Two-photon recordings

  1. Set up the MASTER and STIMULUS computers as described here.

  2. Prepare for a 2-photon recording by setting up according to your lab's setup (VHLab click here)

  3. In the RunExperiment window, make sure the Acquire data checkbox is checked.

  4. In the RunExperiment window, in the "Acquisition list", select the first entry and click "Edit"; change the "type" to prairietp. Also, edit the reference number ref. Other parameters do not matter.

  5. In the RunExperiment window, open the stimulus worksheet that you want to use, such as Ferret Direction.

  6. Find the area you want to image on the 2-photon.

  7. On the 2-PHOTON computer, In the "T-series" panel, select "Data path" and make a new folder in the "Data" directory on the desktop with today's date. For example: 2011-04-01. Use this folder as the Data path.

  8. On the 2-PHOTON computer, set the directory name to be 't00001' (or whatever number is appropriate if you have acquired previous data) and set the directory number to be 1.

  9. On the 2-PHOTON computer, set up the T-series so it has 3 cycles; the number of frames of cycles 1 and 3 should be 1. The number of frames for cycle 2 can be calculated by : NUMBER OF FRAMES = REPS * (DURATION_OF_STIM+INTERSTIMULUS_INTERVAL) * (NUMBER_OF_STIMS_INCLUDING_BLANK) / FRAME_INTERVAL. Add a few extra (20-30) to make sure and enter your result as the number of frames for cycle 2.

  10. On the MASTER computer, on the worksheet, prepare your stimulus. If you want to run a grating, make sure the REPS is set to 12, the ISI is set to 5. In Edit Base, make sure the window is set to fill the screen [0 0 800 600], the spatial frequency is 0.08, and the temporal frequency is 4Hz. Set the number of cycles parameter so the stimuli will have a duration of 5 seconds (20 cycles will take 5 seconds because 5 seconds * 4 cycles / second = 20 cycles).

  11. On the MASTER computer, on the worksheet, click "Run" next to the condition you want to run. It will ask you to make sure acquisition is running. On the Spike2 window, click the "Start" button that is in the lowest toolbar in the middle of the window (NOT the Start button in the upper left window). Finally, with your left hand on the mouse of the the 2-PHOTON computer, prepare to press "Start T-series", and with your right hand on the mouse of the MASTER computer, prepare to press OK to answer "Make sure the acquisition is running". Click the Start T-series on the 2-PHOTON computer first, and then, after you see that it is running, click OK on the MASTER computer. When the stimulus AND the 2-PHOTON have both finished running, click the STOP button on the Spike2 machine (the one in the toolbar next to the START button) and then click "SaveCloseRestart", and just click OK when the dialog box asks you to recluster.