1、Designation: F2998 14Guide forUsing Fluorescence Microscopy to Quantify the SpreadArea of Fixed Cells1This standard is issued under the fixed designation F2998; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revis
2、ion. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This guide describes several measurement and technicalissues involved in quantifying the spread area of fixed cells.Cell spreadi
3、ng and the distribution of cell spread areas of apopulation of cells are the result of a biological response thatis dependent on intracellular signaling mechanisms and thecharacteristics of cell adhesion to a surface. Cell spread area isa morphological feature that can be responsive to alteration in
4、the metabolic state or the state of stress of the cells. Changes incell spread area can also indicate an alteration in the adhesionsubstrate that may be due to differences in manufacturing of thesubstrate material or be in response to extracellular matrixsecretions. High quality measurement of cell
5、spread area canserve as a useful metric for benchmarking and detectingchanges cell behavior under experimental conditions.1.2 The measurement described in this document is basedon the use of fluorescence microscopy imaging of fixed cellsand the use of image analysis algorithms to extract relevantdat
6、a from the images. To produce robust cell spread areameasurements, technical details involved in samplepreparation, cell staining, microscopy imaging, image analysisand statistical analysis should be considered. Several of theseissues are discussed within this document.1.3 This standard is meant to
7、serve as a guide for developingmethods to reliably measure the area to which cells spread at asurface. This surface can be conventional tissue culture poly-styrene or sophisticated engineered biomaterial surfaces. Anexample of a detailed procedure to measure the spreading areaof cells on a tissue cu
8、lture polystyrene surface is provided inthe appendix section.1.4 Cell morphology features such as cell spreading areaand perimeter are generally reported in units of length. Forexample, spreading area per cell (that is, cell spread area) islikely reported in units of m2.Aspatial calibration standard
9、 isrequired to convert between numbers of pixels in a CCDcamera image to m2as an SI unit.1.5 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practice
10、s and determine the applica-bility of regulatory limitations prior to use.1.5.1 Sodium azide is used as a anti-bacterial reagent in theslide mounting media. This preserves the integrity of themounting media. The toxicity of this reagent (for example,MSDS) should be considered before use of this reag
11、ent in largescale slide mounting procedures.2. Referenced Documents2.1 ASTM Standards:E1488 Guide for Statistical Procedures to Use in Developingand Applying Test MethodsF2150 Guide for Characterization and Testing of Biomate-rial Scaffolds Used in Tissue-Engineered Medical Prod-ucts3. Terminology3.
12、1 Definition of Terms:3.1.1 cell morphologythe physical shape properties of acell such as cell volume, cell spread area, and cell perimeter;and the non-unit measures of roundness and circularity.3.1.2 cell spread areathe area that encompasses a2-dimensional (2-D) projection of a 3-dimensional (3-D)a
13、dhered cell.3.1.3 cell objecta single cell or two or more adhered cellsadjacent to each other such that they cannot unambiguously besegmented from one another by cell edge detection techniques.3.2 Definitions of Terms Specific to This Standard:3.2.1 segmentationthe act of classifying pixels in anima
14、ge as cell or non-cell areas and the grouping of adjacent cellpixels into a cell object.4. Summary of Practice4.1 The measurement of a cell morphology feature such ascell spread area in a population of cells in culture can be aquantitative characteristic of cell population behavior and cellpopulatio
15、n state. This document provides guidance on measur-ing the 2-D morphological property (that is, cell spread area)for a population of cells in culture on a material.1This test method is under the jurisdiction ofASTM Committee F04 on Medicaland Surgical Materials and Devices and is the direct responsi
16、bility of SubcommitteeF04.46 on Cell Signaling.Current edition approved Jan. 1, 2014. Published May 2014. DOI: 10.1520/F2998-14.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States14.2 This measurement is typically achieved by seeding apop
17、ulation of cells at low density on a test surface andchemically stabilizing the cellular structure by fixing the cellsat a particular point in time. The cells are then treated with twostains: one that discriminates the cell from non-cellbackground, and another that associates with the nucleus of the
18、cell. The dual-stained cells are then imaged with a fluorescencemicroscope using a low magnification and a high numericalaperture (NA) objective (for example, 10 magnification andNA0.25) that maximizes the number of cells in a singleimage. The magnification and NA should provide sufficientresolution
