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1.1 This guidance document has been developed to facilitate the collection of microscopy images with an epifluorescence microscope 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 quantitative based on sound experimental design and appropriate operation of the digital array detector, such as a charge coupled device (CCD) or a scientific complementary metal oxide semiconductor (sCMOS) or similar camera. Issues involving the array detector and controller software settings including collection of dark count images to estimate the offset, flat-field correction, background 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. This guide is developed around single-color fluorescence microscopy imaging or multi-color imaging where the measured fluorescence is spectrally well separated.

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 may be required to ensure quantitative fluorescence measurements are comparable after changing microscope, sample, and lamp configurations.

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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.

1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

5.1 Overview of Measurement System—Relative intensity measurements made by widefield epifluorescence microscopy are used as part of cell-based assays to quantify attributes such as the abundance of probe molecules (see ASTM F2997), fluorescently labeled antibodies, or fluorescence protein reporter molecules. The general procedure for quantifying relative intensities is to acquire digital images, then to perform image analysis to segment objects and compute intensities. The raw digital images acquired by epifluorescence microscopy are not typically amenable to relative intensity quantification because of the factors listed in 4.2. This guide offers a checklist of potential sources of bias that are often present in fluorescent microscopy images and suggests approaches for storing and normalizing raw image data to assure that computations are unbiased.

5.2 Areas of Application—Widefield fluorescence microscopy is frequently used to measure the location and abundance of fluorescent probe molecules within or between cells. In instances where RIM comparisons are made between a region of interest (ROI) and another ROI, accurate normalization procedures are essential to the measurement process to minimize biased results. Example use cases where this guidance document may be applicable include: