STED Microscope
temporal profile of the emission reveals a new dimension for detection (Figure 4D), where the dyes are now clearly separated. Thus, tion expands
the inclusion of the number of time-resolved detec- simultaneously resolvable
channels (Figure 4B). The resulting multi-color super- resolution image (Figure 4E) demonstrates the ease of performing super-resolution experiments with four targets, all while using only one 775 nm STED laser. Te use of time-resolved detection also improves resolution
for low STED powers, as with the intestinal epithelium micro- villi in Caco-2 cells shown in Figure 5A. To understand how this works, note that the de-excitation effect of the STED beam can be viewed as a modulation of fluorescence lifetime. Since the intensity of the STED beam is not the same everywhere (the donut has a hole), this means that the lifetime is not the same everywhere, that is, measuring the lifetime reveals additional spatial information that can be used to increase the resolution [9] (Figure 5D). Here, a MATRIX array detector was used addi- tionally to clear up background. In short, the MATRIX uses multiple APDs to obtain not only a one-directional, but instead a comprehensive, multi-angle view of the sample, which enables the direct removal of out-of-focus signal and increases optical sectioning (Figure 5C). Low-power imaging of live-cell dynamics. The ability
to image living systems is probably the greatest advantage of light over electron microscopy, as it is the only way to cap- ture detailed physiological processes and dynamics as they happen in their actual environment. The high resolution of STED microscopy provides direct access to subcellular organization and interaction [10]. To realize this potential, the entire process must be as non-invasive as possible. It requires labels able to reach their target without disrupting membrane structures, in addition to being bright, stable, and specific. An excellent example are abberior’s LIVE dyes— organic dyes with optimized chemical modifications, which make them membrane-permeable and useful already at very low concentrations [11–14]. Depending on the residual group, they can be used either for direct labeling of DNA, actin, tubulin, or lysosomes, or attached to a fusion protein, for example a SNAP®-tag. For imaging of a living specimen, it is imperative to mini-
mize cellular stress reactions and phototoxic effects. What the microscope can do here is reduce the light dose by making the most of the photons involved, both going in (STED and excita- tion beams) and coming out (fluorescence) of the specimen. A proven strategy is to apply photons only where and when they are useful. Tis requires pulsed STED lasers, which arrive dur- ing the lifetime of the excited dye states instead of continuous- wave lasers that mostly come too late. It is equally important to adapt the illumination to where sample structures are, for example, by switching it off at exactly those scan positions that contain no labels (Figure 6A). Avoiding useless exposure, in combination with LIVE dyes, facilitates the recording of time series images in living cells. In a series imaging the outer mito- chondrial membrane protein OMP25, no degradation of struc- tures or signal quality was found aſter 50 consecutive frames (Figure 6C). Finally, maximizing detection efficiency with
30
Figure 5: Unlocking sharp imaging in a low-signal high-background sample. Actin in microvilli of Caco-2 cells labelled with abberior STAR RED. (A) Overview image with boxed area compared below. (B) Base STED image. (C) Background reduction by differential detection. (D) Resolution improvement by rejecting out- skirt photons, detail of (A). Scale bars: (A) 5 μm, (B–D) 1 μm. Sample provided by Professor Dorothee Günzel and Jörg Piontek (Charité, Berlin).
APDs, maximizing the signal-to-background ratio with array detectors like the MATRIX, and maximizing resolution by inte- grating lifetime information, high signal quality at low laser power settings is enabled (Figure 6B). All this becomes even more important in cases where multiple exposures are required, such as when imaging a
www.microscopy-today.com • 2022 July
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