MPScope User`s Guide Version 1.1
Contents
1. Al3 goes to PMT4 or Analog Ch 2 Analog outputs AO0 goes to X scan mirror driver replaces output from PCI 6711 AO1 goes to Y scan mirror driver replaces output from PCI 6711 Digital outputs DIOO Stepper motor for Z focus Up recommended DIO1 Stepper motor for Z focus Down recommended DIO2 Digital stimulator output MPScope User s Manual p 10 Digital Z focus control P TTL lines for peripherals Dual monitor PC Outputs General Purpose Laser intensity control USB Counters boards Analog P x Outputs L y B Port RTSI bus a iia PMT of Video Ch 1 Serial Ports PCI 6711 PCI 6711 PCI 6110E J PMT of Video Ch 2 Analog A Inputs 4 7_ PMT of Video Ch3 or Analog In 1 NEAT 300 Micro PMT of Video Ch4 or X Y manipulators Analog In 2 Controller 2 Digital MIPOS 100 Outputs Z lens positioner mirror drivers Laser beam shutter Analog P Analog Out 1 tput euipals L Analog Out 2 Fig 1 Connection layout of MPScan in the original Kleinfeld configuration Items in italic are mandatory The PCI 6110E or PCI 6115E and the PCI 6711 s have their RTSI buses tied together DIO3 Digital stimulator output DIO4 originally reserved Digital stimulator output DIOS originally reserved Digital stimulator output DIO6 originally reserved Digital stimulator output DIO7 originally reserved Digital stimulator output Digital input PFIx any PFIO to PFI9 line frame acquisition TTL trigger PCI2. Arguments Returns Description Example Visual Basic channellndex 1 to 4 framelndex 1 to Frame Count an array of 16 bit signed integer SAFEARRAY short containing either pixel values or analog channel values for a given frame The size of this array is either that of a video frame for channels 1 or 2 or the number of analog samples per frame for channels 3 or 4 Matlab automatically converts this array into a complex matrix Returns frame data Dim frame as Empty frame mpFile ReadFrameData 1l 117 MPScope User s Manual p 39
3. Frame Height parameters have to be entered in the Configuration window e g click on the Configuration button upper pane of the main window A X Offset 0 Y Offset 0 Frame Width 512 Frame Height 512 selects a frame equals to the field of view A 5 4 1 Movie In movie mode MPScan can display up to 512 x 512 pixels from up to four imaging channels The frame rate will depend on the size of the frame Subframes can be chosen by clicking the mouse on a region and drawing a rectangle over it The Configuration dialog is then shown MPScope User s Manual p 15 Pixel offset adjustment when pixels are misaliqned on screen MPScan uses dual direction scans to generate images The laser beam is swept in one direction to scan one line then in the other direction to scan the following line There is a lag between the actual position of the mirror and the voltage command which is approximately 150 us Its exact value depends on your mirror set When the lag is not compensated objects in the image appear duplicated or have hairs resulting from the misaligment of consecutive lines To prevent this effect pixel offset adjustment must be done in all modes except for region scans to compensate for the mirror lag To adjust for pixel lag 1 Set the rotation to 0 Viewer window Raster panel 2 Scan a sample in movie mode 3 Enter a value for instance 150 in the X pixel line shift input box bottom of the Raster panel and
4. MPScan features an Integrated Development Environment IDE hosting the Microsoft VBScript language interpreter available in Internet Explorer Nguyen and Miledi 2003 To start the IDE click on the Experiment Show Script Editor menu The Script window consists of three panels The left one displays the Type Library of MPScan i e information about objects and procedures of MPScan that can be controlled by the script The right panel is the code window and the bottom one displays messages from the IDE When the IDE is displayed additional MPScope User s Manual p 25 buttons are shown at the bottom of the main window These buttons allow starting and stopping the current script or triggering various actions The caption on these buttons can be changed Script Rename Buttons menu item The default script language is VBScript from Microsoft which is a subset of Visual Basic VBScript programs are composed of an Initialization body similar to the main function in a C program and event handlers which are called when a particular event has taken place It is a good idea to declare all global variables in the script in the Initialization section of the script e g Dim MyVariable1l MyVariable2 etc The Initialization section is executed immediatly after the script is started then the script interpreter waits for events to occur An event is triggered when scanning starts scanning ends when a section is done or when a button is clicke
5. Open MPScope User s Manual p 28 MPXYTable MoveToxy Syntax MoveToxY X Y Tells the X Y translation table to go to absolute positions Xand Y in microns ShiftByXxy Syntax ShiftByXY deltaX deltaY Tells the X Y translation table to shiftt by deltaXand deltaY in microns XPosition Reads the current X position of the X Y translation table YPosition Reads the current Y position of the X Y translation table XYCommand Syntax MoveToXY command_string Sends a command string to the XY controller usually via the serial port XYSpeed Syntax XYSpeed new speed Sets the speed of the XY controller 1 lt new_speed lt 20 MPZStepper ZPosition MoveToz ShiftByZ ZSpeed Syntax ZSpeed new speed Sets the speed of the Z stepper 1 lt new speed lt 20 Matlab Start Starts the predefined instance of Matlab PutROI Puts the values of an ROI trace identified by its index from 1 to the number of ROIs into a named Matlab array Execute Executes a Matlab command GPCTR The GPCTR object controls one of the General Purpose Counters GPCTR on the PCI 6110E PCI 6115E The default operation is a pulse train with duty cycle set by the Counter_1 and Counter_2 values However finer control including other operating modes are possible Refer to the NI DAQ reference for GPCTR operations and function parameters MPScope User s Manual p 29 Reset Stops the current operation of the counter Counter 1
6. PCI 6110E or PCI 6115E at precise times with a resolution of 1 ms To set up the stimulation pattern select the Experiment Digital Stimulation menu item A window is displayed Some of the output lines are used by legacy devices Select the appropriate lines and stimulation times Digital stimulation can be armed before scanning or can be initiated before scanning by clicking on the Start Digital Stimulation button in the main window Stopping scanning immediatly stops digital stimulation A 5 12 Analog Stimulator A 5 13 Real time Pixel Histogram window The histogram window displays the count of binned pixel values in incoming frames A running histogram of pixel values in incoming frames is useful to diagnose potential problems MPScope User s Manual p 20 Select the Windows Real Time Pixel Histogram Window menu item in the Video window This option is only available in Movie mode A 5 14 Real time Frame Averaging window A running average of incoming frames is useful when signals are noisy or weak Select the Windows Real Time Frame Averaging Window menu item in the Video window This option is only available in Movie mode Up to 15 frames can be averaged A 5 15 Real time ROI average intensity plot Real time plots of regions of interest is possible only in the Movie scanning mode From the viewer window depress the Rectangular or Elliptical Real Time Plot buttons Select a rectangular or elliptical ROI on t
7. be Base Frame Name Extension ex Frame 13 The Extension is automatically incremented for each frame There is no need to start Matlab with main window button or Matlab menu before exporting frames as this will be done automatically Exporting frames to TIFF files MPView can save consecutive frames of a given video channel into a multi frame TIFF file MPView maps native 12 bit encoded pixel values from 0 to 2047 into 16 bit values ranging from 0 to 65535 If negative native values are encoutered in the frame s they are converted to O and the user is notified of the truncation The ImageDescription tag of each fram contains additional information such as frame rate X Y and Z positions of the stage etc B 3 11 Creating video clips MPView can create uncompressed AVI video files from series of frames The original frame rate can be changed to slow down or accelerate the movie during replay B 3 12 The Matlab Console Matlab can be controlled from within MPScope via the Matlab console Main window Matlab Start Matlab menu MPView prompts the user to use either a Local Server installed on the current machine or a Remote Server located on another machine belonging to a Local Area Network The latter option requires i Matlab to be installed on the remote computer ii knowing the name of the remote computer iii setting up DCOM on both machines This step is mandatory but not trivial Refer to instructions available on the Web f
