Ultima operation Two tabs do have differences the Laser PMT DAQ tab and the Labels tab The Laser PMT DAQ tab has several differences The Pockels section has been replaced with the power sliders for the visible light lasers enabled in the software Both the PMT and the DAQ sections are disabled in SFC mode The Labels tab is altered to include the visible light laser settings and SFC imaging parameters 13 2 SFC Settings Overview SFC Settings Overview cl a D The SFC settings are controlled in the SFC Settings dialog It can be accessed through the Tools menu There are several different tabs for the components of the system and controlling the imaging parameters 13 3 General Tab General Tab nat SPC Settings 1 Siol x Bra Taggers Motors Scan Setting
179. up to four channels Each preamplifier channel also referred to as a blade can receive two inputs a main and a secondary PMT If only one PMT signal is to be received for each channel the left BNC input is used The right BNC is used when averaging in a second PMT signal such as from a sub stage detector 6 11 Galvos amp AOD Galvos amp AOD nao The Prairie Galvanometer Control Box controls a set of galvonometers carefully tuned for best performance Damage can occur if the galvos are run with the wrong drivers CAUTION Caution Switching cables so that the drivers control the wrong galvos can cause galvo damage 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 driven by TriggerSync 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
180. verlays shows the chosen font characteristics for all image window text These options can be changed by choosing Edit at the end of the desired line To the right of the information a preview of the selected font and color is shown Photoactivation Masks specifies the colors used to display masked regions when creating editing Photoactivation masks These colors can be changed by selecting Edit at the end of the desired line The changes made in this dialog box can be saved by selecting Apply or Accept Accept will close the dialog box while Apply will only update the information leaving the dialog open for further changes The changes can be declined and the dialog closed by selecting Cancel 10 4 5 Applications Menu 10 4 5 1 Applications Menu Options Applications Menu Options Lala TriggerSync Linescan Viewer Screen Shot Applications Menu The Applications menu provides links to programs associated with PrairieView This menu can be extended by defining Actions under Tools TriggerSync is explained in this section More information about Linescan Viewer can be found here 10 4 6 Help Menu 10 4 6 1 Help Menu Options Help Menu Options 4a p Keyboard Shortcuts Luigs amp Neumann Technical Note User s Manual Revision History About Screen Shot Help Menu Under the Help menu the user will find supporting material regarding the Ultima including a copy of the user manual and software revision not
181. 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 6 4 Electronics Overview Electronics Overview Barrier Sample Figure Emission Light Path for Dual Channel Detectors Laer Barrier Filters PNT Dichroic Barrier Filters Primary Dichroic PNT Dichroic Dichmic IR Blockine Filter Figure Emission Light Path for Quad Channel Detectors Lisi The Ultima Multiphoton Microscopy System contains many electronic and optical components In this section the electrical rack and peripheral components will be described Pockels Cell Controller 6 dl a Device Controller Galvanometer Flat Panel Displays Controller Keyboard amp Mouse Tray BNC Breakout Boxes PMT High Voltage amp gt p PC Computer Preamp Power Supplies Pockels Cell Controller Main Rack Power Switch Image Ultima Electronics Rack The Ultima electronics rack contains the majority of electronics necessary to operate the Ultima system Monitors Two flat panel displays are standard with each system Workstation Prairie Technologies provides a workstation to control the Ultima and its components and collect data The workstation comes pre loaded with all of the cards drivers and software necessary to control the Ultima system Device Controller The devic
182. ximum Wavelength nm Goto Goto Step Size nen J10 1 A nm Power amp Current Wavelength nm S00 Im Previous Next 810 820 830 840 mee so Need help with Laser Power Calibration Accept Screen Shot Laser Power Calibration Power Calibration The purpose of the Laser Power Calibration is to provide one or more calibration files that will allow the operator to set the desired laser power in mW at the sample via the laser slider control s in PrairieView This option will be in addition to the two operating modes already available Default and Attenuation The actual power calibration is performed by stepping through a range of wavelengths and setting the drive voltage to the proper Vr at each wavelength and after the system has had a few seconds to settle at the new wavelength and drive voltage the laser power at the objective lens is recorded This creates a table like the following Wavelength nm Power mW 700 200 800 400 1050 50 Using the information in the table when in the power mode the laser power to the sample can be controlled between OmW and 50mW from 700nm to 1050nm The actual calibration process is conducted with the Laser Power Calibration dialog within PrairieView 1 Place a power meter below the objective lens slightly above or below the focal plane The meter will probably deliver better results if the laser light isn t focused to such a fine spot size F
183. ysical 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 NeD Calibration File field indicates the calibration file to use for computing the marked point positions when a 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 Browse next to the NeD Calibration File field and select the desired file Uncager Calibration File field indicates 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 Browse next to the Uncager Calibration File field and select the desired file The Pinhole Motion control will only be active on older Prairie systems that include a NeD Pinhole for near field detection The four blue buttons are used to physically move the selected pointing device They are oriented in a diamond pattern to indicate the direction the pointing device should move in the image when the button is pressed For example if the top most blue button is pressed the pointing device should move up or tow

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