Table 1. Troubleshooting table.

Step	Problem		Possible reason	Solution
Procedure 2
3	An excessive amount of virus leaks out of the injection site during/immediately after injection (note that a small amount of ‘backflow’ is acceptable)		Injection speed too fast and/or volume too large	Reduce injection speed and/or volume
Procedure 3a
4	The indicator is dim		The indicator is underexpressing	Increase the concentration and/or volume of the indicator virus injected
	The indicator is very bright, does not appear to change brightness over time and/or fills nuclei		The indicator is overexpressing	Decrease the concentration and/or volume of the indicator virus injected
	The opsin reporter is dim		The opsin is underexpressing	Increase the concentration and/or volume of the opsin virus injected
	Somatically-restricted opsin expression in distal processes (the opsin is no longer somatically restricted)		The opsin is overexpressing	Decrease the concentration and/or volume of the opsin virus injected
	Neurons may have small bright punctate regions on processes in neuropil; expression in the indicator channel is generally very bright and with expression in both cytosol and nucleus		Tissue damage from needle penetration or injection	Ensure that the injection pipette is inserted slowly and carefully. Inject more slowly and/or a smaller volume. Use a thinner, sharper injection pipette
	Both opsin and indicator expressions are dim		The injection failed, the microscope was misaligned or the laser malfunctioned	Check that aliquots/stocks of the indicator/opsin constructs are viable. Avoid excessively diluting constructs and ensure that you inject a sufficient volume to see expression. During surgery, ensure that the correct volume has actually been injected (e.g., with a graduated glass pipette). If the correct volume has left the injection pipette, observe whether you see virus seeping out of the injection site during/immediately after injection. If virus is leaking out, make sure to effectively penetrate the pia (and dura if still intact) and/or try injecting more slowly and leaving the pipette in place after injection for longer Check the microscope alignment and that the laser and the collection system are functioning correctly by imaging a known good sample, such as pollen grains or a similar finely structured, highly fluorescent material
	The opsin and indicator do not co-express		Promoter or serotype conflicts	Different combinations of opsin and indicator work better in different brain regions probably due to serotype and promotor affinities; this may require some trial and error. Expressing one of the constructs transgenically, while expressing the other virally, may help. See Supplementary Table 1 for example published combinations
Procedure 4a
6	Poor sensory-evoked responses	Weak indicator expression, suboptimal injection site, suboptimal stimulus parameters or misalignment of electrical/analysis triggers		The indicator expression may be too low to reliably report the activity evoked by the stimulus; safely increasing the level of expression, by trialing progressively increasing concentrations/volumes of virus, may help. Ensure that stimuli are presented successfully. Ensure that all frame pulses and stimulus triggers are being recorded correctly for alignment during STA. Ensure that the stimulus is appropriate for the brain region being imaged (it is beneficial to confirm area location through widefield response mapping; see Procedure 4a)
Procedure 4b
12	Photostimulation efficacy is poor or failed completely (but expression looks good)	Optical alignment is off, or stimulus parameters are suboptimal		Check alignment of the system (including axial parfocality) and laser power on the sample (under the objective). Most issues can be found by running the spot-burning procedure on a plastic slide. If the optical calibration is correct, the issue may be that constructs are expressing well but that levels are too low for successful stimulation/readout. The stimulation parameters (power, duration and number of repeats) may be suboptimal for the preparation
Procedure 5
20	Low number of cells that are both responsive to photostimulation and responsive to the task/sensory stimulus of interest	Expression is low; stimulus is suboptimal		The number of cells that are ultimately addressable is a product of two independent probabilities: that of being responsive to the sensory stimulus and that of being responsive to the photostimulus. Increasing opsin expression while avoiding signs of ill health will increase the numbers of photostimulatable cells. Depending on the experimental question, the definition of stimulus responsiveness could be relaxed
25	Animals lick constantly	The animal is too water restricted		Remove the animal from the experimental rig and provide it with water to allow the animal to maintain a heavier weight (>80%)
	Animal behavior is inconsistent, and/or animals give up easily when transitioning from behavior boxes to the microscope rig	Animals become frustrated or confused		Ensure that the task design and environment are kept as constant as possible, in temperature, sound proofing and white ambient noise. Keep pauses in the training session (e.g., for setting up imaging acquisitions) as short as possible