Current Progress in Expansion Microscopy: Chemical Strategies and Applications

Abstract

Expansion microscopy (ExM) is a newly developed super-resolution technique, allowing visualization of biological targets at nanoscale resolution on conventional fluorescence microscopes. Since its introduction in 2015, many efforts have been dedicated to broaden its application range or increase the resolution that can be achieved. As a consequence, recent years have witnessed remarkable advances in ExM. This review summarizes recent progress in ExM, with the focus on the chemical aspects of the method, from chemistries for biomolecule grafting to polymer synthesis and the impact on biological analysis. The combination of ExM with other microscopy techniques, in search of additional resolution improvement, is also discussed. In addition, we compare pre-and postexpansion labeling strategies and discuss the impact of fixation methods on ultrastructure preservation. We conclude this review with a perspective on existing challenges and future directions. We believe that this review will provide a comprehensive understanding of ExM and facilitate its usage and further development. CONTENTS R 8. Expansion Isotropy S 9. Conclusion and Outlook S

Enhanced synaptic protein visualization by multicolor super-resolution expansion microscopy

“…Since its first introduction by Chen et al., 20 expansion microscopy has shown impressive results, including the magnified visualization of proteins, lipids, and nucleic acids with fluorescent proteins, antibodies, functionalized sphingolipids, and oligonucleotides, respectively, in cells, tissues, and human clinical specimen 21 , 22 . It is possible to expand samples in series enabling gel expansion factors of 20× and higher 23 but such high expansion factors reduce the labeling density and consequently the signal-to-noise ratio thus requiring highly sensitive imaging methods.…”

Xanthene, cyanine, oxazine and BODIPY: the four pillars of the fluorophore empire for super-resolution bioimaging

“…276 It is worth mentioning here that very recently, expansion microscopy (ExM) has come forth as an alternative imaging modality to obtain sub-diffraction resolution images with conventional optical microscopes wherein the effective resolution of a biological sample is enhanced by physically magnifying the sample evenly through chemical methods. [277][278][279] A particular sample preparation method is followed in this microscopic technique by embedding the biological samples in an evenly swollen hydrogel. The conventional small-molecule fluorophores are not compatible with expansion microscopy as the fluorescent markers need to bind with the hydrogel covalently.…”

Current Best Practices for Analysis of Dendritic Spine Morphology and Number in Neurodevelopmental Disorder Research

“…Indeed, they are particularly powerful when combined with immunohistochemical labeling for synaptic markers, such as postsynaptic density proteins, neurotransmitter receptors, and RNA molecules (reviewed in ref ). To date, however, few studies have employed expansion microscopy to measure dendritic spines in a high-throughput manner, although such morphological interrogation is feasible . Whether such techniques provide a more consistently reliable method to determine spine structure, such as in neurodevelopmental conditions, remains to be seen.…”

“…To date, however, few studies have employed expansion microscopy to measure dendritic spines in a high-throughput manner, although such morphological interrogation is feasible. 69 Whether such techniques provide a more consistently reliable method to determine spine structure, such as in neurodevelopmental conditions, remains to be seen.…”