A Universal Labeling Strategy for Nucleic Acids in Expansion Microscopy

Abstract

Expansion microscopy (ExM) enables nanoscale imaging of ribonucleic acids (RNA) on a conventional fluorescence microscope, providing information on the intricate patterns of gene expression at (sub)cellular resolution and within spatial context. To extend the use of such strategies, we examined a series of multivalent reagents which allow labeling and grafting of DNA oligonucleotide probes in a unified approach. We show that the reagents are directly compatible with third-generation in situ hybridization chain reaction RNA-FISH techniques while displaying complete retention of the targeted transcripts. Furthermore, we validate and demonstrate that our labeling method is compatible with multi-color staining. Through oligonucleotide-conjugated antibodies, we demonstrate excellent performance in ×4 ExM and ×10 ExM, achieving a resolution of ~50 nm in ×10 ExM, both for pre-and postexpansion labeling strategies. Our results indicate that our multivalent molecules enable rapid functionalization of DNA oligonucleotides for expansion microscopy.

Linker Molecules Convert Commercial Fluorophores into Tailored Functional Probes during Biolabelling

“…This allows systematic variations of bioconjugation site, the fluorophore tag or the functional moiety of the linker (Figure 1). While there are other trifunctional linkers described in the literature by us for self‐healing dyes, [9c] the Hofkens lab for expansion microscopy [17b,c] and others, [10a] these require the full assembly of the functional dye before labeling. We thus believe that our concept will be of great importance for many groups in biology, biochemistry and biophysics who are eager to obtain functional fluorophores or modify other properties during biolabelling.…”

Nanoscale fluorescence imaging of biological ultrastructure via molecular anchoring and physical expansion

“…While pre-expansion staining often provides adequate fluorescence signals after the ExM process, it can lead to degradation of the sample fluorescence. For example, organic dyes can be degraded or destroyed during the in situ polymerization step (e.g., degradation of the cyanine dyes by the free radicals) [ 40 , 41 ]. In addition, as described earlier, the fluorescent portion of the antibodies or FPs can be removed during the homogenization step if not properly anchored to the hydrogel network.…”

“…Nucleic acids can be anchored to the hydrogel network via custom-synthesized trifunctional anchors, such as TRITON (also see Sect. 4.4.2 ) [ 40 ]. TRITON is equipped with a hybridization chain reaction initiator for signal amplification, a readout probe which targets the RNA of interest, and a polymerizable acryloyl moiety that helps tether the target RNA to the hydrogel.…”

“…Scale bars, 50 μm ( a , c ) and 10 μm ( b , d ). (Reprinted/adapted with the permission from Wen et al [ 40 ]. Copyright 2021 American Chemical Society) …”

Expansion microscopy with ninefold swelling (NIFS) hydrogel permits cellular ultrastructure imaging on conventional microscope

“…Another approach is through innovations in sample preparation, for example, expansion microscope (ExM) (19)(20)(21), which embeds the specimen into a hydrogel that could expand four times when dialyzed in water, realizing ~70-nm resolution. The ExM technique does not require special machinery or particular data processing, therefore providing affordable approaches for superresolution imaging in most laboratories (22)(23)(24)(25)(26)(27)(28), and has been applied in neuroscience, pathology, and mRNA discovery (29)(30)(31)(32)(33)(34)(35)(36). However, current ExM methods only manage to reach a resolution of about 70 nm, which still lags behind that in modern superresolution instruments (4,16,37,38).…”