Table 2.

Troubleshooting table.

Step	Problem	Possible reason	Solution
1	No laser coming out of fiber	Poor coupling	Use the screws on the laser port to shift the position of the fiber. Change one screw at a time by small increments, where each change should increase the intensity of the light. A good alignment should be qualitatively obvious.
		Damaged fiber	Unlikely, but if so try another fiber.
30	No expression / weak expression / partially functional expression of optical actuator/reporter	Did not wait long enough for in vivo expression	Initially allow very long expression times (6-8 weeks) and then try to shorten them.
		Incorrect injection location	Perform histology, verify location of injection wound, and check other areas for presence of wound and/or expression.
		Nonfunctional virus	Try a different batch/preparation of virus. Focus on seeing robust expression in slices under easier imaging conditions before attempting in vivo.
35	No cellular image	Mirrors are not moving	Try connecting (or forking) your mirror command BNCs to an oscilloscope, to verify that proper “raster” command signals are coming out of the computer.
			Verify that the mirror control boxes are powered “on”.
		Shutter not opening	Verify that you can hear the microscope shutter clicking open and closed when you scan. If not, unhook and re-hook the command lines, and verify that the computer board is not maintaining a “high” signal by resetting the computer.
		Microscope switch settings are incorrect.	Verify that manual shutter is open, that eyepiece toggle is removed, that camera port toggle is allowing laser pass-through, and that the proper dichroic for 2P is selected in the carousel.
		Photomultiplier tube (PMT) not turned up	Verify that the PMTs are turned up. You may even turn them up higher to verify that some auto-fluorescence at least registers a signal.
		Pockels signal set too low	With PMTs set higher than normal, start at low pockels command and then gradually increase until at least an auto-fluorescence signal is registered.
		Objective liquid has evaporated with repeated imaging.	Verify that objective has contact with the preparation and is immersed in liquid.
36	Automatic cell detection does not lock onto cell bodies	Cell detection settings need adjustment	If the cell detection module does not seem to automatically “lock” onto cell bodies, or chooses cell bodies of an incorrect size, follow these steps: A) First, make sure that the “Zoom” and “Magnification” in the lower left of the software are set correctly. B) Second, try adjusting the “Cell Detection” sliders in the upper right to work with your particular fluorescence loading or morphology. If the image is reasonably clear then it should work and if not, image quality may need to be remediated.
39	One of the subsystems does not start	Incorrect triggering or ready state	If one of the subsystems does not start, check that it is connected to the other boards’ triggering cables (see Figure 1), that the subsystems are running and waiting for trigger, and that an error did not occur in the subsystems.
41	No sensory responses	Poor indicator loading	Repeat loading; may need to abort
		Saturated signal	The microscope should be calibrated to detect “gray” cells that are neither too dark (insufficient signal; black) or too bright (too much signal; saturated). If cells are registering at this intermediate “gray” level then there should be sufficient response range remaining to detect transients if the cells are active.
		Anaesthesia too strong	Reduce anaesthesia by 25%, unless animal awakens.
		Incomplete indicator loading	Wait at least an hour for maximal loading of calcium indicators.
		Cells damaged during loading	Calcium indicator should be pressure-loaded as slowly as possible, because any extra pressure will disrupt the neurons and reduce uptake.
		Brain damage	Verify that brain is active with quick loose patch recordings. As one first gets comfortable with evoking sensory responses, electrophysiology (blind loose patch) should be used to ensure that the visual cortex is highly responsive, and to calibrate anaesthesia levels towards this end. Once responses are reliably achievable it is reasonable to switch to imaging wherein the only remaining variables are quality of loading.
43	Fluorescent traces don’t appear to be physiological	Cell IDs in data are mis-attributed to cell areas in the image.	Make sure that IDs in the data traces match the cells in the image. This can be checked by verifying that the portion of the scanpath that gives rise to the data overlaps with the cell’s actual position, and not another cell.
		Mirrors are not fast enough	Verify that mirror commands and positions measured via feedback are closely matched
			Image fewer cells to reduce the number of turns per lap
			Increase the zoom to minimize distance per lap and thus speed
			A simple correction can adjust for mirrors that follow the correct command path but with a delay / skidding. Because the mirror feedback is also recorded during scanning, the actual position of the mirrors is known for every recorded sample and can be assigned to the specific cell sampled at that exact moment (or to no cell if the position is traveling between cells). So, even if the mirrors may not have passed the beam through the cell of interest at the exact microsecond planned, it will likely enter it at a slightly later microsecond, and this precise timing is known. By collecting all the samples when the beam was actually passing through a cell and averaging them, one can resolve an accurate estimate of the fluorescence during that lap around the cells, for each cell in the network.
45	Fiber does not connect to microscope port.	Incorrect adapter	The screw-in nature of this port should ensure a proper coupling. If the terminal does not seem to match, or screwing remains difficult, there may be a mismatch between your configuration and that expected here. In this case, contact your microscope manufacturer for information on proper coupling.
47	After adding a 50/50 mirror, lasers are dim.	50/50 mirror cuts power in half	This is expected after adding this mirror; henceforth double the strength of the laser you are using to achieve the same effective strength coming out of the objective.
53	No light-evoked activity	Intensity is insufficient	Verify that full-field activation is configured and reaching the objective; increase amplitude towards maximum, at 10 Hz and 10 ms pulses.
		Cell does not have detectable optical responses	Verify responsiveness with sensory stimuli
		Optical actuator is insufficient	Wait longer for cellular expression
			Validate that the optical actuator is functional using a reduced preparation, such as culture or slices, with electrophysiology.
53	Brain damaged by laser activation	Light intensity or duration is completely out of range.	In our experience it is less common to damage tissue with photoactivation pulses than to damage it with a recording 2P laser that is set too strongly (>100 mW) in an attempt to gather brighter signal, which could damage tissue. As most photoactivation regimes require fast, sparse pulses, this becomes less of an issue. Damage will likely be obvious, with dark (burned) spots appearing in the tissue where the offending laser was.
54	Interference between optical stimulation and recording	Inherent in experiment	Employ “blanking” in analysis (see “Analysis -Removing Photoartifacts” in Supplementary Note 1).
56	Adapted laser is not centered on lens	Misaligned laser path	Adjust the position of the laser within the microscope light path in broad strokes until it is centered on the back of the objective. Then, use a screw-in target to make sure that what comes out of the objective port is centered.
68	Stimulation is imprecise in X-Y plane	Fiber port aperture is not closed.	Tighten the aperture control knob to minimize the size (though avoid closing it completely).
		Laser intensity is too high	Reduce laser intensity to decrease effective radius.
		Beam calibration is off	Beam re-calibration should not be required if nothing inside the microscope has been changed, but it may be worth checking using Steps 55-61 of the Procedure.
68	Stimulation is imprecise in Z plane	One-photon light has poor Z resolution	Perform a Z scan before targeting a cell to ensure that no other cells are above or below the cell of interest.
			Apply this Procedure using newer optical actuators or stimulation methods that are amenable to 2P stimulation.