Super-Resolution Imaging of Bacteria in a Microfluidics Device

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. © 2013 Cattoni et al.