Chloroplast and Cell Imaging at Submicron Resolution by Two-Photon Excitation

Multi-photon (MPE) NIR excitation of fluorophores--attached as labels to biopolymers like proteins and nucleic acids, or bound at specific biomembrane sites-is one of the most attractive options in biological applications of laser scanning microscopy. Many of the serious problems encountered in spectroscopic measurements of living tissue, such as photodamage, light scattering and auto-fluorescence, can be reduced or even eliminated. FCS can therefore provide accurate in vivo and in vitro measurements of diffusion rates, “mobility” parameters, molecular concentrations, chemical kinetics, aggregation processes, labeled nucleic acid hybridization kinetics and fluorescence photophysics/ photochemistry. Several photophysical properties of fluorophores that are required for quantitative analysis of FCS in tissues have already been widely reported. Molecular “mobilities” can be measured by FCS over a wide range of characteristic time constants from ~10 to 10 ms. At signal levels comparable to 1PE confocal microscopy, two-photon excitation (2PE) reduces photobleaching in spatially restricted cellular compartments, thereby preserving the long-term signal-to-noise during data acquisition. Furthermore, 3PE has been reported to eliminate DNA damage and photobleaching problems that may still be present in some 2PE experiments. Whereas both 1PE and 2PE alternatives are suitable for intracellular FCS observations on thin biological specimens, 2PE can substantially improve FCS signal quality in turbid samples, such as plant cell suspensions or deep cell layers within tissues.