TABLE 3.

Troubleshooting table.

Step	Problem	Possible reason	Solution
8	Nuclei in view are not all in focus	The coverslip is not placed evenly on the skin	Adjust the skin as needed using a pair of plastic tweezers. Position it on the spatula so that it is completely flat Adjust the height of coverslip so that it is just lightly touching the skin
	Change in nuclei shape	Too much pressure from the coverslip	If hypoxic circumstances are prolonged, the nuclei will change shape from an ellipse to a sickle. Lift up the coverslip until blood flow is detected. Wait for ~30 min until the nuclei shapes go back to an ellipse and blood flow can be observed in the veins
	Vibration of skin	Insufficient contact with the coverslip	If contact between the skin and the coverslip is incomplete, the skin will vibrate from respiration. Lower the coverslip until the vibration stops and the imaging plane is even
11	z-axis shifting	Movement of the mouse	If anesthesia is not suitable (the mouse is breathing heavily or awakening) or if the mouse position is not correct (if the spatula is too high or the mouth cone is tilted), there could be movement of the mouse
12	Images are dim and out of focus	Water has dried out on the coverslip	In the case of long imaging times, it is easy to dry out the water on the coverslip. Once the water is nearly evaporated, the imaging will dim and start to become unfocused. Add more water during the interval time, or if the interval time is too short, pause the recording and add more water
13	Images shift	Movement of the stage and/or the platform	If the screws of the spatula of the stage and/or microscope platform are not fastened properly, there could be gradual movement. Fasten all the screws tightly
14	Images are out of focus	Focusing is set to an area outside of the contact area	If the focusing is outside of the contact area, vibration will be observed. Change the x and y axes and find the appropriate contact region
25	Different fluorescence intensity between optical sections	Recorded long range of z-stack without adjusting laser power for depth	A deeper optical plane needs more laser power than the top of the skin. The laser should be attenuated through the stack when recording a long z-stack, such that the top of the stack receives more laser power. This option should increase laser power gradually. For example, in a K14H2BGFP mouse, the power settings are as follows: interfollicular epidermis = 2%, bulge = 3% and hair germ = 4% at 940 nm
	Too much background noise	Laser power is too low	If the laser power is too low, it will not be sufficient to excite the fluorophore, and it will be difficult to distinguish the real signal from the background. Increase the laser power and/or the PMT value to increase the signal
		Laser power is too high	Excessive laser power can cause nonspecific autofluorescence in the hair shaft, in melanin and in dust on the epidermis. Decrease the laser power and keep the imaging region as clear as possible. In non-albino mice, try to image a less-melanin-heavy region
33	Target of laser ablation is not eliminated	Insufficient laser power	Insufficient laser power will not eliminate the target and will cause bleached fluorescence. Bleaching and ablation can be distinguished by autofluorescence. Increase the laser power until the target is successfully ablated, which is recognizable by the generation of autofluorescent protein particle aggregates, organized in a halo-like structure
	Laser ablation causes tissue damage	Excessive laser power	Too much laser power will eliminate not only the target region but also the surrounding area. Start with a low laser power and gradually increase the power until the target area is successfully ablated
36	The hair follicles being revisited are not all visible in the imaging area	Grid size is too small	During the growth phase of the hair cycle, the follicles may extend out of the original area selected for the tiling option. Expand the grid size as necessary in the direction in which the follicles are growing to ensure that all follicles of interest are captured