Multiphoton Microscopy in the Biomedical Sciences

Abstract

We derive an exact steady state solution for a diffusion-coupled reaction localized to a small but open spherical sub-volume in a fluid reaction medium. We show that this leads to a fluorescence-based method for measuring the diffusion constant and the photochemical properties of fluorophores that is simpler and more robust than existing techniques such as fluorescence correlation spectroscopy (FCS). We use this method to study the photobleaching of rhodamine-B labeled protein molecules under different illumination intensities. Together with complementary data provided by FCS, this determines the average number of photons emitted by the fluorophores before photobleaching (~5x104). We demonstrate that this technique can be easily implemented on any confocal or multiphoton microscope or spectrometer and thus it should be adaptable to a variety of biological and chemical problems.