Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation

Multiphoton excitation (MPE) of fluorescent probes has become an attractive alternative in biological applications of laser scanning microscopy becaus e many problems encountered in spectroscopic measurements of living tissue such as light scattering, autofluorescence, and photodamage can be reduced. The present study investigates the characteristics of two-photon excitatio n (2PE) in comparison with confocal one-photon excitation (1PE) for intrace llular applications of fluorescence correlation spectroscopy (FCS). FCS is an attractive method of measuring molecular concentrations, mobility parame ters, chemical kinetics, and fluorescence photophysics. Several FCS applica tions in mammalian and plant cells are outlined, to illustrate the capabili ties of both 1PE and 2PE. Photophysical properties of fluorophores required for quantitative FCS in tissues are analyzed. Measurements in live cells a nd on cell membranes are feasible with reasonable signal-to-noise ratios, e ven with fluorophore concentrations as low as the single-molecule level in the sampling volume. Molecular mobilities can be measured over a wide range of characteristic time constants from similar to 10(-3) to 10(3) ms. While both excitation alternatives work well for intracellular FCS in thin prepa rations, 2PE can substantially improve signal quality in turbid preparation s like plant cells and deep cell layers in tissue. At comparable signal lev els, 2PE minimizes photobleaching in spatially restrictive cellular compart ments, thereby preserving long-term signal acquisition.