19、 to enable accurate counting of the numbers of pixelsassociated with a cell, and allow large numbers of cells to beimaged, thus providing robust population statistics. Images ofboth the nuclei and corresponding cellular areas are collected.These images are then processed and analyzed by imageanalysi
20、s software to achieve both segmentation of the cellobjects from the non-cell background to allow quantification ofcell object spread areas, and segmentation of nuclei fordetermination of number of nuclei in each cell object. Thesetwo metrics allow a value for spread area per cell to begenerated.4.3
21、The spread area per cell measurement can be generatedfor each cell or cluster of cells in an image. These data can beparameterized as average spread area per cell or as a probabil-ity distribution. Each of these reported values are characteris-tics of the cell population adhered to a defined substra
22、te.Statistical methods applicable to average values or distributioncomparisons can be used to identify statistically significantchanges.4.4 It is important to note that the cell spread area measure-ment described here can be influenced by a large number ofexperimental factors that can influence cell
23、 state and theadhesion substrate. These include variations in reagents usedduring culturing of cells (such as serum, substrate vendor,media) and differences in how the cells are handled duringmaintenance and storage. Significant robustness testing andsensitivity analysis of these factors on the cell
24、 spread areameasurement will be required to develop this measurement intoa test method for benchmarking a particular cell culturesystem.5. Significance and Use5.1 Under well-controlled conditions, the quantitativeevaluation of morphological features of a cell population canbe used to identify change
25、s in cellular behavior or state. Cellmorphology changes may be expected when, for example,there is a response to changes in cellular cytoskeleton organi-zation (1), a response of cells to toxic compounds, changes indifferentiation state, and changes in adhesion properties of cellsto a substrate by e
26、ither chemical or mechanical-inducedextracellular matrix-based (ECM-based) signaling pathways(2, 3). Typically, populations of cells exhibit a range ofmorphologies even when the cells are genetically identical andare in a homogeneous environment (4). This biological varia-tion in cell response is du
27、e to both cell-cycle variations andstochasticity in the cellular reactions that control adhesion andspreading in cells. By using cell-by-cell, microscopy-basedmeasurements and appropriate statistical sampling procedures,the distribution of cell morphologies such as cell spreadingarea per cell can be
28、 measured. This distribution is highlycharacteristic of the culture and conditions being examined.5.2 It is important to note that the use of this technique forcells on or in a 3-D scaffold materials can complicate theinterpretation of the data. The topographic transforms of thecells on a 3-D materi
29、al may require full volumetric imagingand not just wide-field fluorescence imaging as described here.5.3 the following are several examples of how this measure-ment can be used in a laboratory:5.3.1 Quantify Cellular Response to a BiomaterialThemeasurement of cell spread area can be used to characte
30、rize theresponse of cells to biomaterials. For example, spreading ofmost cell types is extremely sensitive to the stiffness of theculture substrate (5), (6). It is important to note that cellresponse to an ECM may be dependent on the preparation ofthe matrix. For example, the same ECM proteins prepa
31、red in afibrillar or non-fibrillar surface coating can result in differentmorphology response5.3.2 Quality Control Metric for General Cell CultureConditionsCell spread area may be a useful metric formonitoring a change in cell culture conditions (that is, due to aserum component, pH, passage number,
32、 confluence, etc.). Cellmorphology is often altered when cells are stressed andproceeding through cell-death related processes (that is, apo-toposis).5.3.3 Quality Control Metric for Biomaterial FabricationCell spread area measurements may be useful for generatingspecifications for a biomaterial. Th
33、ese specifications maystipulate how a particular cell line responds to a fabricatedbiomaterial.5.3.4 Quality Control Metric for Cell Line Integrity andMorphology BenchmarkingThe morphology characteristic ofa cell line grown under specified conditions should ideally bethe same over time and in differ
34、ent laboratories. Thus, cellspread area measurements may be useful for validating that nosignificant changes in the cell cultures have occurred. Thismeasurement provides a benchmark that is useful for estab-lishing the current state of the cell culture and a metric that canbe charted to increased co
