Expansion Microscopy for Beginners: Visualizing Microtubules in Expanded Cultured HeLa Cells - PubMed (original) (raw)

Expansion Microscopy for Beginners: Visualizing Microtubules in Expanded Cultured HeLa Cells

Chi Zhang et al. Curr Protoc Neurosci. 2020 Jun.

Abstract

Expansion microscopy (ExM) is a technique that physically expands preserved cells and tissues before microscope imaging, so that conventional diffraction-limited microscopes can perform nanoscale-resolution imaging. In ExM, biomolecules or their markers are linked to a dense, swellable gel network synthesized throughout a specimen. Mechanical homogenization of the sample (e.g., by protease digestion) and the addition of water enable isotropic swelling of the gel, so that the relative positions of biomolecules are preserved. We previously presented ExM protocols for analyzing proteins and RNAs in cells and tissues. Here we describe a cookbook-style ExM protocol for expanding cultured HeLa cells with immunostained microtubules, aimed to help newcomers familiarize themselves with the experimental setups and skills required to successfully perform ExM. Our aim is to help beginners, or students in a wet-lab classroom setting, learn all the key steps of ExM. © 2020 The Authors.

Keywords: HeLa cells; antibody; beginner; clearing; expansion microscopy; fluorescence microscopy; hydrogel; imaging; immunocytochemistry; immunohistochemistry; microscopy; microtubules; super-resolution microscopy.

© 2020 The Authors.

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Figures

Figure 1

Figure 1

Key steps in expanding fixed HeLa cells with immunostained microtubules. AcX, 6‐((acryloyl)amino)hexanoic acid, succinimidyl ester; ProK, proteinase K.

Figure 2

Figure 2

Construction of the gelation chamber and humidified tip box (protocol steps 31‐37).

Figure 3

Figure 3

Digestion, expansion, and mounting of the sample (steps 40‐54).

Figure 4

Figure 4

Imaging of HeLa cells with immunostained microtubules before and after expansion. Imaging was performed on an Andor spinning‐disk confocal microscope with a 40×, numerical aperture (NA) 1.15 water‐immersion objective. (A) Confocal image of HeLa cells with immunostained microtubules, imaged at a single xy plane at the bottom of the cells. The inset in the upper left zooms in on the small box at the middle right. (B) Confocal image of a ∼4.5× linearly expanded HeLa cell with immunostained microtubules, imaged at a single xy plane at the bottom of the cell. The inset in the upper left zooms in on the small box at the bottom left. Scale bars in (B) indicate post‐expansion scales.

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