8. electrode up and down A 5 18 Laser Control If laser control was enabled in the Hardware configuration window laser power and with certain types of laser systems wavelength can be adjusted from the X Y Z Laser panel Intensity control can be achieved with lasers that are not computer controlled as described in Nguyen et al 2006 J Neurosci Methods Connect the GPCTR1 output line of the PCI 6110E or PCI 6115E main acquisition board to the servo In the Hardware Options window choose Phil s power control and input the parameters that determine the duration of MPScope User s Manual p 24 the pulse width coming from the GPCTR counter The pulse width controls the rotation angle of the servo mechanical output Because of hysteresis in this arrangement two sets of parameters are needed one for rotation in one direction Power Increase and one for the reverse direction power decrease A 5 19 Z objective lens piezo electric positioner MPScan provides the capability to perform fast X Z imaging thanks to a PiezoJena MIPOS 100 Z objective lens piezoelectric position To enable the MIPOS 100 one PCI 6711 board has to be installed and assigned as the Optics Control Board Main menu gt Hardware Options The RTSI buses of the PCI 6110E or PCI 6115E and that of the PCI 6711 have to be connected In the Hardware Options dialog in the Z Piezo page the type has to be set to MIPOS 100 Set the list box to the desired analog ou
9. peripherals General Purpose Laser intensity control Counters Analog ei x mirror drivers Outputs 7 y USB Ports RTS bus PMT of Video Ch 1 Serial Ports PCI 6711 PCI 67 11 PCI 6110E J PMT of Video Ch 2 Analog al Inputs 7 PMT of Video Ch3 or Analog In 1 MP 285 Micro PMT of Video Ch4 or Micro manipulators Analog In 2 manipulator 2 controlling MIPOS 100 X Y Z focus Z lens positioner Analog P Analog Out 1 Outputs Spaa Ly Analog Out 2 Fig 2 Connection layout of MPScan in the Denk or Svoboda scopes Items in italic are mandatory The PCI 6110E or PCI 6115E and the PCI 6711 s have their RTSI buses tied together PCI 6110E or PCI 6115E Digital outputs any digital output 8 lines total can be used to deliver a TTL compatible signal In addition one digital output line can be used to control the laser beam shutter COM Ports One COM port can be used to connect to the MP 285 microelectrode positioner that controls the X Y Z focus in some microscope realizations In the Hardware configuration panel MPScope User s Manual p 12 choose the Invert Z motion check box if the MP 285 moves the preparation rather than the objective Prior to starting MPScan set the MP 285 microstep resolution to coarse i e 0 2 um microstep A 4 3 Connecting optional equipments TTL trigger The digital trigger uses a digital high TTL level on any PFI line to start a scan Digital outputs could be TTL low MPC 2
10. 6711 board 1 Analog output AO0 not used or used as output 0 10 V to drive the MIPOS 100 Z objective positioner MOD input PCI 6711 board 2 Analog output AO0 not used or used as Analog stimulator output channel 1 AO1 used as Analog stimulator output channel 2 Digital output DIOO goes to laser beam shutter If any PCI 6711 board is to be used connect its RTSI lines to that of the PCI 6110E or PCI 6115E The RTSI bus will carry signals to synchronize all the boards COM Ports COM port number assignement will depend on the configuration COMx Danaher NEAT 300 X Y controller MPScope User s Manual p 11 COMy Sutter MP 285 motorized micromanipulator 1 COMz Sutter MP 285 motorized micromanipulator 2 USB ports A variety of optional peripherals can be connected to the USB ports USB x a PMD 1608 FS low cost 8 channel 16 bit data acquisition USB module MPScan provides an ActiveX library allowing scripts to measure up to 8 different analog voltages USB y Sutter MP 385 USB controlled micromanipulator 1 USB z Sutter MP 385 USB controlled micromanipulator 2 A 4 2 Other microscope hardware configurations MPScan is compatible with the Denk or Svoboda two photon laser scanning microscope design Figure 2 Very little modifications are required to run MPScan Refer to the previous section for most connections Differences are listed below Digital Laser beam shutter i Outputs Dual monitor PC upy i TTL lines for
11. 85 microelectrode positioner MPScan can control up to two MPC 285 microelectrode positioners Sutter Instruments for automatic placement of sharp or patch clamp pipettes or to move the microscope field of view in the X Y Z directions Connect the MPC 285 controller to an unused serial port on the PC If no serial port is available USB serial port converters can be used ESP 300 stage controller MPScan supports the Newport ESP 300 three axes controller to move the focus in the X Y and Z directions Connect the ESP 300 to the computer and enter the appropriate information in the Hardware Configuration dialog box in MPScan Phil Tsai s laser control Laser intensity can be controlled easily by connecting a cheap servo mechanism 25 used in model boats to the zero order half wave plate rotating device Laser light attenuation goes like the cosine of the plate rotation angle Connect the command input of the servo to the GPCTR1 Output of the PCI 6110E or PCI 6115E board See the Laser Control section in this manual Photon integrator Photons detected by the PMTs can be integrated during the dwell time of the laser above each pixel A TTL compatible pixel clock is available at the SCANCLK_LINE output of the PCI 6110E or PCI 6115E Z Axis objective lens positioner Connect the AOO output of the PCI 6711 board 1 to the MOD input on the controller of the MIPOS 100 A 5 Using MPScan A 5 1 Starting and Exiting MPScan Important In the Kleinfe
12. File is MPFile Data Example Visual Basic mpFile CreateObject MPFile Data MPFile features three methods OpenMPFile ReadParameter and ReadFrameData C 4 2 OpenMPFile Method Argument filename Returns 0 if no error or a numeric code corresponding to a standard Win32 error Description Opens an MPScope file for reading Example Visual Basic openresult mpFile OpenMPFile c test mpd C 4 3 ReadParameter Method Argument parameter name Returns the value of the parameter as a string or an empty string if there is no match Description Reads the value of a named parameter All parameter names are case sensitive A II MPScope files store the following parameters Scan Mode X Frame Offset Y Frame Offset Channel Name 1 Enabled 1 Input Range 1 Channel Name 3 Enabled 3 Input Range 3 Channel Unit 3 Channel Prefix 3 Conversion Factor 3 Offset 3 Data Point Per Frame 3 Frame Count X Position Y Position Z Position Stack Count Frame Width Comments Frame Height Pixel Clock Channel Name 2 Enabled 2 Input Range 2 Channel Name 4 Enabled 4 Input Range 4 Channel Unit 4 Channel Prefix 4 Conversion Factor 4 Offset 4 Data Point Per Frame 4 z Interval Averaging Count Repeat Count Magnification Rotation MPScope User s Manual p 38 Example Visual Basic frameCount mpFile ReadParameter Frame Count C 4 4 ReadFrameData Method
13. Frame size 512 lines x 512 pixels down to 100x10 Dual direction scans for the fast mirror Five acquisition modes Movie 512 x 512 pixels 4 video channels with streaming up to 5 fps 256 x 256 pixels 4 video channels with streaming up to 10 fps 30 fps for smaller frames Stack Frame averaging possible for each section Linear or exponential automatic constant illumination control time series of stacks Option to close shutter between section Generates a scripting event each time a section is acquired Fast Stack 200 microns in z 1 micron per section 256 x 256 in 20 seconds 600 microns in z 1 micron per section 256 x 256 in 90 seconds 1000 microns in z 1 micron per section 256 x 256 in 240 seconds Linear or exponential automatic constant illumination control Time series of stacks Line Scan as fast as 2 6 kHz per line Region Scan Select multiple rectangular elliptic or polygonal regions MPScan will create a raster pattern and scan it Display Subframe selection with mouse Frame magnification on screen MPScope User s Manual p 6 Black and white false colors custom color LUT for each video channel Frame overlay for targeted patch clamping On line PMT offset correction Rotation zoom While you scan software rotation and zoom Real time display Real time histogram of binned pixel values Real time frame averaging window Real time ROI Region Of Interest plotting Scrolling plot of ave
14. GB RAM A National Instruments PCI 6110E OR PCI 6115E multifunction board MPView does not need Labview but requires Traditional NI DAQ 6 5 or up to be installed The microscope should include at minimum A TTL triggered laser beam shutter Two galvanometric scan mirrors e g Cambridge Technologies 6210 for X and Y scans with their MicroMax 670 driver boards Up to four PMT detectors providing a voltage output usually via a current voltage converter The voltage outputs have to be low pass filtered at a cut off frequency ranging from 150 to 300 kHz Multipole Bessel filters are preferred Alternatively a high speed photon integrator can be usedl Optional microscope equipments include A stepper motor to change focus see Tsai et al 2002 highly recommended A Neat 300 X Y translation table see Tsai et al 2002 A preferred solution is to control X Y and Z motions using a Newport Instruments ESP300 three axis controller with associated translation stages Alternatively a Sutter MP285 microelectrode manipulator can be used to move the focus along the X Y and Z axes MPScope User s Manual p 9 Up to two National Instruments PCI 6711 analog output board The Kleinfeld configuration uses these boards to generate the X and Y scan voltages MPScan uses one board to output up to two analog voltages to drive stimulation inputs of microelectrode amplifiers The other board can be used to control addit
