Fluorescent probes are commonly used in biological fluorescence microscopy for tracking specific structures and sub-cellular compartments, and for indicating cellular ionic conditions. Recent development in multi-photon fluorescence microscopy has greatly expanded the usage of fluorescent probes in biomedical research. Considering its non-linear nature, two-photon excitation may generate very different fluorescence spectral response in the sample when compared with single photon excitation. It is thus necessary to measure the two-photon spectra of various fluorescent probes, so that two-photon fluorescence microscopy may be operated effectively and the images properly interpreted. This report represents the first installment of a continued effort in characterizing the multi-photon fluorescence spectra of commonly used bio-probes.Two-photon fluorescence spectra excited with near infrared at 780nm were obtained with a SpectraPro-500 spectrophotometer (Acton Research) equipped with a TE-cooled PMT and coupled to a Spectra-Physics Tsunami Ti-sapphire laser pumped by a Coherent Verdi solid-state laser operated at 85MHz, l00fs pulse.
Label-free near-infrared reflectance microscopy as a complimentary tool for two-photon fluorescence brain imaging
