Quantitative Fluorescence Confocal Laser Scanning Microscopy (CLSM)

Abstract

The confocal imaging geometry provides a dramatic optical advantage for fluorescence microscopy by discriminating against out-of-focus background with minimal loss of image-forming signal. Significant enhancement of both axial and lateral imaging resolution is also available but only with substantial signal loss. Because of these optical advantages, the confocal laser scanning microscope (CLSM) can clearly image thin optical sections from within thick fluorescence-labeled living specimens. A stack of optical sections is easily combined to reveal three-dimensional (3D) fluorescent marker distributions with diffraction-limited spatial resolution. When bright stable fluorophores are available, cellular dynamics can be measured by recording a time series of CLSM images.