15. Laser control A 5 19 Z objective lens piezo electric positioner A 6 ActiveX Automation Features A 6 1 Server Features A 6 2 Scripting Features A 6 3 Automation Reference A 6 3 1 Predefined objects A 6 3 2 ActiveX Automation Server Objects Properties and Methods A 6 3 3 ActiveX Automation Events A 7 Known Bugs B MPView B 1 Summary of features B 2 System Requirements B 3 Installation B 4 Using MPScope B 4 1 Opening a data file B 4 2 Viewing frames B 4 3 Examining the data file parameters B 4 4 Reviewing a data file B 4 5 Opening a workspace B 4 6 Copying and averaging frames B 4 7 X Y and Z projections B 4 8 Measuring pixel intensities B 4 9 Creating and Plotting Regions of Interest B 4 10 Exporting frames B 4 11 Creating video clips B 4 12 The Matlab Console C MPFile C 1 System Requirements C 2 Installation and registration C 3 Using MPFile in Matlab C 4 MPFile Reference C 4 1 Instantiating MPFile C 4 2 OpenMPFile Method C 4 3 ReadParameter Method C 4 4 ReadFrameData Method INTRODUCTION MPScope is a suite of programs to acquire and analyze data from ultrafast laser scanning microscopes MPScope was originally written for the setup developed in David Kleinfeld s laboratory at the Physics Department UCSD Detailed description of this microscope can be found in Tsai et al 2002 However MPScope can be used with other systems based on identical scanners and data acqu
16. MPScope User s Guide Version 1 1 April 2006 Quoc Thang NGUYEN Physics Department UCSD qnguyen physics ucsd edu DISCLAIMER The programs described in this manual are available as is The author takes no responsability in personal injury loss of data material or time that could result from using MPScope Please report bugs or suggestions to qnguyen physics ucsd edu Copyright Quoc Thang NGUYEN 2005 2006 All Rights Reserved INTRODUCTION A MPScan A 1 Summary of features A 2 System Requirements A 3 Installation A 4 Hardware connections A 4 1 Original Kleinfeld configuration A 4 2 Other microscope hardware configurations A 4 3 Connecting optional equipments A 5 Using MPScan A 5 1 Starting and Exiting MPScan A 5 2 Login A 5 3 Setting the hardware configuration A 5 4 Choosing a scan configuration A 5 4 1 Movie A 5 4 2 Stack A 5 4 3 Fast Stack A 5 4 4 Line Scan A 5 4 5 Region Scan A 5 4 6 Starting and stopping a scan A 5 4 7 Frame rotation and zoom A 5 4 8 Acquisition parameters A 5 5 Display A 5 6 Saving data to a file A 5 7 Scan configuration A 5 8 Saving and retrieving a configuration A 5 9 X Y Z Control A 5 10 Waypoints A 5 11 Digital Stimulator A 5 12 Analog Stimulator A 5 13 Real time Pixel Histogram window A 5 14 Real time Averaging window A 5 15 Real time ROI average intensity plot A 5 16 Analog inputs A 5 17 Controlling motorized micromanipulators A 5 18
17. PFile were written in the Matlab language However MPFile could be used in any language supporting Activex Automation such as C C Delphi or Visual Basic Matlab or LabView C 1 System Requirements MPFile requires a PC running Windows 2000 or Windows XP If using Matlab as the Automation Client MPFile will work with Matlab release R14 or later If using R13 apply the solution described at the Mathworks Web site in Technical Solutions 1 1B2JW www mathworks com support solutions data 1 1B2dW html C 2 Installation and registration Copy MPFILE OCX to the directory of your choice Open a console window and type regserv32 path MPFILE OCX to register the component with Windows C 3 Using MPFile in Matlab This example is written in Matlab Refer to the Automation Client documentation of Matlab for further details f figure 300 300 500 500 mpfile actxcontrol MPFile Data 0 0 500 500 f openResult invoke mpfile OpenMPFile c MCC 050105 006 MPD openResult 0 frameCount invoke mpfile ReadParameter Frame Count frameCount 150 ActiveX controls reside in figure windows The first line opens such a figure The MPFile OCX is instantiated in the window The OpenMPFile takes the full file name as its parameter and returns 0 if everything is OK The ReadParameter returns a string C 4 MPFile Reference C 4 1 Instantiating MPFile MPScope User s Manual p 37 The ProgID of MP
18. Write only The duration of the first interval in a train A multiple of the time base 50 ns 20 MHz From 1 to 27 1 Counter 2 Write only The duration of the second interval in a train A multiple of the time base 50 ns 20 MHz From 1 to 27 1 Start Prepare and arms the counter SelectSignal SetApplication Control Example This script snippet generates a pulse train on the GPCTR 0 output line With GPCTRO Reset Counter 1 1000 50 microseconds for first interval Counter 2 10000 500 microseconds for second interval Start End With MPLaser Power Adjustable from 0 to 100 in Wavelength A 6 3 3 ActiveX Automation Events Code can be automatically executed by MPScan in response to the following events One of the 10 user available buttons at the bottom of the main window has been clicked OnAcquisitionStart Triggered immediatly before frame acquisition starts OnAcquisitionEnd Triggered when frame acquisition has ended OnSectionEnd Triggered each time a new section in a stack was completed OnNewFrames Triggered each time frames have been received You can use this event to send ROI values to Matlab in near real time To perform an action during an event add code to the appropriate event handler Be sure to check the Enable Event check box in the Script Editor MPScope User s Manual p 30 A 7 Known Issues and Bugs 1 Stack averaging with count of 1 produces inv
19. alibrate distances button displays a window Calibration consists of placing the tip of the electrode at four different points in space forming the summits of a cube and registering the position of the electrode The pipette tip filled with a fluorophore is to be placed at the center of the field indicated by the intersection of crosshairs by moving the field of view in the X Y or Z directions Once calibration is done MPScan can calculate the motions of the pipette with respect to the position of the targeted cell Click on the Calibration button to show a the Calibration window MPScan will ask first to position the pipette tip at the center of the crosshairs and register its position Position 1 in Fig 4 MPScope will then move the pipette to the other positions and will ask to position the tip at the crosshairs of the screen using the translation table and z stepper controls NOT with the micromanipulator motion devices Once 4 points have been registered MPScan can map any coordinate in the X Y Z space of the microscope with a coordinate in the x y z space of the micromanipulator l PERR T position Microscope translation axes Fig 4 Calibration principle MPScope User s Manual p 23 Approach Descent Descent Position Contact Position A Target Home 1 X Z Y Descent Position Contact J Target Contact Position Fig 5 Home Descent and Contact positions At this stage the user can regis
20. alog outputs Seal Test starts after the corresponding button is depressed and stopped when the user clicks again on the button MPScan outputs a 200 ms 50 duty cycle square pulse 50 ms of baseline at 0 mV or 0 nA 100 ms at the user selected level and 50 ms baseline at 0 mV or O pA via the selected output channel and records one or two channels depending on the analog input configuration The seal or electrode resistance is calculated by averaging and subtracting the baseline values from the last 20 ms of the step interval Negative values for the user selected levels can also be entered if for instance one wishes to measure the membrane resistance of a given cell In all modes scanning Free Run or Seal Test the display can be adjusted by the user Colors and grid lines can be set by selecting the Settings menu item under the Display menu title To alter Y magnification or Y offset from the Analog window select the channel to modify Ch 1 or Ch 2 then move the scroll bar to change the offset or click on the arrows to change magnification Y Magnification ranges from 1 to 32 To reset the offset to 0 click on the scroll bar and type 0 zero During scanning analog inputs are acquired only if the scanning mode is set to Movie Line scan or Region Scan The time base i e the duration of the X axis for any analog channel is set to the duration of a frame Each screen will display all the analog data tak
