Heat denaturation enables multicolor X10-STED microscopy at single-digit nanometer resolution

Abstract

Expansion microscopy (ExM) improves imaging quality by physically enlarging the biological specimens. In principle, combining a large expansion factor with optical super-resolution should provide extremely high imaging precision. However, large expansion factors imply that the expanded specimens are dim and are therefore poorly suited for optical super-resolution. To solve this problem, we present a protocol that ensures the 10-fold expansion of the samples through high-temperature homogenization (X10ht). The resulting gels exhibited relatively high fluorescence intensity, enabling the sample analysis by multicolor stimulated emission depletion (STED) microscopy, for a final resolution of 6-8 nm. X10ht offers a more thorough homogenization than previous X10 protocols based on enzymatic digestion, and thereby enables the expansion of thick samples. The better epitope preservation also enables the use of nanobodies as labeling probes and the implementation of post-expansion signal amplification. We conclude that X10ht is a promising tool for nanoscale resolution in biological samples.

Visualizing proteins by expansion microscopy

“…X10 expansion of cultured cells was performed using proteinase K exactly as described in the following protocol article: 28 . X10 expansion relying on autoclaving (X10ht 69 ) was performed as follows. The samples were incubated with 0.3 mg/ml Acryloyl-X (SE; #A-20770, Thermo Fisher Scientific) in alkaline buffer (150 mN NaHCO 3 , pH 6.4), overnight, at room temperature.…”

“…ONE analysis of brain slices. a, Images of a 200 µm-thick rat brain section before (left) and after (right) expansion, relying on autoclaving for homogenization 69 . The scale bar does not take the expansion factor into consideration.…”

Expansion Microscopy: Super-Resolution Imaging with Hydrogels

“…The earliest example of this was the combination of the original 4-fold expanding expansion microscopy protocol with STED microscopy, for a combined resolution below 10 nm . Almost all major optical super-resolution modalities have now successfully been combined with various expansion microscopy techniques. − ,,− …”

“…The main pioneering method of this approach is MAP, but the principle was later also adopted in techniques such as ZOOM and MAGNIFY. , As pointed out above, protease treatment risks destroying epitopes and makes proteins inaccessible to most antibodies and similar affinity probes . MAP and other protocols thus replace proteolytic digestion with denaturation via heat and chaotropes, such as SDS or urea. ,,, This principle is well-known from SDS-PAGE, for example. Not all antibodies still recognize their epitopes in proteins treated this way, but most do and it is typically possible to find a working one for any given protein. ,,,, This direct access to native proteins poses several advantages.…”

“…Since expansion microscopy is an entirely sample preparation-based technique, it can even be combined with classical optics-based super-resolution methods. This has pushed the attainable resolution into the single-digit nanometer range, , and recently beyond, to below 1 nm …”