STED Microscope
with a distorted donut, where the central hole is partially filled, inevitably leads to loss of signal and resolution. Since one cannot finely adapt the immersion and embed-
ding media for each sample, room temperature, and focusing depth, the only solution is to pre-compensate the distorted wavefronts to counteract any deviations they pick up in the sample on their way to the focus plane [18,19]. In astronomic telescopes, this has long been done with deformable mirrors that cancel atmospheric streak and turbulence, and the same is available for microscopy. A deformable mirror in the beam path effectively restores nearly perfect conditions in the focus plane as is clearly demonstrated by a drastically improved sig- nal quality at increased imaging depth (Figure 7A). Especially when comparing to reference conditions, it becomes clear that for imaging depths of more than 40 μm, it is almost indispens- able to apply a countermeasure against sample-induced dis- tortions (Figure 7B). Once again, MATRIX detection can be applied additionally to reduce out-of-focus background (Figure 7B, right column).
Optimizing for Spatial Resolution with MINFLUX With the techniques presented here, resolution in the dou-
ble-digit nanometer range can be reached in most cases. Full maximization of the resolving power in STED can yield up to 20 nm resolution with good sample preparation and acquisi- tion conditions. Research questions that require even higher resolution call for another method: MINFLUX, which achieves a new technical standard as it reaches spatial resolutions in the range of the size of a molecule and can be used to track emit- ters with tens of thousands of localizations and unprecedented speed [20–23]. It does this with a new imaging approach, which increases
Figure 7: Unlocking super-resolution imaging in thick tissue samples. (A) 3D-STED overview image along the z-axis using MATRIX differential detection and Adaptive Optics. Areas at two depths (at 20 μm and 40 μm labeled b and c, respectively) are compared below. (B, C) Comparison between uncorrected 3D-STED and the addition of countermeasures to retrieve signal quality at 20 μm (B) and 40 μm (C) imaging depth. Scale bars: (A) 10 μm, (B and C) 2 μm. Sample provided by Abdelali Jalil, PhD (Saints Pères Paris Institut for Neuroscience, SPPIN, Paris) during a live demo at MiFoBio 2021.
a technical point of view. After all, the sample is the last part in the optical train of the microscope, and it does make a large difference when light must travel through one hun- dred microns or more of optically irregular tissue before it is focused. Even when the sample itself is relatively homo- geneous, there is usually a step-like change in refractive index (optical density) between the immersion medium and the sample. This results in spherical aberrations, which become worse for greater refractive index mismatches and with greater distance from the cover slip. For thin samples, one often gets away with using a water immersion lens for aqueous samples, but the resulting mismatch is too large for focusing deep inside a sample. When imaging with super- resolution, the situation becomes worse, since all available methods highly depend on clean light distribution in the focus plane. For example, trying to do STED microscopy
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the information content of the collected photons. MINFLUX allows individual emitters to be located much more quickly and accurately, by finding the minimum of their intensity dis- tribution instead of the maximum as with PALM/STORM. To this end, a donut-shaped excitation beam is placed in the vicinity of the emitter and slightly wiggled to find the posi- tion for which emission becomes minimal. When this is the case, the donut is exactly centered on the molecule, and since the position of the donut is known with great precision, so is the location of the molecule. Equally important, only a few photons are spent in this process. Commercial capabilities of MINFLUX systems are evolving at a fast pace and cur- rently include two-color single-molecule imaging in all three dimensions. Dyes optimized for MINFLUX, such as the FLUX family from abberior, open new ways to characterize protein complexes and other organizational units of the cell on the nanometer scale.
Summary In the past decade, super-resolution microscopy has
developed into a mature field and is now readily available and easy to use. Nevertheless, the borders of what can be done are continually being pushed further. STED provides straightforward super-resolution imaging of delicate sam- ples and deep into thick tissue, and MINFLUX delivers the ultimate spatial resolution of the size of a molecule. With these possibilities, it can be expected that new chapters
www.microscopy-today.com • 2022 July
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