21. aved and restored from disk Pulse train generation Script control of the two General Purpose Counters GPCTR on the PCI 6110 6115E Can be used to generate digital pulse trains with 50 ns accuracy during scripts MPScope User s Manual p 7 Electrophysiological acquisition Two analog channels inputs 20 10 5 2 1 V selectable input ranges Gap free during frame acquisition Always synchronized with video channels Sampling rate reduced by decimation Displays data in separate oscilloscope like window Two non imaging modes free run and seal test Seal test can also be done during imaging Analog stimulator via one PCI 6711 2 x 10 V 12 bit analog channel outputs Constant steps trains seal test and incremental amplitude mode Triggered by frame acquisition Analog stimulation patterns can be saved and restored from disk Micromanipulator control for two motorized micromanipulators Sutter MP 285 Manual control of X Y Z and X Z or Y Z diagonal axes Targeted patch clamping mode Overlay mode in display Advanced file format Combined optical electrophysiological data streaming during acquisition Advanced NTFS based file system 64KB sector writing for faster disk streaming Event driven scripting Allows users to write their own programs to control the microscope ActiveX Scripting Integrated Development Environment editor debugger and type library viewer Users can write event driven programs in the Microsoft VBScript la
22. ce of scripts It is unnecessary to reference them in order to invoke their methods or access their properties i e MPScope User s Manual p 26 MultiPhoton LoadProtocol myProtocol PRT can be abbreviated as LoadProtocol myProtocol PRT In addition MPScan can control the two General Purpose Counters GPCTR available in the PCI 6110E PCI 6115E board These counters are represented by the obejcts GPCTRO and GPCTR1 Shortcut methods and properties are available to generate TTL compatible pulse trains on the GPCTRO and GPCTRO outputs Other more general methods can provide script writers with finer control over counter functions These methods are identical to those in the NI DAQ application programming interface A 6 3 2 ActiveX Automation Server Objects Properties and Methods Browse the Type Library Left panel of the Script Window for information on objects properties and methods MultiPhoton AddFileProperty Adds a custom file property value pair to the Summary Information stream of a data file This pair can be subsequently read either in MPView or can be inspected by clicking the file icon with the right mouse button then selection Properties and the Custom tab Syntax AddFileProperty propname propvalue where propname and propvalue are strings Example AddFileProperty Laser Power CStr laserPower FileComments Laser Shutter Predefined object LoadConfiguration SetLaserBeamTo Syntax SetLaserB
23. computers are too fast to communicate with the MP 285 and could get erroneous position information back from the controller In this case increase the Read Delay parameter until the x y Z positions are correct A 5 4 Choosing a scan configuration MPScan offers the following scan modes Movie Stack Fast Stack Line Scan and Region Scan These modes can be selected in the Video window in the Scan panel Full frames are acquired by the following 511 modes Movie Stack and Fast Stack The entire field of view determined by the maximal mirror deflection voltage Settings Hardware Options then Mirrors panel and the zoom factor Viewer window Raster panel spans 512 x 512 pixels Any rectangle inside this region can be scanned Cambridge Technology 6210 scanners provide a 20 deflection for input voltages spanning 10V Typically the maximal mirror deflection voltage is 2 or 2 5 V Zoom factors adjustable from the Viewer window can be set between 1 and 10 The zoom factor reduces the field of view by decreasing the mirror deflection mirror voltage zoom 0 X Offset 128 Y Offset 128 256 of field ns lt D D I 0 E i rm Frame Width 256 511 f Fig 3 Configuration parameters for a 256 x 256 pixels frame Fig 3 shows the layout of a 256 x 256 frame centered in the field of view To scan the grayed region in Fig 3 the appropriate values for X Offset Y Offset Frame Width and
24. d Each time a event happens the code associated with this event is executed A program terminates when the user clicks on the Stop Script button or calls the EndScript procedure Script source code can be saved to disk recalled or printed Browser Open Type Library opens a type library for browsing If Matlab is installed in your computer try opening for instance C MATLAB6p5 bin win32 mlapp tlb Browser Load Reference reloads the type library of MPScan Example Enter the following line in the main window MsgBox Hello World Click on the Start Script button A window with the text Hello World should appear Click on OK then on Stop Script To enter code for a given event select the event from the list box on the Script Editor window and enter the event code Typically the main section will contain initialization code The script will then wait for events On line help The editor features an on line help Highlight a VBScript keyword and click on F1 to open the VBScript help file For complete help click on the Help VBScript Reference menu item Saving retrieving and printing scripts Scripts can be saved to files reloaded from data files or printed See the File menu in the main window A 6 3 Automation reference A 6 3 1 Predefined objects The MPScan Automation Server defines and instantiates the following global objects MultiPhoton MPLaserShutter MPXYTable MPZStepper These names are part of the namespa
25. eamTo X Y where X and Y are integer values from 0 to 511 This command sets the laser beam to absolute position X and Y by moving the mirrors StartDigitalStim StopDigitalStimulation StartScan StopScan StartStreaming StopStreaming SetDigitalBit MPScope User s Manual p 27 NewDataFile Syntax NewDataFile or NewDataFile lt filename gt Ex NewDataFile c mydata myfile mpd The lt filename gt argument is optional If omitted MPScan chooses a default filename yymmdd_xxx MPD where yymmdd is the year month and date using two numerals xxx is an index from 1 to 999 Wait XY_Table Predefined object Z Stepper Predefined object LastFrameIndex The index of the last frame acquired Matlab A predefined instance of Matlab Scanning 0 if not scanning 1 when scanning DoEvents The analog of Visual Basic DoEvents Use it with Scanning to wait for the completion of a scan like Waiting for a scan to finish While Scanning lt gt 0 DoEvents Wend YScanOffset Reads or sets the the value of the Y offset of the scan pattern EndScript Immediatly ends the script Same as if user would end script manually Use this procedure to terminate a program GPCTRO General purpose counter 0 GPCTR1 General purpose counter 0 Laser Laser control for wavelength and power WriteMessage Syntax WriteMessage message string Writes a message string to the Message window of the Script Editor MPLaserShutter Close
26. en during a frame It is also possible to run a seal test while scanning is in progress in Movie or LineScan This mode is useful when making visual contact on a neuron Start a scan then click on the Seal Test button This will temporarily stop scanning initiate the seal test and restarts the scan This sequence is necessary to synchronize scan data and analog output During the subsequent scan a seal test waveform is delivered every frame and the pipette resistance is displayed In this mode the entire Seal test pulse lasts exactly one frame and has a 50 duty cycle A 5 17 Motorized micromanipulators Up to two Sutter Instruments MP 285 and in the future MP 385 micromanipulators can be controlled either manually or automatically to perform two photon patch clamping The user needs to register the initial position of the pipette using the Calibration procedure and the position of the targeted cell Targeted patch clamping is only possible in Movie mode User interface to control the micromanipulators is located in the Manipulators panel of the main window Manual control This mode allows positioning of the electrode anywhere along the four axes of the micromanipulator Clicking on the buttons will move the manipulator by a distance specified in the Increment input box at a given speed In coarse mode speed cannot exceed 2900 microns s while in fine mode speed is limited to a maximum of 1310 microns s For motions across a fourth axi
27. erted frames Description If stacks are performed with an average of 1 alternate frames are shown inverted Fix Use an even average count This bug is being investigated 2 Very rare random crashes during acquisition due to network activity Description The computer displays a Blue Screen of Death or resets during acquisition This happens extremely rarely and is due to a bug in the National Instruments driver This bug not only affects MPScan but also Labview programs Fix During acquisition it is recommended to disconnect the computer from the network 3 Error message after creating a new directory Description When creating a new data file if the user creates a new directory in the file creation dialog and selects it to place the new data file a Windows error message appears Fix Disregard the wrongful error message and select the directory anyway MPScope User s Manual p 31 B MPView MPView is the analysis program of MPScope It is designed to let users to review and analyze their files efficiently Further frame post processing is eased by the ability to rapidly export raw or processed frames either to Matlab or ImageJ B 1 System Requirements MPView requires a PC running Windows 2000 or Windows XP If desired MPView can export frames directly to Matlab The Mathworks Natick MA B 2 Installation Copy MPVIEW EXE to the directory of your choice MPView is designed to interface seamlessly with Matlab B
