The advent of laser scanning confocal microscopy solves the dilemma of studying thick specimens with optical microscopy by creating optical slices less than 1 μm in thickness. Increasingly, confocal microscopy is an essential analytical tool for studying the structure and physiology of living cells. Because confocal microscopy collects light from only a fraction of the specimen volume, greater illumination is required. Consequently, photodamage and photobleaching are greater considerations, especially for study for living cells where repeated measurements over time are desired. To minimize photodamage, laser intensity should be attenuated by 100-1000 fold, photomultiplier circuits should be operated at highest sensitivity, and stable fluorophores should be used. When these conditions are met, literally hundreds of high resolution confocal images can be obtained from single cells loaded with parameter sensitive fluorophores.The number of parameter-specific fluorophores useful for observing single living cells by confocal microscopy is large and increasing. By labeling with calcein and collecting serial images, the volume, shape and surface topography of single living cells are reconstructed with results rivaling scanning electron micrographs.
Simultaneous quantification of multiple endogenous biothiols in single living cells by plasmonic Raman probes