Comparison between fresh and fixed human biopsies using spectral and lifetime measurements: Fluorescence analysis using one and two photon excitations

The purpose of this study is to make a comparison between the fluorescence emissions of fresh extracted human biopsies and fixed human biopsies, in order to evaluate the impact of fixation on autofluoresence signal. Our group is developing an endomicroscope to image brain tissues in-vivo, however to date, in order to validate our technology the easiest type of samples we can access are fixed samples. However, the fixation is still challenging. For that, we aim through this study to determine whether we should pursue to work on fixed samples or we should shift to work on fresh biopsies. Data were collected on spectroscopic, lifetime measurement and fluorescence imaging set-ups with visible and two-photon excitations wavelengths. Five fresh and five fixed samples are involved in the experiment. Endogenous fluorescence of fixed biopsies were calculated. Experimental results reveal that at 405 nm and 810 nm, the fresh samples have an intensity of fluorescence two times higher than that of fixed samples. However, for each fluorophore and each excitation wavelength, the lifetime for fresh samples is shorter than that for fixed samples. Still, further studies and investigations involving the comparison between different samples are required to strengthen our findings.