35、nfidence for within and betweenlaboratory comparisons of cellular measurements (7).6. Test Considerations6.1 Fluorophore Reagents:6.1.1 For high quality cell morphology measurements, it isimportant to collect images of both the cell spreading area andimages of the cell nuclei in corresponding frames
36、. This allowsthe calculation of the normalized metric, average spread areaper cell. An excellent method for collecting these images iswith two-color fluorescence microscopy imaging. Fluorescentstains are used to highlight specific features of the cell. The useof high quality cell stains will optimiz
37、e the contrast differencesbetween the background and the cell features being probed.6.1.2 It is critical that the cell feature stains exhibit highsignal-to-background ratios. For the case of cell edge detectionit is important to consider that the measurements are extractedfrom cell images with image
38、 analysis procedures. Reliable cellF2998 142edge detection algorithms function best with cells that exhibithigh-contrast edge staining around cell features (8). Severalcell stains (see Table 1) can be used to provide contrast at thecell and nuclear edge. It is important to note that empiricalevaluat
39、ion of samples treated with several stains may berequired to evaluate the best staining strategy that optimizescellular and nuclear edge detection (9). This is most importantfor cell edge stains because nuclear stains such as 4,6-diamidino-2-phenylindole (DAPI) and Hoechst 33342 typi-cally perform v
40、ery well.6.1.3 It is important to consider the spectral properties of thestains that are used to identify the edge features and the nuclearfeatures of a cell. To retain independence between the cellobject area measurement and the nuclei count measurement, itcan be useful to minimize bleed-through fl
41、uorescence (oftentermed crosstalk) between the nuclear stain and the cell objectstain. Bleed-through is minimized by choosing fluorophoreprobes that have significantly different excitation and emissionproperties. For example, if a nucleus was stained with Hoechst33342 (exciting wavelength = 350 nm,
42、emission wavelength =461 nm) and the cell edge was stained with a Texas Red basedfluorophore (exciting wavelength = 590 nm, emission wave-length = 620 nm), it is unlikely that the emission from theHoechst 33342 fluorophore would interfere with the respectiveTexas Red fluorophore-based images. The ap
43、propriate controlfor bleed-through is to evaluate a sample stained with only onefluorophore in each filter channel at the appropriate integrationtime, binning, and gain settings. Morphology measurementsfrom cell edge and nuclear staining are often not highlysensitive to fluorescence bleed-through bu
44、t the issue should beconsidered when identifying a pair of staining reagents.6.1.4 In general, the staining reagents should not directlyinteract with the material on which the cells are adhered. If useof the staining reagent with a test material results in ahigh-level fluorescent background, it is p
45、ossible that contrast atthe cell edge will not be sufficient for edge detection withimage analysis techniques. Control experiments that evaluatethe level of fluorescent background on materials after stainingcan aid in selecting the most suitable staining reagents for thesemeasurements.6.1.5 It is de
46、sirable that the fluorophore labels are notsensitive to cell fixing and handling conditions, and that thestain does not exit the cell after extended periods of time. Thiscriteria significantly improves the robustness of the morphol-ogy measurement as cell can be prepared and stained at onepoint in t
47、ime and analyzed in another point in time.6.1.6 It is desirable that the fluorophore labels are relativelyphotostable so that a sample can be reimaged with little changein the microscope settings. This property improves the reliabil-ity of the image analysis procedures that are used to extractdata f
48、rom the images.6.2 Cell Sample and Substrate PreparationTest cells aretypically removed from a maintenance flask by trypsinizationor other de-adhesion procedures and then seeded on the testsurface. The cells are allowed to adhere to the test surface fora controlled number of hours before the cells a
49、re fixed, stained,and imaged.6.2.1 Care must be taken to ensure that a suspension ofsingle cells (and not clumps of cells) is achieved duringde-adhesion/harvesting to increase the probability that cellsseeded on the test substrate at low density will be isolated fromother cells. This condition will provide the most accurate singlecell morphology measurements. If large numbers of cellclusters are observed on the substrate, the ability to measurethe distribution of cell spread areas may be compromised, butit should still be possible to measure the average spread areaper cell
