New fluorescence microscopy methods for microbiology: sharper, faster, and quantitative

Abstract

In addition to the inherent interest stemming from their ecological and human health impacts, microbes have many advantages as model organisms, including ease of growth and manipulation and relatively simple genomes. However, the imaging of bacteria via light microscopy has been limited by their small sizes. Recent advances in fluorescence microscopy that allow imaging of structures at extremely high resolutions are thus of particular interest to the modern microbiologist. In addition, advances in high-throughput microscopy and quantitative image analysis are enabling cellular imaging to finally take advantage of the full power of bacterial numbers and ease of manipulation. These technical developments are ushering in a new era of using fluorescence microscopy to understand bacterial systems in a detailed, comprehensive, and quantitative manner.

Two different approaches to breaking the diffraction limit. A. STED microscopy uses two different lasers, a conventional excitation laser (left) and a torroidal doughnut-shaped STED laser (middle). The STED laser deactivates the fluorescence outside of a small central zone, thereby reducing the effective excitation area (right). B. Single molecule localization microscopy methods such as PALM and STORM use photoactivatable fluorophores to successively image the localization of a small number of molecules at a time at high precision by finding the molecule’s centroid. The many resulting images (4 are shown in the example illustrated here) are then superimposed to generate the final super-resolution image.