Criteria for Quantitative Dynamical Confocal Fluorescence Imaging

摘要

Scanning confocal microscopy (SCM) offers a dramatic instrumental advantage forfluorescence microscopy through discrimination against out-of-focus background fluorescence, through inherent resolution perpendicular to the plane of focus and improved in-plane resolution. Quantitative measurements of fluorescence intensity in such images can provide precise image determinations of fluorescence marker distributions in three dimensions. These features promise significant improvement over conventional imaging methods, but they have yet to be realized with quantitative results from fluorescence SCM. The necessary criteria to achieve useful results are considered and the limitations and promise of scanning confocal fluorescence microscopy are appraised here. This reprint offers preliminary analyses of these problems, presents some preliminary measurements of crucial parameters, reports the relevant properties of one typical commercial laser scanning confocal instrument (BioRad MRC-500) to illustrate the problems and finally suggests some approaches to take maximum advantage of scanning confocal imaging for quantitative fluorescence image analysis. (jhd)