28. es are essentially similar to data files at the exception that they contain frame data from only one channel Workspaces can be saved as data files B 3 6 Copying and averaging frames MPScope User s Manual p 33 Destination for these operations must be an empty workspace B 3 7 X Y and Z projections X Y and Z maximal projections can be performed on contiguous frames The result is stored in a workspace If the operation fails due to a problem with frame size create a new blank workspace and try again with the new workspace B 3 8 Applying gamma correction Destination for these operations must be an empty workspace B 3 9 Measuring pixel intensities To obtain a pixel statistics of an area click on the Operations Area statistics menu item and select a region to analyze by clicking and dragging the mouse above the region A dialog box will appear with the statistics of the area To obtain a pixel intensity plot of across a line click on the Profile button on the Viewer then draw a line by clicking the mouse and dragging it and releasing the mouse A Profile window will appear B 3 10 Creating and plotting regions of interest Three types of regions of interest ROIs can be created Users can select either a rectangular or an elliptical ROI with the mouse by clicking and dragging In case two channels were acquired MPView will ask if the same region should be created in the adjacent channel Once a rectangular
29. h viewer has navigation buttons lt lt lt Stop gt gt gt corresponding to menu items under the Frame menu To go to the first frame last frame or a particular frame in the file click on the Frame First Frame Frame Last Frame or Frame Go to Frame menu items Keyboard shortcuts include Left Arrow Next Frame Right Arrow Prev Frame Ctrl G Got to Frame The menu item Frame Comments allows users to add a comment to the current frame Users can choose to display frames either in Gray Scale default in False Colors or using custom colors View Gray Scale View False Colors View Custom Colors Custom color schemes are selected using the dialog shown under View Custom Colors Look Up Table Pixel values below 0 are shown in green or black in gray scale or false colors respectively Frames can be enlarged or reduced View Increase Zoom View Decrease Zoom B 3 3 Examining the data file parameters To examine the data file parameters click on the File File Information menu item B 3 4 Reviewing a data file Multiple independent views of the same data file can obtained by selecting the View New Frame Viewer menu item Commands include fast forward and reverse next frame previous frame and go to frame B 3 5 Opening a workspace Users can create workspaces File New Worskpace File which are groups of frames residing in memory Workspaces are usually created to store results from frame operations Workspac
30. he frame by clicking and dragging the mouse over a region of interest Once the left mouse button is released a dialog window will appear The ROI color in the Plot window and whether the ROI is set as background can be selected by the user Each new region of interest will create a new subplot in the ROI Live Plot window The ROI Live Plot window will display the averaged pixel value of each ROI corrected with the value of the background ROI for the appropriate channel if such ROI was selected by the user When multiple channels are enabled MPScan will ask if a given ROI should be replicated in the other channels The ROIs can be used to generate a real time plot of FRET or ratiometric calcium concentration A 5 16 Analog inputs Analog inputs can be visualized in the Analog window These inputs have to be enabled in the Configuration window Up to two channels of analog inputs can be set up using the Al2 and Al3 inputs of the main acquisition board These inputs are renamed Analog Ch 1 and Analog Ch 2 To use the analog input channels go to the Configuration window enable the Al2 or Al3 inputs but uncheck the Use as Imaging Ch box It is recommended to give the channels a meaningful name and units Because analog inputs usually do not require a high acquisition rate these signals can be subsampled by inputting a value in the Data Points Frame input box This will correspond to an acquisition frequency calculated below Norma
31. ing Edition VBScript MPView the analysis program can perform Frame operations such as viewing frame X Y or Z stacking averaging and linear combination Frame export either as bitmap or multi frame TIFF files Region area or line statistics Automatic object detection and ROI plotting Extended support for Matlab via ActiveX Automation including direct export Creation of AVI movies MPFile is an ActiveX control designed to read MPScope data files It is intended to be used in programming environments such as Matlab or Visual Basic MPScope and its source code is available free of charge to researchers in academic institutions However because of the preliminary nature of MPScope this software package should not be distributed without the consent of the author Acknowledgement of the use of MPScope in published work will be appreciated Nguyen Tsai and Kleinfeld 2006 J Neurosci Methods in press MPScope was developed thanks to the support of the National Institute of Mental Health MH 071566 to Quoc Thang Nguyen and the Kleinfeld laboratory Special thanks to Philbert S Tsai UCSD for testing and suggesting features and to Chris Schaffer and Nozomi Nishimura Cornell University for helpful discussions MPScope User s Manual p 4 REFERENCES For more information on the microscope hardware refer to P S Tsai N Nishimura E J Yoder E M Dolnick G A White and D Kleinfeld Principles design and cons
32. ional optical components such as a PiezoJena MIPOS 100 Z objective positioning piezoelectric actuator to provide a fast X Z scanning capability In case any of the extra boards are used they need to have their RTSI bus connected in hardware to the main board Up to two MPC 285 motorized micromanipulators from Sutter Instruments for two photon patch clamping Note The rotator described in Tsai et al 2002 is not needed by MPScope and can be bypassed A 3 Installation Copy MPSCAN EXE and SCRPT56 CHM to the directory of your choice A 4 Hardware connections Hardware connections depend on the way the microscope was built If the microscope is a copy of the one described in Tsai et al 2002 refer to section A 3 1 otherwise follow instructions in section A 3 2 MPScan can interface with other devices via the output lines of the digital port on the PCI 6110E PCI 6115E such as stimulators via a serial port when using the MPC 285 microelectrode positioner or can be externally started via a TTL trigger Refer to section A 3 3 for wiring instructions A 4 1 Original Kleinfeld configuration Text in bold refers to mandatory hardware modifications to the Kleinfeld design Text in italic highlights optional modifications to accomodate new features Refer to fig 1 for general wiring layout PCI 6110E or PCI 6115E Analog inputs AIO goes to PMT1 Al1 goes to PMT2 Al2 goes to PMT3 or Analog Ch 1 ex cellular amplifier etc
33. isition board In particular MPScope should be compatible with the multiphoton systems developed by Winfried Denk MPI Heidelberg and Karel Svoboda HHMI MPScope is specifically designed for in vitro ex vivo or in vivo systems neurophysiology It is a turnkey professional looking software with superior performance enhanced functionalities and intuitive user interface Many of these advantages derive from advanced features found in Windows 2000 and XP MPScope consists of two programs MPScan and MPView and one utility MPFile These executable files are compiled and stand alone i e they do not require run time libraries or specific programming environments to run MPScan is the acquisition program of MPScope This program features Several scanning modes including fast frame stack fast stack line scan and arbitrary region scanning X Y and Z control including waypoints storage and recall Real time display of average values of regions of interest ROIs Live display of frames averaged frames and frame pixel intensity histograms Buit in frame rotation and optical zoom Simultaneous gap free acquisition and live display of up to two analog channels A dual output analog stimulator and an 8 bit digital stimulator Automated two photon targeted patching for up to two electrode micromanipulators An ActiveX scripting environment allowing users to write their own programs from within MPScan in Microsoft Visual Basic Script
