1.1 This guidance document has been developed to facilitate the collection of microscopy images with epifluorescence microscopy that allow quantitative fluorescence measurements to be extracted from the images. The document is tailored to cell biologists that often use fluorescent staining techniques to visualize components of a cell-based experimental system. Quantitative comparison of the intensity data available in these images is only possible if the images are made quantitative with experimental design and proper CCD camera operation. Issues involving CCD camera and controller software settings, collection of dark count images, flat-field correction, benchmarking of the excitation lamp and the fluorescent collection optics are considered. 1.2 This document is developed around epifluorescence microscopy, but it is likely that many of the issues discussed here are applicable to quantitative imaging in other fluorescence microscopy systems such as fluorescence confocal microscopy. 1.3 Fluorescence intensity is a relative measurement and does not in itself have an associated SI unit. This document does discuss metrology issues related to relative measurements and experimental designs that maybe required to ensure quantitative fluorescence measurements are comparable after changing microscope configurations. This document does describe the measurements of radiant flux which has the SI unit of watts. 1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
To perform quantitative biology with data collected from epifluorescence microscopes, it is important that experimental design of the image collection strategy be considered.
The title and scope are in draft form and are under development within this ASTM Committee.
Citing ASTM Standards
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