Principles of fluorescence for quantitative fluorescence microscopy

Continuing developments in fluorescence microscopy are providing increasingly sophisticated research tools that support detailed noninvasive investigations of molecular level functions in biological systems, including the capability to investigate molecular dynamics, interactions, and structure with exquisite sensitivity and specificity. Many of the important fluorescence microscopy-based research methods employ sophisticated data collection and analysis procedures, and a clear understanding of the fundamental principles of molecular fluorescence is a prerequisite for their successful implementation. This chapter therefore aims to introduce the basic photophysical properties of fluorescent molecules and to discuss some of the ways in which these properties influence experimental design and analysis in quantitative fluorescence microscopy. The emphasis is on fundamental principles rather than specific methods or applications.