34. ld configuration if the X and Y scan signals AOO and AO1 go through the rotator box before going to the mirror controllers set the Tandem value to 2048 and zero the offsets of rotator Before starting MPScan make sure that Labview is not running Exiting MPScan is done by clicking on the File Exit menu item MPScope User s Manual p 13 A 5 2 Login MPScan allows users to have different settings such as their private data directory Login can be either as Default User or by entering one s name Click on the MPScan icon to start Only one copy of MPScan is allowed to run at any time A login window will be displayed Enter the name of the user Each user has the following parameters which are stored privately last scanning configuration used data directory protocol directory contains scanning configurations analog or digital stimulation parameter files and scripts position of some windows MPScan will display either the successful connection or the failure to detect hardware such as the X Y table Click on OK The main window of MPScan includes several permanent windows The Video window which shows frames from up to two video channels The Analog window which displays incoming analog signals in an oscilloscope like screen The Histogram window The Averaging window The X Y Z control panel Some of these windows are minimized or hidden depending on the scanning configuration If this is
35. lly analog inputs are displayed in the Analog window in an oscilloscope like manner during frame acquisition However it is possible to acquire these analog inputs without scanning Two analog acquisition modes independent of scanning are available The first one called Free Run allows viewing analog inputs in a scrolling display Sampling rate and maximal duration of the display Time Base can be set by the user The sampling rate can be adjusted as low as 0 05 ms corresponding to a sampling frequency of 20 kHz or as high as 1 ms 1 kH with increments of 0 1 ms Start Free Run by clicking on the button in the Analog Input window The Time Base can be set between 1 s and 10s MPScope User s Manual p 21 The second mode is Seal Test which automatically calculates the resistance of a microelectrode or a membrane seal A microelectrode amplifier is driven by one output of the analog stimulation board PCI 6711 see Fig 1 The board has to be present in the computer to enable Seal Test MPScan automatically determines if the amplifier is used in voltage clamp or current clamp by checking if output commands are in mV and analog input are in pA or nA voltage clamp or if the output is in pA or nA and analog inputs are in mV current clamp To set up the units correctly use the Configuration window to enter units and conversion factors for analog inputs and the Analog Stimulator window to enter the appropriate parameters for the an
36. n menu item in the main window MPScan maintains a list of the 10 previous configurations To restore a previous configuration click on the Experiment Previous Scan Configuration menu item A 5 8 Saving and retrieving a configuration MPScope User s Manual p 19 Scanning configurations can be permanently stored on disk in a directory implicitely specified by the user See the File Open Configuration File Save Configuration File Save As Configuration menu items in the main window When MPScan is closed the program will prompt the user to save the current configuration if it was changed from the original one during the session A 5 9 X Y Z Control The X Y Z panel controls the X Y translation table and the Z stepper device The remote control style buttons are self explanatory A 5 10 Waypoints A waypoint consists of a set of X Y and Z coordinates that can be stored alongside with an image of the frame at that position and recalled even during scanning Waypoints can be stored sequentially by clicking on the Save Waypoint button Waypoints pane main window A meaningful description of the waypoint can be entered in the text box just above the Save Waypoint button Waypoint descriptions are listed in the X Y Z panes The user can display the image of the waypoint and can delete all the waypoints A 5 11 Digital Stimulator The digital stimulator is capable of delivering TTL pulses on the digital port of the
37. n blue To change the color scheme click on the various options of the View menu of the Viewer window The Overlay Channel 1 on Channel 2 menu item is useful during two photon targeted patch clamping The pixel offset for each channel can be changed by selecting a value in the Channel 1 Channel 2 Channel 3 or Channel 4 input boxes on the Video window The latest frame can be saved as a bitmap by executing the View Save as bitmap menu item in the Video window When a subframe is selected for scanning frames may appear very small Use the Increase Zoom or Decrease Zoom menu commands under the Zoom menu in the Video window to adjust the viewing size of the frame This zoom is different from the Magnification factor in the Raster panel A 5 6 Saving data to a file MPScope User s Manual p 18 MPScan always maintains a data file ready The name of the file is automatically chosen by MPScan or can be overridden by clicking on the New File button or by choosing the File New Data File menu item in the main window MPScope data files have a MPD extension The default name chosen by MPScan is YYMMDD_XXX MPD where YY is the year MM is the current month DD is the current day and XXX is a numeral incremented each time a new data file is created Data can be saved to file anytime during acquisition by clicking on the Start Streaming button on the Video window Stopping streaming can be done the same way Streaming can start immediatl
38. n the Stack control panel main Viewer features three input boxes to enter the initial power usually at the top of the stack and the final power at a prescribed z distance enter a negative value for stacks going down inside the tissue The user can opt either to turn constant illumination off or use a linear or exponential relationship MPScope User s Manual p 16 A 5 4 3 Fast Scan Fast Scan is a mode consisting of taking a movie at high frame rate while focus is continuously changed Select the Fast Stack from the Scan panel go back to Movie first if the Fast Stack is button disabled The Fast Stack panel will appear Select first by how much the stack depth enter a negative value to go from top to bottom move the slider to select the travel speed this will give also the travel time and the repeat count If the repeat count is gt 0 the Fast Stack will repeat by going in the backward direction then forward etc until the number of repeats has been reached Frame size and frame rate can be adjusted in the Configuration window Fast Stack can be used with laser intensity automatic control see Section A 4 4 2 Click on the Start Scan button to start Fast Scan You can pre arm streaming to disk by clicking on the Start Streaming button beforehand During a Fast Scan the z stepper control panel X Y Z Laser will indicate that the stage is moving At the end of a Fast Stack operation the z focus is brought back to its origi
39. nal position the laser power is set to its initial value if laser control of stack was enabled A 5 4 4 Line Scan Use the configuration dialog to set the pixel clock compatible with line scan In Movie mode go to the View menu of the Viewer window and select Linescan Trajectory A horizontal line yellow by default will appear in the middle of the screen This line shows where linescans will take place Optimize the placement of the line scan by zooming rotating ect the desired region in Movie mode first i e place the cells of interest under the line scan Select line scan mode from the Scan panel in the Viewer window The configuration dialog will appear Frame Height can be left at 512 lines per frame A 5 4 5 Region Scan To select a region scan you must be in movie scan mode with X and Y frame offsets 0 and Frame Width and Frame Height 512 Select the Region scan mode by clicking on the Region scan button in the Scan panel of the Viewer window A window will appear Regions can be either rectangles ellipses or polygons It s OK to draw regions that overlap When regions overlap pixels in the common areas are scanned only once Choose to draw either a rectangle ellipse or polygon For the first two choices position the mouse depress the left button drag the mouse across the area to select and release the left button For polygons click with the left mouse i e depress release quickly on each vertex of the polyg
