Characterization and Calibration in Wide Field and Sectioned Fluorescence Microscopy SIPcharts

Abstract

A fluorescence image calibration method is introduced based on the use of standardized uniformly fluorescing reference layers. Crucial to the approach is that these layers are highly uniform. It is demonstrated to be effective for the correction of non-uniform imaging characteristics across the image (shading correction) as well as for relating fluorescence intensities between images taken with different microscopes or imaging conditions. The approach can be used both in wide field or regular and sectioned (see the section on fluorescence microscopy). In wide field it is shown that in addition the variation of the illumination intensity over the image can be determined on the basis of the uniform bleaching characteristics of the layers. This permits correction for the latter and makes bleach-rate-related imaging in wide field microscopy practical. The significant potential of these layers for calibration in quantitative fluorescence microscopy is illustrated with a series of applications. The approach is also shown to be valuable for general microscope testing and characterization. Specifically in sectioning, specifically confocal, microscopy a set of parameters derived from through-focus datasets of such layers can be used to define a number of properties relevant to sectioned imaging. The main characteristics of a particular imaging situation can then be summarized in a sectioned imaging property chart (SIPchart), which turns out to be a very useful tool for characterizing the properties of particular sectioned imaging systems.