F04.46 - Cell Signaling

SIGNIFICANCE AND USE
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:  
5.2.1 Characterization of cell cycle distribution by quantifying the abundance of DNA in individual cells (1).7  
5.2.2 Measuring the area of positively stained mineralized deposits in cell cultures (ASTM F2997).  
5.2.3 Quantifying the spread area of fixed cells (ASTM F2998).  
5.2.4 Determining DNA damage in eukaryotic cells using the comet assay (ASTM 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 (ASTM F2944).
SCOPE
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 ...

SIGNIFICANCE AND USE
5.1 Under well-controlled conditions, the quantitative evaluation of morphological features of a cell population can be used to identify changes in cellular behavior or state. Cell morphology changes may be expected when, for example, there is a response to changes in cellular cytoskeleton organization (1), a response of cells to toxic compounds, changes in differentiation state, and changes in adhesion properties of cells to a substrate by either chemical or mechanical-induced extracellular matrix-based (ECM-based) signaling pathways (2, 3). Typically, populations of cells exhibit a range of morphologies even when the cells are genetically identical and are in a homogeneous environment (4). This biological variation in cell response is due to both cell-cycle variations and stochasticity in the cellular reactions that control adhesion and spreading in cells. By using cell-by-cell, microscopy-based measurements and appropriate statistical sampling procedures, the distribution of cell morphologies such as cell spreading area per cell can be measured. This distribution is highly characteristic of the culture and conditions being examined.  
5.2 It is important to note that the use of this technique for cells on or in a 3-D scaffold materials can complicate the interpretation of the data. The topographic transforms of the cells on a 3-D material may require full volumetric imaging and not just wide-field fluorescence imaging as described here.  
5.3 the following are several examples of how this measurement can be used in a laboratory:—  
5.3.1 Quantify Cellular Response to a Biomaterial—The measurement of cell spread area can be used to characterize the response of cells to biomaterials. For example, spreading of most cell types is extremely sensitive to the stiffness of the culture substrate (5), (6). It is important to note that cell response to an ECM may be dependent on the preparation of the matrix. For example, the same ECM proteins prepared in a fibrillar or...
SCOPE
1.1 This guide describes several measurement and technical issues involved in quantifying the spread area of fixed cells. Cell spreading and the distribution of cell spread areas of a population of cells are the result of a biological response that is dependent on intracellular signaling mechanisms and the characteristics of cell adhesion to a surface. Cell spread area is a morphological feature that can be responsive to alteration in the metabolic state or the state of stress of the cells. Changes in cell spread area can also indicate an alteration in the adhesion substrate that may be due to differences in manufacturing of the substrate material or be in response to extracellular matrix secretions. High quality measurement of cell spread area can serve as a useful metric for benchmarking and detecting changes cell behavior under experimental conditions.  
1.2 The measurement described in this document is based on the use of fluorescence microscopy imaging of fixed cells and the use of image analysis algorithms to extract relevant data from the images. To produce robust cell spread area measurements, technical details involved in sample preparation, cell staining, microscopy imaging, image analysis and statistical analysis should be considered. Several of these issues are discussed within this document.  
1.3 This standard is meant to serve as a guide for developing methods to reliably measure the area to which cells spread at a surface. This surface can be conventional tissue culture polystyrene or sophisticated engineered biomaterial surfaces. An example of a detailed procedure to measure the spreading area of cells on a tissue culture polystyrene surface is provided in the appendix section.  
1.4 Cell morphology features such as cell spreading area and perimeter are generally reported in units of length. For example, spreading area per cell (that is, cell spread area) is likely reported in units of µm2. A spatial ...