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

ABSTRACT: Superresolution microscopy enables probing of cellular ultrastructures. However, its widespread applications are limited by the need for expensive machinery, specific hardware, and sophisticated data processing. Expansion microscopy (ExM) improves the resolution of conventional microscopy by physically expanding biological specimens before imaging and currently provides ~70-nm resolution, which still lags behind that of modern superresolution microscopy (~30 nm). Here, we demonstrate a ninefold swelling (NIFS) hydrogel, that can reduce ExM resolution to 31 nm when using regular traditional microscopy. We also design a detachable chip that integrates all the experimental operations to facilitate the maximal reproducibility of this high-resolution imaging technology. We demonstrate this technique on the superimaging of nuclear pore complex and clathrin-coated pits, whose structures can hardly be resolved by conventional microscopy. The method presented here offers a universal platform with superresolution imaging to unveil cellular ultrastructural details using standard conventional laboratory microscopes.

SUBMITTER: Li H

PROVIDER: S-EPMC9067917 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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

Li Hongxia H Warden Antony R AR He Jie J Shen Guangxia G Ding Xianting X

Science advances 20220504 18

Superresolution microscopy enables probing of cellular ultrastructures. However, its widespread applications are limited by the need for expensive machinery, specific hardware, and sophisticated data processing. Expansion microscopy (ExM) improves the resolution of conventional microscopy by physically expanding biological specimens before imaging and currently provides ~70-nm resolution, which still lags behind that of modern superresolution microscopy (~30 nm). Here, we demonstrate a ninefold ...[more]

PMID: 35507653

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Project description:Expansion microscopy (ExM) is an innovative approach to achieve super-resolution images without using super-resolution microscopes, based on the physical expansion of the sample. The advent of ExM has unlocked the detail of super-resolution images for a broader scientific circle, lowering the cost and entry skill requirements for the field. One of its branches, ultrastructure expansion microscopy (U-ExM), has become popular among research groups studying apicomplexan parasites, including the acute stage of Toxoplasma gondii infection. Here, we show that the chronic cyst-forming stage of Toxoplasma, however, resists U-ExM expansion, impeding precise protein localization. We then solve the in vitro cyst's resistance to denaturation required for successful U-ExM. As the cyst's main structural protein CST1 contains a mucin domain, we added an enzymatic digestion step using the pan-mucinase StcE prior to the expansion protocol. This allowed full expansion of the cysts in fibroblasts and primary neuronal cell culture without disrupting immunofluorescence analysis of parasite proteins. Using StcE-enhanced U-ExM, we clarified the localization of the GRA2 protein, which is important for establishing a normal cyst, observing GRA2 granules spanning across the CST1 cyst wall. The StcE-U-ExM protocol allows accurate pinpointing of proteins in the bradyzoite cyst, which will greatly facilitate investigation of the underlying biology of cyst formation and its vulnerabilities.ImportanceToxoplasma gondii is an intracellular parasite capable of establishing long-term chronic infection in nearly all warm-blooded animals. During the chronic stage, parasites encapsulate to form cysts predominantly in neurons and skeletal muscle. Current anti-Toxoplasma drugs do not eradicate chronic parasites, leaving a reservoir of infection. The cyst is critical for disease transmission and pathology, yet it is harder to study, with the function of many chronic-stage proteins still unknown. Ultrastructure expansion microscopy, a new method to overcome the light microscopy's diffraction limit by physically expanding the sample, allowed in-depth studies of acute Toxoplasma infection. We show that Toxoplasma cysts resist expansion using standard protocol, but an additional enzymatic digestion with the mucinase StcE allows full expansion. This protocol offers new avenues for examining the chronic stage, including precise spatial organization of cyst-specific proteins, linking these locations to morphological structures, and detailed investigations of components of the durable cyst wall.

Dataset Information

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

Publications

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

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