Laser Scanning Microscopy

Abstract

This chapter provides a basis for understanding laser scanning microscopy (LSM) and the approaches that may be used to apply high-resolution digital microscopy to the study of bacteria. Additional reviews of image analysis, digital imaging, and LSM for microbiology applications are also provided in this chapter. The major focus of most courses is cell biology, although the information is of use and techniques are broadly transferable across disciplines. In practice, one of the appealing aspects of fluorescence microscopy is the lack of a requirement for fixation, dehydration, and the air drying of samples required by some other techniques. One of the goals of this approach is to examine living bacterial cells, aggregates, and biofilms. Changes in any of the microscope settings will alter the section thickness, the brightness, and the apparent size of the objects being imaged. The major advantage of LSM is the capacity to collect a series of images that allow the user to obtain 3-D spatial information. The chapter gives general protocol and considerations of lectin staining. A number of approaches have been applied to study diffusion in bacterial biofilms and serve to illustrate the application of kinetic analyses using LSM systems. The major limitation of all light microscopic systems and particularly LSM is poor axial resolution. In LSM there are both 2-D- and 3-D-related considerations regarding sampling. Application of LSM techniques results in the creation of vast image and data sets.