Actin restructuring duringSalmonella typhimuriuminfection investigated by confocal and super-resolution microscopy

Jason J Han,Yuliya A Kunde,James H Werner,Elizabeth Hong-Geller

doi:10.1117/1.jbo.19.1.016011

Abstract

We have used super-resolution optical microscopy and confocal microscopy to visualize the cytoskeletal restructuring of HeLa cells that accompanies and enables Salmonella typhimurium internalization. Herein, we report the use of confocal microscopy to verify and explore infection conditions that would be compatible with super-resolution optical microscopy, using Alexa-488 labeled phalloidin to stain the actin cytoskeletal network. While it is well known that actin restructuring and cytoskeletal rearrangements often accompany and assist in bacterial infection, most studies have employed conventional diffraction-limited fluorescence microscopy to explore these changes. Here we show that the superior spatial resolution provided by single-molecule localization methods (such as direct stochastic optical reconstruction microscopy) enables more precise visualization of the nanoscale changes in the actin cytoskeleton that accompany bacterial infection. In particular, we found that a thin (100-nm) ring of actin often surrounds an invading bacteria 10 to 20 min postinfection, with this ring being transitory in nature. We estimate that a few hundred monofilaments of actin surround the S. typhimurium in this heretofore unreported bacterial internalization intermediate.

Highlights

IntroductionOver the past few decades, a number of far-field super-resolution (SR) optical microscopies have been developed that enable fluorescence imaging of cellular structure with unprecedented subdiffraction-limited spatial resolution.[1,2,3,4,5,6,7,8] The three primary SR optical configurations are stimulated emission depletion (STED) microscopy,[8] which is a confocal scanning–based technique that achieves resolution enhancement by limiting the spatial distribution of excited fluorophores in the sample; structured illumination microscopy (SIM),[5] which uses patterned, or structured, light to encode normally inaccessible high-spatial frequency information into the fluorescence signal; and single-molecule localization microscopy (SMLM),[1,2,3] which relies on high-precision centroid localization of spatially isolated single-point emitters
We note that while there has been some progress in extending single-molecule localization microscopy (SMLM) methods from 2-D to 3D,13,34 many of these methods are limited in their depth of field to Æ500 nm[13] or are equipment intensive.[34]
Due to the degradation in localization precision that comes from having a high background,[12] SMLM is generally better suited to looking at thin samples to minimize the background and increase the localization precision

Summary

Introduction

Over the past few decades, a number of far-field super-resolution (SR) optical microscopies have been developed that enable fluorescence imaging of cellular structure with unprecedented subdiffraction-limited spatial resolution.[1,2,3,4,5,6,7,8] The three primary SR optical configurations are stimulated emission depletion (STED) microscopy,[8] which is a confocal scanning–based technique that achieves resolution enhancement by limiting the spatial distribution of excited fluorophores in the sample; structured illumination microscopy (SIM),[5] which uses patterned, or structured, light to encode normally inaccessible high-spatial frequency information into the fluorescence signal; and single-molecule localization microscopy (SMLM),[1,2,3] which relies on high-precision centroid localization of spatially isolated single-point emitters. For a densely labeled sample, the relative number of molecules in the bright (Nbright) and dark (Ndark) states must be carefully controlled and maintained such that Nbright ≪ Ndark during the entire course of the imaging experiment. This requirement is due to the fact that in SMLM, it is essential that molecules are imaged individually, such that in any given image, the point spread function of two molecules in the bright state are not overlapped. In SMLM, individual molecules are localized with high precision (typically 10 to 20 nm, limited by the total number of photons detected and background noise12), with a computer-generated image of all centroids detected during the course of the experiment being used to construct a high-resolution map of the underlying molecular density of the fluorophore used for fluorescent labeling

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