Abstract
In a stimulated emission depletion (STED) microscope the region in which fluorescence markers can emit spontaneously shrinks with continued STED beam action after a singular excitation event. This fact has been recently used to substantially improve the effective spatial resolution in STED nanoscopy using time-gated detection, pulsed excitation and continuous wave (CW) STED beams. We present a theoretical framework and experimental data that characterize the time evolution of the effective point-spread-function of a STED microscope and illustrate the physical basis, the benefits, and the limitations of time-gated detection both for CW and pulsed STED lasers. While gating hardly improves the effective resolution in the all-pulsed modality, in the CW-STED modality gating strongly suppresses low spatial frequencies in the image. Gated CW-STED nanoscopy is in essence limited (only) by the reduction of the signal that is associated with gating. Time-gated detection also reduces/suppresses the influence of local variations of the fluorescence lifetime on STED microscopy resolution.
Highlights
Far-field fluorescence microscopy is a powerful imaging tool for investigating cells due to its non-invasive access to the cellular interior, the specific and sensitive detection of cellular features through fluorescence tagging, and the simple sample preparation
The gCW-Stimulated emission depletion (STED) images of the point-like objects (FNDs and beads) reveal their nominal size (35– 40 nm), which indicates that we have reached a spatial resolution of 35 nm or below
These image conditions have been reached at comparatively low average continuous wave (CW)-STED powers of PSTED = 250 mW for the fluorescent nano-diamonds (FNDs) and 230 mW for the crimson beads
Summary
Far-field fluorescence microscopy is a powerful imaging tool for investigating (living) cells due to its non-invasive access to the cellular interior, the specific and sensitive detection of cellular features through fluorescence tagging, and the simple sample preparation. Stimulated emission depletion (STED) microscopy [2,3] overcame the diffraction barrier and increased the spatial resolution of fluorescence microscopy for the first time by a large factor; in principle it can reach resolution at the molecular scale. For this purpose, STED microscopy (or nanoscopy) uses stimulated emission to inhibit fluorescence emission at predefined sample coordinates such that adjacent features emit sequentially in time. STED currently provides the fastest subdiffraction resolution recordings [21]
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