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Abstract:
We characterize a novel fluorescence microscope which combines the high
s
patial 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.
