Hardware required for LSPS mapping experiments

See Shepherd et al 2003; 2005 for details

Setting up for LSPS mapping experiments

Before doing mapping experiments for the first time two calibration steps have to be completed.

1) The position and control of the laser beam have to be aligned with the CCD image; this is achieved using the imagingSys GUI

2) The laser power has to be calibrated using the photodiode GUI

Calibrating beam position and control with ImagingSys

1) Place a piece of paper on the recording chamber and focus the camera image on the paper.

2) Open the laser shutter and using the neutral density graduated filter reduce the laser power to minimal values in order to obtain a sharp small image of the laser spot on the paper.

3) Set the TestScan pull-down menu to Stationary, and hit Start

4) Set the Horizontal and Vertical Offset to values that position the laser spot right on the center of the field of view (as viewed through the camera). Check/uncheck 'Invert' as needed so that positive values give rightward (for the horizontal channel) or upward (for the vertical channel) offsets. To confirm that the spot is right at the center save an image of the spot and open it wit the "Grab Video" button of the Mapper GUI (see below). The laser spot should be right at the center, were the axis cross.

5) Now select 'square' or 'circle' from the TestScan pulldown menu

6) Set the Horizontal and Vertical amplitudes so that the beam moves up to the borders of the field of view 

7) If necessary, using the 'square' setting, rotate the camera so that its field of view is perfectly aligned with the trajectory of the beam.

8) Set Horizontal and Vertical 'Span' to the size of the camera field of view in microns.  

Calibrating the laser power readout with the photodiode GUI

1) Hit clear

2) Fill in your initials in the 'User' field. (tick) Tip: also append the amplifier's gain setting.

3) Place the power meter either directly under the objective lens, or if necessary remove the objective and place the power meter in the beam path.

4) Increase the beam intensity to roughly the normal working level (e.g. 20 mW). Enter the value of the readout of the power meter on the 'mW' field and hit enter. (tick) Tip: If using a Pockels cell, temporarily increase the transmission by changing the bias voltage; this can be done either manually, or from the command line (see Useful Utilities).

5) Change the intensity of the laser (e.g. with the rotating graded filter) and repeat #3. Three to four measurements are usually enough. Points should lie on line, and the power values should span the powers that will be used in experiments.

6) If power was measured under the objective set 'Transmission' to 100%. If it was measured on the objective back focal plane (i.e., with the objective removed), set 'Transmission' to the measured transmission of the objective for that wavelength (e.g. 80% for UV).

(For further information, see 'Descriptions of all programs > photodiode'.)

Running a mapping experiment

  • Calibrate the pockels cell by hitting 'Calibrate Pockels' (This might not be necessary as the program does it automatically when it is booted) . You should obtain a smooth monotonically increasing curve.

    Tip

    A convenient way to run the calibration routine is to occlude the beam path (somewhere downstream of the photodiode) with a Post-It. Then manually open the shutter and adjust the circular neutral density filter setting to maximize the transmission. Close the shutter. Now run the calibration routine; the Post-It prevents the slice from being illuminated. Finally, remove the Post-It.

  • Grab a picture of the slice using the 'Grab Video' button. Typically this is an image you just saved with your rig's video software. The image with an overlay of the currently selected mapPattern should appear in the videoImg window. Orient the stimulus grid to the slice landmarks as you like, using the rotation and offset tools.

  • Select pulses for the shutter and  pockels in the stimulator GUI, and for the amplifier (i.e a test pulse) in the ephys GUI. Typically the pockels pulse will be 1 msec in duration, with 100 msec delay. The amplitude should be 100 (units are in 'percent transmission'). The shutter pulse should bracket the pockels step; for example, duration of 4 msec, delay of 98 msec, amplitude 5000 (units are in mV; i.e., for TTL).
  • You also need to select pulses for the mirrors. Make a dummy pulse that puts out 0 and select it in both the xmirror and ymirror channels. The xMirorr and yMirror channels also need to be on.
  • Enter a gain of 1 for each channel in the stimulator.
  • The mapper gui needs to be configured to know which map patterns folder to use. Create a 'mapPatterns' folder in your Ephus settings directory (unless you already copied the 'standard user directories' from the Model Data File Archives), then download the 'standard map patterns' from the Model Data File Archives, and place these files in the 'mapPatterns' folder. Next, using the mapper, use File>Open to select this mapPatterns folder. In the mapper gui, the map patterns listbox will show all the patterns available in the folder.

  • Specify the mapPattern and the spacing between positions. Use the '=' tick to set the X and Y spacing to the same value. The mapping grid should appear overlaid on the videoImg window.
  • Position the map, both offset and rotation ,  using 'dX' 'dY' and 'deg'  
  • Set mode to  'DAQ' in the loopGUI.
  • Set the interval between positions in the mapper GUI ('ISI' in the upper  right corner of the mapper GUI). The interval between positions equals the trace length of each acquisition in DAQ mode. Set the same value as specified in the mapper for the trace length in the loopGUI, acquirer, stimulator and ephys.
  • Use the '1' button in the 'soma [SvobodaLab:um]' box in the mapper to mark the soma position. Right-click on the videoImg window.
  • Hit Map to start a map.

Here is what should happen

  1. The shutter should be clicking at the correct speed
  2. The Acquisition window should be updating
  3. A red circle should be hooping across the image on the Videoimage
  4. The position window in the Mapper should update monotonically
  5. At the end of the map you should be queried for a filename; or is Autosave is checked on the xsg program then a file should be saved automatically as indicated on the Matlab command line
  6. After the conclusion of the map the map number should increment

More notes on mapping

Note that you do NOT need to select 'External' in ephys, acquirer, or stimulator, because the mapper launches a user function that automatically sets these and a number of other parameters.

Note also that the xsg settings change with various map modes. Maps are put into their own subdirectories. Flashes (see next) go in a 'flashes' directory. This will be done in the most orderly way if you select the 'Concat' option.

Flash/Mouse/Re-mouse -- you can flash a single location at the current mirror position (which can be set to the center of the image by hitting 'zero mirrors') by hitting 'Flash'. Or, you can select sites to flash using the mouse. To do so, hit mouse, and RIGHT-click at a desired location on the slice image. To do N sites, LEFT-click on all but the Nth site, which you then RIGHT-click on. To repeat, hit 'Re-'.

Online mapper -- this gets launched (it's a user function) when you hip 'Map'. Go to the online map analysis gui to change the analysis parameters.

Explain the DAQ mode
Explain pulse creation

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