Table 2.

Troubleshooting table.

Step	Problem	Possible reason	Solution
6	Force-displacement relationship is not linear	Bending displacement of the needle tip is too large	Use lighter weights or prepare a stiffer needle
11	Needle tips vibrate or fluctuate during calibration	Air currents interfering with measurement	Shut off any possible sources of air flow or perform calibration in water
12	Calibration error is huge	The difference in stiffness between two needles is too large (>10-fold)	Prepare a more flexible reference needle, or prepare another needle which has an intermediate stiffness between those two needles such that the difference is less than 10-fold
21	Agarose surface looks bumpy and has uneven thickness	Agarose is concentrated when warmed The temperature of agarose solution during coating process is too low	Keep the agarose concentration exact (0.1% final) Turn up the temperature of agarose solution to 80°C while keeping the agarose concentration
22	Agarose is patchy on coverslip surface	Hydrophilic surface has deteriorated	Reduce the time of surface drying
44	Spindle rotates and moves in chamber	Extract solution has not fully settled	Spread the extract with even thickness on the coverslip and layer the oil on top very gently
46	Spindle collapses over time	Bad extracts Photodamage	Use fresh and high-quality extract Use minimal exposure of fluorescence light by optimizing camera sensitivity, binning, interval of time-lapse, filter set, etc