Time-resolved fluorescence microscopy

Abstract

Fluorescence imaging techniques are powerful tools in the biological and biomedical sciences, because they are minimally invasive and can be applied to live cells and tissues. The fluorescence emission can be characterized not only by its intensity and position, by also by its fluorescence lifetime, polarization and wavelength. Fluorescence Lifetime Imaging (FLIM) in particular has emerged as a key technique to image the environment and interaction of specific proteins in living cells. Using a time-correlated single photon counting (TCSPC)-based FLIM set-up, we find that the fluorescence lifetime of GFP-tagged proteins in cells is a function of the refractive index of the medium the cells are suspended in. In addition, combining Fluorescence Recovery After Photobleaching (FRAP) of fluorescently labeled proteins of different sizes in sol gels with time-resolved fluorescence anisotropy measurements, we demonstrate that we can measure their lateral and rotational diffusion. This allows us to infer the size and connectivity of the pores in the sol gel matrix. Moreover, wide-field photon counting imaging, originally developed for astronomical applications, is a powerful imaging method because of its high sensitivity and excellent signal-to-noise ratio. It has a distinct advantage over CCD-based imaging due to the ability to time the arrival of individual photons. The potential of time-resolved widefield photon counting imaging with a fast CMOS camera applied to luminescence microscopy is demonstrated.

Two-Color Two-Photon Excitation Using Femtosecond Laser Pulses

“…Furthermore, pulsed excitation furnishes the basis for excellent time resolution using time-gated 5 or time-correlated single-photon-counting (TCSPC) 6 detection systems. Hence, a variety of time-resolved fluorescence techniques such as fluorescence lifetime imaging (FLIM) 5,[7][8][9] or fluorescence anisotropy imaging (Tr-FAIM) 8 have been established.…”

Time-resolved and two-photon emission imaging microscopy of live cells with inert platinum complexes

“…Recent advances in fluorescence-lifetime detection techniques have made fluorescence lifetime imaging microscopy (FLIM) a reality (7). To date, FLIM has been based primarily on endogenous molecules, such as the aromatic amino acid tryptophan (8), and on fluorescent dyes including GFP-tagged proteins and fluorescein derivatives (9), all of which have lifetimes of a few nanoseconds.…”

Setup and characterization of a multiphoton FLIM instrument for protein–protein interaction measurements in living cells

“…7 for a recent review). The correct use of FLIM calls for the proper characterization of the instrument.…”