40. nguage Interoperable with Matlab Events 10 user programmable buttons when frame acquisition starts ends when section in stack is done when new frames are acquired Methods to position X and Y mirrors to add parameters and comments to data file to control X Y Z position to send average pixel intensities from ROIs to Matlab in real time to control multipurpose hardware timers on the data acquisition board ActiveX component for Measurement Computing USB 1608FS USB based acquisition board for extra analog input channels Compiled multithreaded Windows optimized code Object oriented design fully compiled Extensive use of threads and Microsoft COM based architecture Uses Windows specific application programming interfaces APIs to boost performance MPScope User s Manual p 8 Stand alone program No need for Labview environment or run time MPScope data files Experimental parameters Stores up 40 default parameters magnification X Y Z position comments Custom parameter name values can be added programmatically to file by script or ActiveX enabled application Self documenting files Parameters in file can be read by right clicking on the file name in the Windows shell e g Explorer A 2 System Requirements The minimal computing configuration for MPScan requires A PC preferably with two processors running Windows 2000 or XP The computer in our laboratory is a dual 1 GHz processor with 1
41. on To finish creating the polygon click on the right button of the mouse The Configuration dialog will appear Select an approprite pixel clock and click on OK You are now ready to scan MPScope User s Manual p 17 A 5 4 6 Starting and stopping a scan Use the Scan Start Scan Scan Stop Scan menu items in the main window or click on the Start Scan button on the top left corner of the Video window to start or stop frame acquisition Scans can be automatically stopped after a preset number of frames was collected In the Video window Scan panel unselect No frame limit and enter the number of frames in the Stops after input box A 5 4 7 Frame rotation and zoom Frames can be rotated by an arbitrary angle from the Rotation group in the Raster panel in Video window Similarly frames can be magnified by reducing the amplitude of the scan pattern by a factor shown in the Magnification group Raster panel Magnification results from reducing the maximal deflection angle of the scan mirrors A 5 4 8 Acquisition Parameters Acquisition parameters such as frame rate frame size and channel assignments can be changed from the Configuration window A 5 5 Display The Video window displays frames either in gray levels false colors or using a custom look up table LUT In the first mode all pixels with values less than O are displayed as black all values larger than 2047 are displayed as red and mid level level 1024 is shown i
42. or system administrators Once Matlab is running enter commands from the caret prompt Users wanting to use Matlab Mathworks Natick MA to analyze frames have two possibilities They can export frames directly to an instance of Matlab either running locally or on a remote server or use a library to open and read MP files The library is packaged into an ActiveX DLL called MPFILE DLL see chapter C in this document B 3 13 Binary Frame Operation MPView supports creating a linear combination of 2 frames The output of the operation MPScope User s Manual p 35 can be described as result gain1 Frame1 gain2 Frame2 where gain1 and gain2 vary between 1 and 1 To perform a binary operation open the file s containing Frame1 and Frame2 then create an empty workspace menu or Ctrl N Select from any Viewer menu Operations Binary Frame Operations Select the file or workspace the frame index and the gain to apply for Frame1 and Frame2 Click on Create Result Frame to set up an empty frame in the destination workspace then click on Update Result Frame to see the result of the operation each time you want to change parameters such as gains MPScope User s Manual p 36 C MPFile MPFile is an ActiveX control OCX designed to be used in programs other than MPView to read MPScope data files MPFile includes high level functions that do not require to deal with the internal layout of MPScope files Examples in section C 2 Using M
43. or elliptical ROI is created MPView will query for the starting frame and ending frame of the plot A plot of pixel averages across frames will be created Alternatively threshold based objects can be detected automatically by MPView Once objects are classified users can double click on them to generate a plot In case two video channels exist in the file the user can create the same ROI in the opposite channel Plot values can be transferred to the clipboard as a character based TAB separated array of frame index ROI average pixel value pairs Such arrays can be easily pasted into other programs B 3 10 Exporting frames MPScope User s Manual p 34 MPView can directly export series of video frames directly into a local or remote instance of Matlab Alternatively MPView allows saving individual frames as bitmap files Also MPView can save groups of frames in a multi frame TIFF file This kind of file can be opened directly in ImageJ These operations are accessible from the Viewer menu bar Exporting frames as bitmaps Saved bitmaps are 24 bit RGB Exporting frames in Matlab Exported frames are exported as arrays of double To export frames to Matlab from a viewer select the Operations Export Frames to Matlab menu item In the Export Frames to Matlab dialog box select the channel if two channels are in the file the starting frame and ending frame Each frame will be exported to a different Matlab array which name will
44. rage pixel intensity from user defined regions of interest during scanning Rectangular or elliptical ROIs Tile Overlay display User can define one ROI as background other ROIs are background corrected ROI values are available for real time scripting Laser control Front panel laser control for illumination power Can be controlled by scripts Acquisition configurations Keep a list of the 10 most recent configurations for fast settings Configurations can be saved and restored to from disk X Y Z control Controls NEAT300 X Y translation table controller or interfaces with Newport ESP 300 and Sutter MP 285 three axes controllers Move to X Y or Z during acquisition Absolute or relative coordinates Waypoints can be named stored with an image of the frame and returned to even during frame acquisition Z piezoelectric objective lens positioner via one PCI 6711 Supports the PiezoJena MIPOS 100 Z piezoelectric fast positioner up to 100 um displacement Position controlled by an analog output from a PCI 6711 board Z displacement by fixed increment can be done for every line scan or for every frame Digital stimulator Up to 8 independent digital TTL output lines 1 ms resolution timer Two level pulses of digital bits can be repeated for a finite or infinite number of times Pulses can be preceded by a prepulse Digital stimulation is triggered internally by frame acquisition or by user Digital stimulation patterns can be s
45. s select X Z or Y Z Pipette Angle corresponds to the actual tilt MPScope User s Manual p 22 of the electrode During Motions the simulated LED at the top right corner of the Manual Control panel changes from green to red indicating that no commands can be issued to the micromanipulator Two photon electrode placement is done in several steps Once a pipette is placed in the setup a calibration step is performed The user can then move the pipette to a Home position which can be recorded The user will register the coordinates of the cell of interest by placing it in the crosshairs and clicking on the Set Target button Automatically placement of the pipette in the immediate vicinity of the target cell is done by successively activating the Approach and Descent motions Once the pipette is within a short distance from its target the user can manually Contact the cell by moving the electrode by a few microns at a time The Approach Descent and Contact parameters can be set up by clicking on the respective Settings buttons Calibration is necessary to map the coordinate system of the position 3 micromanipulator with that of the X Y Z translation device Fig 4 Micromanipulator axes of s ipette picropipet R meen positio oe 100 um position 2 Calibration must be performed each time the orientation of the micromanipulator axes changes for instance after changing pipette Clicking on the C
46. ter the Home position of the pipette To register the position of the target cell the user will move the cell at the center of the crosshairs using the microscope translation controls and click on the Register Target button Approach is an automatic motion to move the pipette from its current position usually but not necessarily the Home position to the Descent position Fig 5 The Approach trajectory follows straight lines along the X Y and Z axes of the micromanipulator The order of the motions i e X first then Y and finally Z motions can be programmed in such a way to prevent the pipette to collide with an object such as the objective In Fig 5 Z is executed before Y and Z motions to prevent the pipette from colliding with the objective To execute Approach click on the Approach tab The Descent trajectory Fig 5 is a motion that follows a straight line in the X Z or Y Z plane from the Approach position to the Contact position at an angle determined by the Pipette Angle Typically Descent will correspond to the motion of the pipette inside the tissue before it reaches the cell of interest The Descent phase will stop before the target by a distance specified by the user in the Descent settings Final Contact with the cell Fig 5 is controlled by the user typically by monitoring the access resistance of the patch clamp pipette or by measuring the potential Click on the up or down buttons in the Contact panel to move the
