Autofluorescence in zebrafish - Which wavelengths do zebrafish NOT autofluoresce (Apr/02/2008 )

Trying to do some fluorescence/confocal imaging of novel markers in zebrafish, but their autofluorescence is interfering. Does anyone know of a wavelength at which zebrafish have reduced autofluoresence? I can do my imaging on live fish, but would rather on fixed fish (4%PFA). I've tried reducing autofluorescence of fixed fish with sodium borohydride, but the quenching is not strong enough.

Can you get access to a spectrofluorometer? If so, put some embryos into the cuvette and run some excitation wavelength scans at various emission wavelength settings, and perhaps a few emission wavelength scans with some fixed excitation wavelengths *(fixed where your first runs found especially effective excitation wavelengths). You might have some light-path problems with the 90 degree excitation-emission light path, but zebrafish embryos are reasonably transparent so you might get good transmittance of the emitted light. It's worth a shot, you could get a lot of data rapidly for a few hours of work.

or bring the fish to the microscope and run a lambda (?) scan, i.e. excite at all possible wavelengths and see where you get the least signal.