Super-Resolution Imaging of Bacteria in a Microfluidics Device

Diego I Cattoni,Jean-Bernard Fiche,Tâm Mignot,Marcelo Nöllmann,Alessandro Valeri,Dirk-Jan Scheffers

doi:10.1371/journal.pone.0076268

Abstract

Bacteria have evolved complex, highly-coordinated, multi-component cellular engines to achieve high degrees of efficiency, accuracy, adaptability, and redundancy. Super-resolution fluorescence microscopy methods are ideally suited to investigate the internal composition, architecture, and dynamics of molecular machines and large cellular complexes. These techniques require the long-term stability of samples, high signal-to-noise-ratios, low chromatic aberrations and surface flatness, conditions difficult to meet with traditional immobilization methods. We present a method in which cells are functionalized to a microfluidics device and fluorophores are injected and imaged sequentially. This method has several advantages, as it permits the long-term immobilization of cells and proper correction of drift, avoids chromatic aberrations caused by the use of different filter sets, and allows for the flat immobilization of cells on the surface. In addition, we show that different surface chemistries can be used to image bacteria at different time-scales, and we introduce an automated cell detection and image analysis procedure that can be used to obtain cell-to-cell, single-molecule localization and dynamic heterogeneity as well as average properties at the super-resolution level.

Highlights

Bacteria have evolved complex, highly-coordinated, multicomponent cellular engines, such as the apparatus responsible for chromosome segregation/cell division/separation, the flagellar motor, the transcription/replication machines, or secretion/ conjugation machineries, to achieve high degrees of efficiency, accuracy, adaptability, and redundancy [1]
Single-molecule based super-resolution microscopy methods, such as photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) or other variants based on the same principles [4,5,6,7,8], have the advantage that they reach the highest resolutions (20–40 nm) and are applicable to live cells
A field of view containing tens of cells was first imaged by bright-field microscopy, the cell contour was imaged by detecting the fluorescence signal emitted by FM4-64 (Figure 1A-iii), and a complete PALM dataset comprising of,20000 frames was acquired (Figure 1A-iv)

Summary

Introduction

Highly-coordinated, multicomponent cellular engines, such as the apparatus responsible for chromosome segregation/cell division/separation, the flagellar motor, the transcription/replication machines, or secretion/ conjugation machineries, to achieve high degrees of efficiency, accuracy, adaptability, and redundancy [1]. Conventional fluorescence microscopy methods enable non-invasive observation of protein organization and localization in live cells with high specificity, and have played an important role in the investigation of these processes. The maximum resolution attainable by these methods is intrinsically limited by light diffraction and is several orders of magnitude lower than for X-ray or electron tomography. This limitation is considerably acute for bacteria, as the maximal resolution (,250 nm) is comparable to the size of the cell (typically ,1– 2 um). Recent advances in fluorescence microscopy have led to the development of several conceptually independent super-resolution methods that break the intrinsic resolution limit imposed by the diffraction of light [2,3]. Single-molecule based super-resolution microscopy (smSRM) methods, such as photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) or other variants based on the same principles [4,5,6,7,8], have the advantage that they reach the highest resolutions (20–40 nm) and are applicable to live cells

Methods

Results

Conclusion