Table 3 |.

Troubleshooting table

Step	Problem	Possible reason	Solution
Procedure 1
3	Unclear bacterial fluid after sonication	Insufficient sonication	Dilute the bacterial fluid and increase sonication cycles
Procedure 2
1	Poor cell attachment	Glass-bottom surface is not ideal for cell attachment	Coat the glass-bottom plate or dish with poly-D-lysine, fibronectin or collagen
		Too high or too low cell confluency	Confirm cell viability and proper confluency
		Rough handling	Pipette carefully during washing or medium changing
2A(iv)	Failure in autofocus	Overexposed particles	Plate cells at proper density and confirm cell viability
			Avoid fields containing dead cells or cell debris, which could result in overexposed particles
		Vibrations	Place the whole equipment setup on a shockproof platform
	Cell death during long-term imaging	Excessive exposure time	Reduce exposure time, especially when a short wavelength excitation light source is utilized
		Unstable culture environment	Check temperate control, CO2 injection and water supplementation during the whole imaging process
2B(ii-iv)	Blurry images	Image out of focus	Pipette carefully
		AFC is not activated	Open AFC before imaging
		3D structure of different subcellular compartments	Choose independent focal plane and pinhole size for specific subcellular components
2B(v)	Low fluorescence responses in cells treated with chemicals	Reagent solutions are not fully mixed with cell culture medium	Mix fully after adding reagents
Procedure 3
15	Dim fluorescence of cell microcapsules	Low-level FiLa expression	Choose a stable monoclonal cell line with high expression of FiLa sensors
		Low cell viability	Control the preparation time of sodium alginate-cell suspension, and preheat buffer to room temperature in advance
		Inappropriate exposure time	Adjust the exposure time to improve image quality
		Low injection dose of microcapsule	Increase the injection dose appropriately
		or ruptured microcapsules	Reduce time of depolymerization or transplant the microcapsules with 10 mL syringe
Procedure 4
3	Low fluorescence signal in mouse muscle tissue	Inappropriate serotype of AAV	Choose the appropriate tissue or cell specific serotype of AAV
		Poor quality of AAV	Improve the titre and purity of AAV
		Low fluorescence intensity	Increase the injection dose of virus or increase expression time after virus injection
		Too deep an injection	Inject virus at the shallow surface of the gastrocnemius muscle
7	The field of view moves	Image out of focus	Inject insulin carefully and avoid touching the mouse or the microscope stage
		The mouse moves during the injection or imaging process	Anesthetize the mouse sufficiently by adjusting the dose of sodium pentobarbital
Procedure 5
3	Abnormal values	Plasma or serum samples are contaminated by red blood cell rupture	Contain blood samples in sterile tubes
			Avoid oscillating samples before centrifugation
		Degradation of the substrate to be tested	Divide samples into aliquots, store in ultra-low temperature freezer and avoid repeated freezing and thawing
		Evaporation of water in samples	Seal the tubes of samples with parafilm
6	The background value is too high	Unsuitable dilution factor	Increase the dilution factor or use the sensor with high affinity
9	Sample values are out of the sensor detection range	Unsuitable dilution factor	Test dilution factor in advance
		Use of sensor with inappropriate affinity	Use sensor with appropriate affinity
Box 1, step 8	Cells detach after washing with assay medium	Aspirating or adding the medium too hard	Remove or add medium carefully
	Low or high signal detection for measurements	The assay is not sensitive enough or the detection range is exceeded	Test cell density in advance and adjustment for different cell lines
Box 1, step 9	Did not yield the anticipated results	Drugs are not loaded properly into the injection ports, or not all injection ports are fulfilled with drugs or medium	Loaded drugs or medium should cover the injection ports properly to ensure enough air pressure. Make sure to pipette carefully, in one stream and avoid air bubbles
		Injection ports are clogged	Make sure that drugs are dissolved sufficiently without crystallization
		Incubation time is too short	Increase measurement points and wait for curve to flatten out
		Drug concentrations are not optimal	Test drug concentrations in advance and adjust for different cell lines
Box 1, step 10	Abnormal values	Data are disrupted by abnormal values of background correction wells	Check the values of the background correction wells and exclude abnormal values
		Cell density is not uniform in some of the wells	Trypsinize the cells into single-cell suspension and thoroughly mix before seeding
			Exclude the outliers