Expansion Microscopy with a Thermally Adjustable Expansion Factor Using Thermoresponsive Biospecimen–Hydrogel Hybrids

Abstract

Expansion microscopy (ExM) is a technique in which swellable hydrogel-embedded biological samples are physically expanded to effectively increase imaging resolution. Here, we develop thermoresponsive reversible ExM (T-RevExM), in which the expansion factor can be thermally adjusted in a reversible manner. In this method, samples are embedded in thermoresponsive hydrogels and partially digested to allow for reversible swelling of the sample–gel hybrid in a temperature-dependent manner. We first synthesized hydrogels exhibiting lower critical solution temperature (LCST)- and upper critical solution temperature (UCST)-phase transition properties with N-alkyl acrylamide or sulfobetaine monomers, respectively. We then formed covalent hybrids between the LCST or UCST hydrogel and biomolecules across the cultured cells and tissues. The resulting hybrid could be reversibly swelled or deswelled in a temperature-dependent manner, with LCST- and UCST-based hybrids negatively and positively responding to the increase in temperature (termed thermonegative RevExM and thermopositive RevExM, respectively). We further showed reliable imaging of both unexpanded and expanded cells and tissues and demonstrated minimal distortions from the original sample using conventional confocal microscopy. Thus, T-RevExM enables easy adjustment of the size of biological samples and therefore the effective magnification and resolution of the sample, simply by changing the sample temperature.

Expansion Microscopy: Super-Resolution Imaging with Hydrogels

“…As seen throughout this review, expansion microscopy heavily borrows from established molecular biology and chemistry methods in its various modules. Hydrogels used in various expansion microscopy methods ,,, adapt polymer chemistries from basic material science studies. , Homogenization approaches often borrow from established methods to lyse model organisms for DNA extraction, ,, transfection, − , and immunostainings. , To anchor different biomolecules to the hydrogel and make them accessible for readout, various chemistries have been adapted, from off-the-shelf reagents targeting proteins via NHS-ester chemistry , or RNA bases to click chemistry for universal biomolecule access . Expansion gels can further be combined with optical super-resolution imaging methods − ,,− (see Combining Expansion Microscopy and Optical Super-Resolution Approaches).…”

“…One of the main interests in swellable hydrogels from a material science perspective has traditionally been their application as sensors, e.g., of pH, ion concentration, ethanol concentration, or temperature. , Kang et al have applied some of the tools developed in this context to create T-RevExM ( T hermoresponsive Rev ersible Ex pansion M icroscopy), using the comonomer N -alkyl acrylamide to create a hydrogel/sample hybrid that begins to expand below a critical temperature, , and the comonomer methacryloyl sulfobetain to create a hydrogel/sample hybrid that begins to expand above a critical temperature . T-RevExM is the only expansion microscopy technique to date that does not include any anionic comonomer component.…”

“…The rapid development of expansion microscopy techniques highlighted in this reviewwith myriad gel chemistries ,,− ,,,, and adaptations to access different biomolecules ,,,,− ,, and biological specimens ,− ,, being introduced on an almost weekly basisshould not make us forget that this is still a young technology. Significant challenges remain, in many areas.…”

“…As seen throughout this review, expansion microscopy heavily borrows from established molecular biology and chemistry methods in its various modules. Hydrogels used in various expansion microscopy methods 1,4,26,73 adapt polymer chemistries from basic material science studies. 79,86 Homogenization approaches often borrow from established methods to lyse model organisms for DNA extraction, 1,4,29 transfection, [37][38][39]156 and immunostainings.…”

Advances in ultrasound-responsive hydrogels for biomedical applications

“…Compared with traditional hydrogels, intelligent hydrogels can achieve multiple, variable, controllable and reversible changes in their shape and properties in a manner of space or time, thus greatly adapting to the complex application environment in vitro and in vivo. [37][38][39][40][41] Recently, various intelligent hydrogels, such as light-responsive hydrogels, [42][43][44][45][46] thermal-responsive hydrogels, [47][48][49][50][51] pH-responsive hydrogels, [52][53][54][55][56] ultrasound-responsive hydrogels, [57][58][59] ion-responsive hydrogels, [60][61][62] etc., have been developed and showed amazing biomedical applications. Among them, as a new class of intelligent hydrogels, ultrasound-responsive hydrogels have already exhibited irreplaceable application prospects.…”