Effect of handling and fixation processes on fluorescence spectroscopy of mouse skeletal muscles under two-photon excitation

We investigated the effects of handling and fixation processes on the two-p hoton fluorescence spectroscopy of endogenous fluorophors in mouse skeletal muscle. The skeletal muscle was handled in one of two ways: either section ed without storage or sectioned following storage in a freezer. The two-pho ton fluorescence spectra measured for different storage or fixation periods show a differential among those samples that mere stored in water or were fixed either in formalin or methanol. The spectroscopic results indicate th at formalin was the least disruptive fixative, having only a weak effect on the two-photon fluorescence spectroscopy of muscle tissue, whereas methano l had a significant influence on one of the autofluorescence peaks. The two handling processes yielded similar spectral information, indicating no dif ferent effects between them. (C) 2000 Optical Society of America. OCIS code s: 170.6510, 170.6280, 170.6930, 170.4580, 170.2520, 170.7160.