Fluorescence Microscopy and the Reactions of Single Molecules

Abstract

The majority of applications of fluorescence reported in this book deal with fluorescence in solution. In many of them high spatial resolution is not required and therefore detectors for extreme sensitivity or extreme temporal accuracy can be used. However, for a number of applications in chemistry, biology, and medicine, images from microscopes are required and their resolution should ideally be close to the diffraction limit. Modern cameras allow high spatial resolution and high sensitivity at moderate temporal resolution: Single photon counting cameras with a sensitivity of better than a nanolux achieve the sensitivity of individual photomultipliers. Cooled CCD cameras are one to two orders of magnitude less sensitive (10 to 100 nanolux) but have a better spatial resolution and are easier to handle. The temporal resolution is poor as compared to photomultipliers or avalanche photodiodes. Fortunately, a large number of applications in chemistry, biology, and medicine do not need higher temporal resolution. In the present contribution we will report on a few such applications: single molecule detection and handling as well as the observation of single molecule reactions in the fluorescence microscope. This microscope will also be used to look into individual cells and to study the distribution of their chromosomes and finally to detect how calcium waves direct the coordinated beating of the heart and heart tissues.