Molecular Dynamics in Living Cells Observed by Fluorescence Correlation Spectroscopy with One- and Two-Photon Excitation

TLDR

Multiphoton excitation reduces scattering, autofluorescence, and photodamage in living tissue, while fluorescence correlation spectroscopy provides a powerful tool for measuring molecular concentrations, mobilities, kinetics, and photophysics. The study investigates the characteristics of two‑photon excitation compared with confocal one‑photon excitation for intracellular fluorescence correlation spectroscopy. The authors outline FCS applications in mammalian and plant cells and analyze the photophysical properties of fluorophores necessary for quantitative FCS in tissues. Measurements in live cells and membranes are feasible down to single‑molecule concentrations, with mobilities spanning 10⁻³ to 10³ ms; 2PE improves signal quality in turbid tissues and reduces photobleaching, enabling long‑term acquisition.

Abstract

Multiphoton excitation (MPE) of fluorescent probes has become an attractive alternative in biological applications of laser scanning microscopy because 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 excitation (2PE) in comparison with confocal one-photon excitation (1PE) for intracellular applications of fluorescence correlation spectroscopy (FCS). FCS is an attractive method of measuring molecular concentrations, mobility parameters, chemical kinetics, and fluorescence photophysics. Several FCS applications in mammalian and plant cells are outlined, to illustrate the capabilities of both 1PE and 2PE. Photophysical properties of fluorophores required for quantitative FCS in tissues are analyzed. Measurements in live cells and on cell membranes are feasible with reasonable signal-to-noise ratios, even 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 approximately 10(-3) to 10(3) ms. While both excitation alternatives work well for intracellular FCS in thin preparations, 2PE can substantially improve signal quality in turbid preparations like plant cells and deep cell layers in tissue. At comparable signal levels, 2PE minimizes photobleaching in spatially restrictive cellular compartments, thereby preserving long-term signal acquisition.