One-photon versus Two-photon Laser Scanning Mic roscopy and Digital Image Analysis of Microbial Biofilms

Abstract

Publisher Summary Laser scanning microscopy (LSM) represents a standard optical technique in life sciences. LSM can be employed in many different modes including reflection and fluorescence, single and multichannel, one-photon and two-photon, with data collection via intensity and lifetime imaging. This chapter focuses on one-photon and two-photon imaging of microbiological samples and digital-image analysis of the resulting LSM data sets. A structured approach to stepwise examine a new, unknown biofilm sample has been outlined in the chapter. In this approach, advantage is taken of the intrinsic sample properties such as reflection and autofluorescence. Simple but specific staining for cellular and polymeric biofilm constituents and their distribution is applied and probes for cell identity, cell viability as well as cell and enzyme activity are discussed. The chapter describes various probes for the microenvironment with the exception of reflection imaging. Most of the fluorochromes can be used for two-photon excitation although their excitation/emission response will be different from that in one-photon LSM (1P-LSM). Another option in two-photon LSM (2P-LSM) is that several of the fluorochromes may be combined and excited with one two-photon wavelength only and the resulting emission signals individually collected in separate channels.