47. the first time you run MPScan you need to setup the hardware configuration first see A 4 3 A 5 3 Setting the hardware configuration Setting the hardware configuration is usually done the first time MPScan is run after which the configuration is saved permanently in the Registry However the harware configuration can be changed anytime You can also run the hardware configuration to attempt to connect to a device that was disconnected when MPScan started Click on the Settings Hardware Options menu item to display the Hardware Options dialog Make sure that the board indices match those found in the NI DAQ Measurement and Calibration program on the Desktop the COM port and COM speed assignments are valid for the NEAT 300 translation table or for any serial port dependent devices e g MP 285 ESP 300 etc See the Read Delay instructions for the MP 285 below MPScope User s Manual p 14 Click on OK or Cancel to resume The Hardware Status dialog will be displayed in case OK was chosen Review the hardware status and click on OK You can display the Hardware Options dialog again if there was a mistake in hardware assignment Optional hardware Motorized micromanipulators Up to two computer controlled motorized manipulators can be added to the microscope system Default transmission rate for MP 285 is 9600 bauds In case axes orientations of the micromanipulators are inverted check the appropriate boxes Many
48. tput command channel that will control the Z position In general AOO is the default channel Connect this analog output to the MOD input of the MIPOS controller Typical parameters in the Configuration panel include From Z To z and Increment For instance corresponding values of 0 100 and 10 indicate that the Z actuator position will start at 0 ms end at 100 um and incremented at either every line or frame by 10 um A 6 ActiveX Automation Features MPScan was designed from its inception to be programmable by end users with the shortest learning curve and the maximal efficiency as possible This ruled out forcing the users to delve into Matlab source code or to modify a Labview program The widespread usage of the ActiveX technology in many Windows applications such as Excel or Matlab provides a great deal of flexibility to the user In addition numerous ActiveX controls can be used in Activex scripts to control hardware An ActiveX control to interface with a USB based 8 channel 16 bit data acquisition board PMD 1608FS Measurement Computing Middleboro MA is available from the author A 6 1 Server features MPScan exposes many internal objects and methods to ActiveX Automation controllers In other words it is quite easy to write a program in Visual Basic Object Pascal or C C to control the microscope via MPScan Refer to section A 5 3 for description of Activex Automation objects in MPScan A 6 2 Scripting features
49. truction of a two photon laser scanning microscope for in vitro and in vivo brain imaging Chapter 6 in In vivo optical imaging of brain function R D Frostig Ed pp 113 171 CRC Press Boca Raton 2002 A PDF reprint of this book chapter is available in the Publications section of http neurophysics physics ucsd edu More details on MPScan can be found in Q T Nguyen P S Tsai and D Kleinfeld MPScope a versatile software suite for multiphoton microscopy 2006 J Neurosci Methods in press Background on VBScript and COM in general Q T Nguyen and R Miledi e Phys a suite of electrophysiology programs integrating COM Component Object Model technologies J Neurosci Methods 2003 128 21 31 About VBScript Many books available in general bookstores deal with VBScript Tutorials and references can be found either on line at www msdn microsoft com or can be consulted from the Help menu in MPScan MPScope User s Manual p 5 A MPScan A 1 Summary of features Multi user capability Login window sets user preferences Hardware MPScope is compatible with Denk Svoboda or Kleinfeld custom multiphoton microscope designs No specialized hardware necessary Typical configuration one Windows PC one National Instruments data acquisition board interfacing two galvanometric mirrors Frame acquisition Up to four video channels two can be used as analog channels 20 10 5 2 5 1 25 V selectable input ranges
50. validate this value by clicking on the Apply button 4 Observe the screen and try to move duplicate pixels on top of each other by adjusting the X pixel line shift value There should be little or no hairs caused by misaligment of the forward and backward scans 5 Enter the same value in the Y pixel line shift input box validate and rotate the scan by 90 Adjust the Y pixel line shift value to get a sharp image Pixel shifts are stored in protocols The future version of MPScan should automatically align lines created by forward and backward scans using a feedforward algorithm A 5 4 2 Stack When this mode is selected the Stack panel is displayed in the Video window A stack consists of a number of sections Each section is separated by a z interval A negative z interval indicates that the microscope focus is moved down after each section A section consists of one or several frames that are averaged Stacks can be repeated for a number of times selected by the user to obtain stack time series The stack mode can be selected only if the previous mode was Movie The laser shutter can be closed after each section to prevent overexposing the sample to laser light The corresponding setting can be set in the Hardware Configuration dialog When imaging inside tissue MPScan can provide constant illumination as focus is set deeper At present laser intensity control is provided by a servo mechanism see section A 3 3 The bottom box i
51. y default MPView tries to start Matlab from the same computer it is running from However it is possible to run Matlab on another computer using DCOM Distributed COM You will have to set DCOM on the server and client sides Refer for instance to http www opcactivex com Support DCOM_Config DCOM_ClientSettings dcom_clientsettings html for instructions on setting up DCOM B 3 Using MPView B 3 1 Opening a data file From MPView After starting MPView click on the Open button on the main tool bar or on the File Open File menu item Select a MPScope file extension MPD for MPscope Data file and Click OK on the button of the Open Data File dialog box You can open only one instance of a data file From the Shell Single or multiple data files can also be opened by MPView by dragging and dropping their icons from an Explorer file directory window or from the Desktop in the main window of MPView MPView associates MPD files with MPView by setting registry values the first time it is run Next time Windows is restarted MPD data files will have the same icon as MPView Clicking on the file icon will start MPView and immediatly open the data file Note If MPScan is running MPView cannot open the current data file of MPScan MPScope User s Manual p 32 B 3 2 Viewing frames Each data file is associated with one or several Viewer windows To add a new Viewer click on the View New Frame Viewer menu item of a Viewer Eac
52. y during scanning by setting Start Streaming before initiating frame acquisition When scanning ends streaming ends automatically A new empty data file is created each time a the scan configuration is modified by the user When the old data file is committed to disk the file comments main window menu bar are written to the file Check for the change in the data file name in the caption of the Video window and for a beep indicating that changes in the previous file were committed to disk MPScope data files are self documenting by containing the following parameters Type of data file Movie Stack Line Scan Progressive Line Scan or ROI Scan X Y position of the franslation table in microns Z focus position File comments TIP To view all the file parameters outside MPView for instance from Explorer click on the file icon with the right mouse button A property sheet will appear Select the Custom tab All parameters and their corresponding values will be listed Frame data in MPD files are stored in a custom binary format Applications other than MPView can use the MPFile ActiveX control described in this document to access data in MPD files A 5 7 Scan configuration The Scan Configuration dialog allows users to set up various scan parameters such as number of video and analog channels gains etc This window can be either automatically displayed or explicitely called by clicking on the Experiment Scan Configuratio
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