Make sure the objective you want to use is on the microscope.
The 20x objective does not need a spacer, but all other objectives do.
The 5x objective is an air objective; all other objectives are immersion objectives.
Choose your chamber. Use M3 screws to secure the chamber onto the metal platform.
Set up the Gibson pump by stretching the tubing (there are two plastic holders that have to be positioned) and locking the upper two squeezing arms in place.
Turn on Warm-U by connecting the battery.
Connect the inflow tubing to the chamber. Place inflow tubing in H2O first, then turn on the pump and make sure that the chamber is not leaking.
Open the vacuum valve and make sure the vacuum pipette is not clogged.
Tune Warm-U while measuring the solution in the chamber with a thermocouple.
Turn on the nitrogen tank and make sure the air table is floating properly.
Place air tubing in your solutions (ACSF, etc.) and turn on the carbogen tank.
Acquisition
Turn on the master switch for all 3i equipment (white switch on the top of the glass rack).
Wait until the yellow blinking light on the Photometric camera turns off. Then turn on the computer. Note that this sequence is necessary for the computer to detect the camera.
Open SlideBook6 (x64) on the computer.
Turn on the laser safety shutter (a key).
Hit Focus for camera and laser setup:
Choose the camera (C13440 is the regular one) you want to use
Choose the correct objective
Define a Z stack (for ephys experiments, take 1~3 stacks)
Hit Live to test image.
Open Bright to turn on brightfield. Make sure the exposure time is low (1-5 ms).
Find the region of interest in the slice
Open Alt to turn on the laser (alternative light source). Increase the exposure to 100-300 ms, depending on the expression level.
Adjust the region of interest to get maximum expression.
Go back to brightfield, adjust focus and hit Snap to take an image (name it as brightfield).
Go back to laser, adjust focus and hit Snap to take an initial image and name it as initial.
Hit Capture for protocol setup:
Select the laser (488 Flash for GCaMP)
Select the desired acquisition type
Input the desired time interval and number of samples. Note that the time interval should be greater than or equal to the exposure time.
Save the setup with a specific protocol name
Go back to Focus, then go to the 6D tab.
(Optional) Click on autofocus.
Add the protocol(s) of interest or update protocol if the imaging plane is changed.
Change the comment field to recording.
Hit 6D and make sure the protocol settings are passed into 6D.
If the exposure time is the same as the sampling interval, choose Freerun capture to prevent any lag in acquisition.
Hit start to begin acquisition.
Cleanup
Close SlideBook6.
Turn off the laser safety shutter (a key).
Turn off the master switch to the microscope.
Turn off the carbogen and nitrogen tanks.
Turn off the power strip for the stimulator if you used it.
Disconnect the battery to Warm-U.
Run H2O through both inflow tubing for 15 minutes (5 minutes for one, then switch to the other for 10 minutes).
Run air through the inflow tubing for 5 minutes.
Turn off and reset the Gibson pump so that the tubing can relax when not used.
Empty the vacuum flask.
Close the vacuum valve.
Clean objectives with lens cleaning solution and lens paper. Use 2 pieces of lens paper: one with cleaning solution and one without.
Remove and store away chamber and stimulating electrode.
Copy data files to the server or the CONFOCAL_ANALYSIS computer.