Abstract
We characterize a novel fluorescence microscope which combines the high spatial discrimination of a total internal reflection epi-fluorescence (epi-TIRF) microscope with that of stimulated emission depletion (STED) nanoscopy. This combination of high axial confinement and dynamic-active lateral spatial discrimination of the detected fluorescence emission promises imaging and spectroscopy of the structure and function of cell membranes at the macro-molecular scale. Following a full theoretical description of the sampling volume and the recording of images of fluorescent beads, we exemplify the performance and limitations of the TIRF-STED nanoscope with particular attention to the polarization state of the laser excitation light. We demonstrate fluorescence correlation spectroscopy (FCS) with the TIRF-STED nanoscope by observing the diffusion of dye molecules in aqueous solutions and of fluorescent lipid analogs in supported lipid bilayers in the presence of background signal. The nanoscope reduced the out-of-focus background signal. A lateral resolution down to 40-50 nm was attained which was ultimately limited by the low lateral signal-to-background ratio inherent to the confocal epi-TIRF scheme. Together with the estimated axial confinement of about 55 nm, our TIRF-STED nanoscope achieved an almost isotropic and less than 1 attoliter small all-optically induced measurement volume.
Highlights
The monitoring of biologically relevant structures and reactions down to the single-molecule level with fluorescence tagging has become one of the most promising approaches for understanding a variety of phenomena in biophysics, biochemistry and life science
Thereafter, if confining the measurement to a surface is acceptable, i.e. far-field wave propagation is given up along the optical axis, one can resort to total internal reflection (TIR) as a means to reduce the axial extent of the measurement volume
We carried out fluorescence correlation spectroscopy (FCS) experiments to further characterize the performance of the TIRF-stimulated emission depletion (STED) nanoscope by monitoring the intensity fluctuations due to fluorophores diffusing in and out of the observation volume
Summary
The monitoring of biologically relevant structures and reactions down to the single-molecule level with fluorescence tagging has become one of the most promising approaches for understanding a variety of phenomena in biophysics, biochemistry and life science. Since the advent of the confocal laser scanning microscope (LSM), fluorescence microscopy and spectroscopy are often performed on the very same instrument It offers a small observation volume for spectroscopic measurements together with the ability to quickly image the three-dimensional structures of interest with high spatial resolution and minimal invasion. When it comes to detecting fluorophores dwelling close to the interface, as for instance labeled constituents of cell membranes, illuminating a large area should be avoided because photo-bleaching would bleach most fluorophores long before they ever reach the measurement volume In this case, an annular laser illumination can provide an evanescent excitation confined within a central peak that is surrounded by weakening side lobes (Bessel beam like pattern) [26,27]. An almost isotropic observation volume of
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