Table 1.

Troubleshooting table.

Problem	Possible reason	Solution
STEP 1
Glial cell invasion	Meninges and/or white matter contamination	Make sure that meninges and white matter are completely removed
Neuronal cell death	Presence of L-polylysine	Increase the number of washes after the coating step
	Low cell density	Increase the number of cells per dish Choose a different lot of horse serum
STEP 3
No transfected neurons	Traces of antibiotic during transfection	Increase the number of washes before the transfection
STEP 6
Absence of spines	Neuronal cultures are not mature	Make sure neuronal cultures are at least 18 DIV when performing such experiments
	Coverslips stay to long outside of the medium	Quickly mount the coverslip in the holder before the imaging
STEP 6
Swollen dendrites	Neurons dried in the early stage	Ensure the coverslip does not dry at any time
STEP 7
Neurons are red before photoconversion	The 488 nm light intensity is too high	Decrease the 488 nm laser light intensity
STEP 9
Photoconversion outside the ROI and/or bleached the adjacent shaft area	Calibration is not set properly	Repeat the calibration process
	The photoconverted region is too large	Reduce the size of photoconversion ROI
	Movements of the sample	Make sure the insert remains steady throughout the imaging session
	The Focus stabilizing device is not on	Switch the Focus stabilizing device on