Part 1 deals with Acquisition, Part 2 deals with Analysis
1.1 How is synchronization between the 2-photon and other devices handled?
One can choose from several synchronization methods, as defined in twophotoncalibration.m. The most common choice is 'Fitzpatrick', which is to use the synchronization system used in the Fitzpatrick lab Prairie Technologies 2-photon system. In this system, we typically connect the frame trigger of the 2-photon acquisition system to the CED 1401 so the trigger times can be extracted. The 2-photon can be configured to record frames in response to triggers, or to record asynchronously and generate triggers at each frame. Typically, we have recorded asynchronously, and the Prairie system generates triggers. The type of triggers that are generated varies with the Prairie Time Series acquisition mode. If max speed is unchecked, then there is a trigger generated at the onset of each frame (note: SV is _pretty sure_ it is the onset, and not the offset), so the number of triggers will equal the number of frames. Note that the non-max speed mode has about a 300ms pause between frames for PrairieView to convert from its block file format to a .TIFF, which it does on the fly. If max speed is checked, then PrairieView only generates a trigger at the beginning of each Cycle; for this reason, we always specify a minimum of 3 cycles per record, to ensure we get at least 3 frames that are time stamped. In this mode, the number of triggers is equal to the number of Cycles. The timing of individual 2-photon frames is computed by linearly interpolating between the times of all frames reported by the Prairie computer (recorded in the xml header file for that record) and the trigger times that are also recorded on the CED 1401. The analysis software calculates the time each pixel was recorded (using information in the xml header file regarding dwell time, line period, and frame period times, which include the acceleration/deceleration times of the mirrors), so we know whether each pixel corresponds to stimulated time or unstimulated time. We don't chunk whole frames, we do it at the pixel level (so, the software "knows" that the upper left is recorded about 1s earlier than the lower right, for example; it knows the time precisely for each pixel).
1.2 Where on the Prairie system is the trigger signal extracted?
It is a BNC signal off of the top ("A" in some systems) BNC 2090 board, the pin number <FILL THIS IN>. On the Fitzpatrick lab Rm 430. rig, this connects to CED 1401 port 2.
2.1. My "preview image" in analyzetpstack is messed up; I fixed the problem with my data or settings, but the preview image is still messed up.
When analyzetpstack firsts open a directory, it saves a copy of the preview image in MYDIR/analysis/scratch/preview_DIRNAME_CHANNEL.mat, where MYDIR is your experiment directory, DIRNAME is the directory name (e.g., t00001), and CHANNEL is the channel number. The workaround is to 1) close analyzetpstack, 2) delete the appropriate cached preview file, and then 3) reopen analyzetpstack. The preview image will be recomputed from the image series on disk.
2.2. When I select different slices in analyzetpstack, the preview image that is displayed doesn't change or changes to the wrong image. What's wrong?
This situation typically arises when the user begins clicking in analyzetpstack before Matlab has finished loading all of the data for the window. Matlab is a bit odd in that it will allow processing of button clicks before the "initialization" commands have completed. When this is the case, the solution is to close the analyzetpstack window without saving, and then re-open the analyzetpstack window. Wait until all of the processing has stopped. On Windows, you can tell when it is all stopped when you can click in the command window and the analyzetpstack window is no longer immediately re-raised to be the front window. On Unix (includes Mac OS X), you can tell by entering some dummy command like A=5 on the command line; when Matlab can finally process this command and prints "A = 5" you know it is ready to use.
2.3. What do I do if I need to rotate images that I have already acquired?
If you are using PrairieView as your Platform, you can use the function
manipulate_prairie_data(dirname, channel, shift, rotation)
Make a copy of the directory you wish to modify (let's call it t00xxx-001), and give it a name like t01000-001 (something unused). Also copy its corresponding data directory t00xxx and call it t01000.
Type manipulate_prairie_data('e:\mypath\t01000', channel, 0 0], rotation), channel is the channel number (e.g., 1), and rotation is the rotation angle in degrees.
Open the directory in the analyzetpstack window by clicking Add new slice, and check the quality of the rotation. If you're not happy with it, click Remove slice, delete the new directory, and start over. If you are happy with it, click Remove slice and proceed.
Neuter the original data directory by renaming it something like t00xxx-original; then rename the reference.txt inside the t00xxx-original directory as reference-original.txt.
Finally, rename the new directories (maybe you chose t01000 and t01000-001) so it has the same name as the the original directory t00xxx.
2.4. How can I find out the rotation of an image series within Prairie?
Look it up in the header file
A header file is saved for each experiment E:\........\t00005-001\t00005-001.xml
Use the find tool to locate "rotation" in the file. The following number is the rotation in degrees.
2.5. When a cell is recorded right at stimulus onset, so that half of the image of the cell is recorded before stimulus onset, and half of the image of the cell is recorded after stimulus onset, is this included in data analysis?
The default analysis settings will ignore this frame data for any such cells; it will not be considered "spontaneous", nor will it be considered "stimulus". Other cells in the same image that were recorded completely either before the stimulus or during the stimulus will be included.
2.6. What do the associate abbreviations 'TP', 'SP', 'FE', 'ME', 'Ach', 'S', 'L' mean?
TP means "this is 2-photon data"
SP means "this is spiking cell data"
FE - "flash" exposure
ME - "motion" exposure
Ach - achromatic (sometimes we use S for S-cone-isolating, or L for L-cone isolating for color work)
S - S-cone-isolating
L - L-cone isolating