Laser Scanning Microscopy of Yersinia pestis Infected Tissues

Laser scanning microscopy (LSM) is a technology that allows for direct observations of host-pathogen interactions during infection. Two of the most available forms of LSM are confocal and two-photon LSM. In addition to high resolution and contrast, these two technologies also provide high excitation penetrance in unsectioned samples. High penetrance allows for imaging of layers of tissue that are difficult to image with other more conventional microscopy approaches. Thus, confocal and two-photon LSM open the possibility of observing infection in a three-dimensional context, where the natural architecture of a tissue is preserved. Few studies have used LSM technology to gain insights into Yersinia pestis pathogenesis in the mammalian host. The use of LSM in the plague field has an enormous potential for the discovery of the mechanisms that lie behind key aspects of pathogenesis such as colonization, dissemination, and tissue damage. This chapter provides guidance for the implementation of confocal or two-photon LSM to study Y. pestis interactions with the host in unsectioned tissues. This document provides specific instructions applied to imaging of Y. pestis, and also discusses relevant aspects of imaging, such as the operation of laser scanning microscopes and the use of fluorescent probes.