Optical methods have always played an important role in the study of physiological phenomena. Light microscopes have been used for more than one hundred years to gain insight into the structure of biological systems in general and the nervous system in particular. Anatomical studies had given us a first understanding of shapes and types of nerve cells. Another example is the ubiquitous and rarely
mentioned use of the light microscope to guide electrophysiological recording electrodes toward their targets. Only in the last two decades, however, has light microscopy become widely used to gather
quantitative dynamic information about physiological processes. The list of examples includes: the study of diffusion in membranes with fluorescence methods;3–6 the detection of transmembrane potential changes using intrinsic signals7 or extrinsic fluorophores (for reviews see Refs. 8, 9, and 10); the measurements of nanometer displacements;11–14 and the quantification of changes in intracellular ion concentration using fluorescent indicator dyes (for a review see Ref. 15). More recently, light has also been used to trigger physiological processes by applying biological effector molecules with high spatial and temporal resolution via photochemical
release, commonly called ‘‘uncaging.’’16–19 In this article, I review the contribution that the combination of two-photon absorption with laser scanning20 has made to functional biological imaging in recent years.