Defining the Tools: an Analysis of Laser Scanning Confocal and Wide-Field/Restoration Fluorescence Microscope Imaging

Abstract

Digital fluorescence microscopy is now a standard tool for determining the localization of cellular components in fixed and living cells. Two fundamentally different imaging technologies are available for imaging fluorescently labelled cells and tissues, in either the fixed or living state. The laser scanning microscope uses a diffraction-limited focused beam to scan the sample and develop an image point by point. in addition, a pinhole placed in a plane confocal to the specimen prevents emitted out-of focus fluorescence from reaching the photomultiplier tube (PMT) detector. By combining spot illumination and selection of infocus fluorescence signal, the laser scanning confocal microscope (LSCM) creates an image of the specimen largely free of out-of-focus blur. By contrast, a wide-field microscope (WFM) illuminates the whole specimen simultaneously and detects the signal with a spatial array of point detectors, usually a charge-coupled device camera (CCD). This approach collects an image of all points of the specimen simultaneously and includes all the out-of-focus blurred light. Subsequent restoration by iterative deconvolution generates an estimate of the specimen, largely free of out-of-focus blur. While many other fluorescence imaging modalities exist, these two methods represent the majority of the fluorescence imaging systems currently in use in biomedical research.