Standard Guide for Performing Quantitative Fluorescence Intensity Measurements in Cell-based Assays with Widefield Epifluorescence Microscopy

Importancia y uso:

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 Practice 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 ensure 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:

5.2.1 Characterization of cell cycle distribution by quantifying the abundance of DNA in individual cells (1).8

5.2.2 Measuring the area of positively stained mineralized deposits in cell cultures (Practice F2997).

5.2.3 Quantifying the spread area of fixed cells (Guide F2998).

5.2.4 Determining DNA damage in eukaryotic cells using the comet assay (Guide E2186).

5.2.5 The quantitation of a secondary fluorescent marker that provides information related to the genotype, phenotype, biological activity, or biochemical features of a colony or cell (Practice F2944).

Subcomité:

F04.46

Referida por:

F3510-21, F3088-22, F3504-21, F2997-21, F2944-20

Volúmen:

13.02

Número ICS:

07.100.01 (Microbiology in general)

Palabras clave:

camera offset; CCD bias; cell measurement; dark count correction; fluorescence intensity; fluorescence microscopy; image normalization; instrument benchmarking; instrument qualification; linear dynamic range; nonuniform field; quantitative microscopy;