Mechanically resolved imaging of bacteria using expansion microscopy.

Youngbin Lim,Margarita Khariton,Anthony L Shiver,Kerwyn Casey Huang,Samuel R Bray,Keara M Lane,Jian Qin,Bo Wang,Katharine M Ng,Raghuveer Parthasarathy

doi:10.1371/journal.pbio.3000268

Youngbin Lim, Margarita Khariton + Show 8 more

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https://doi.org/10.1371/journal.pbio.3000268

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Abstract

Imaging dense and diverse microbial communities has broad applications in basic microbiology and medicine, but remains a grand challenge due to the fact that many species adopt similar morphologies. While prior studies have relied on techniques involving spectral labeling, we have developed an expansion microscopy method (μExM) in which bacterial cells are physically expanded prior to imaging. We find that expansion patterns depend on the structural and mechanical properties of the cell wall, which vary across species and conditions. We use this phenomenon as a quantitative and sensitive phenotypic imaging contrast orthogonal to spectral separation to resolve bacterial cells of different species or in distinct physiological states. Focusing on host–microbe interactions that are difficult to quantify through fluorescence alone, we demonstrate the ability of μExM to distinguish species through an in vitro defined community of human gut commensals and in vivo imaging of a model gut microbiota, and to sensitively detect cell-envelope damage caused by antibiotics or previously unrecognized cell-to-cell phenotypic heterogeneity among pathogenic bacteria as they infect macrophages.

Highlights

Imaging of heterogeneous bacterial populations has broad applications in understanding the complex microbiota that exist on and within our bodies, as well as complex host–microbial interfaces, yet remains a significant challenge due to the lack of suitable tools for distinguishing species and identifying altered physiological states [1,2,3]
Expansion provides quantitative imaging contrast to distinguish bacterial species To test whether the cell wall restricts expansion in expansion microscopy (ExM), we imaged a mixture of two common symbiotic bacteria isolated from the gut of the fruit fly Drosophila melanogaster, Acetobacter tropicalis, and Lactobacillus plantarum [24] using a standard ExM protocol (Fig 1A, top) [25]
Our findings suggest that expansion microscopy of microbes (μExM) both improves imaging resolution, as shown in previous studies [19,20,21,25], and provides an additional imaging contrast associated with cell wall mechanical properties that is orthogonal to spectral separation commonly used in fluorescence microscopy

Summary

Introduction

Imaging of heterogeneous bacterial populations has broad applications in understanding the complex microbiota that exist on and within our bodies, as well as complex host–microbial interfaces, yet remains a significant challenge due to the lack of suitable tools for distinguishing species and identifying altered physiological states [1,2,3]. While prior studies have focused on achieving uniform expansion of samples, primarily in mammalian cell and animal tissues [19,20,21], we posited that differences in the expandability of the bacterial cell wall, which would not be digested through typical ExM protocols, could provide imaging contrast that reflects its molecular structure and distinct mechanical properties.

Results

Conclusion