Troubleshooting

	PROBLEM	CAUSE	SOLUTION
Problems caused during imaging	No SRS signal	“Pump” or “Stokes” beam wavelength used is incorrect Light is not focused on the sample AOM is not working Photodiode is improperly functioning	Re-calculate difference frequency. Check that the lasers are tuned to vibrational resonance. Calibrate with pure reference chemical (dodecane/triolein) prior to imaging samples. Scan a range of wavelengths (± 2 nm). Ensure proper mirror alignment and condenser placement (critical). Ensure that AOM is functioning (check with a fast photodiode). Re-check settings of lock-in amplifier, reference to right modulation frequency. Removing excitation filter; photodiode should obtain a strong signal from the modulated excitation beam.
	SRS signal too weak/too high	Laser is not aligned to the center of the scanning area Laser power is too low/too high Amplifier settings are incorrect Low/high lipid levels in the sample	The laser should be aligned to the center of the scanning area using dodecane oil and by adjusting the mirror angles, before imaging samples. If changing the laser power or amplifier settings cannot increase the signal, then calibrate the setup again. On the image acquisition software, set the pixel intensity such that the background will be almost zero. Also avoid excess saturated pixels. A general rule of thumb is to image wild-type/control samples first, arrange the settings accordingly, and keep those parameters constant during the analysis of mutant/treated samples.
	High background	Amplifier/detector settings are incorrect Transferred excess bacteria when picking worms Cell culture medium generates background signal	Minimize transfer of bacteria while picking the worms, as this may contribute to high background. Replace with alternate medium/PBS. Image as soon as possible.
	Cannot focus on the sample	Objective lens may be dirty	Make sure the lenses are clean. Clean only with lens cleaner. Use immersion oil for higher magnification objectives.
Problems caused during sample preparation	Worms are still mobile in the anaesthetizing solution.	The concentration of the anaesthetizing solution is insufficient	Wait for several minutes after adding worms to the anesthetizing solution. Increase the concentration of sodium azide or levamisole modestly. If worms must be recovered after imaging, avoid highly concentrated solutions as they may kill worms.
	Worms burst frequently.	The samples waited too long for imaging after preparation Volume of the anaesthetizing solution is too low or its concentration too high Concentration of the agarose solution used for the pad is too high	Whole worm samples need to be imaged within approximately 30 minutes. Preservation at 4°C for a few more hours longer is possible, but samples cannot be recovered. If worms burst even within 30 minutes, the agarose pad or the anaesthetizing solution might be causing the problem. Lower the concentration of the agarose or the anaesthetizing solution. Also try increasing the volume of anesthetizing solution.
	Worms scatter too much on the agarose pad.	Volume of anaesthetizing solution is too high The agarose pad is too large	Place anaesthetizing solution onto the coverslip. Drop a smaller volume of agarose solution to make the pad.