ASTM F3294-18
(Guide)Standard Guide for Performing Quantitative Fluorescence Intensity Measurements in Cell-based Assays with Widefield Epifluorescence Microscopy
Standard Guide for Performing Quantitative Fluorescence Intensity Measurements in Cell-based Assays with Widefield Epifluorescence Microscopy
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 ...
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Standards Content (Sample)
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.
Designation:F3294 −18
Standard Guide for
Performing Quantitative Fluorescence Intensity
Measurements in Cell-based Assays with Widefield
1
Epifluorescence Microscopy
This standard is issued under the fixed designation F3294; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 1.5 This international standard was developed in accor-
dance with internationally recognized principles on standard-
1.1 Thisguidancedocumenthasbeendevelopedtofacilitate
ization established in the Decision on Principles for the
the collection of microscopy images with an epifluorescence
Development of International Standards, Guides and Recom-
microscope that allow quantitative fluorescence measurements
mendations issued by the World Trade Organization Technical
to be extracted from the images. The document is tailored to
Barriers to Trade (TBT) Committee.
cell biologists that often use fluorescent staining techniques to
visualize components of a cell-based experimental system.
2. Referenced Documents
Quantitativecomparisonoftheintensitydataavailableinthese
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2.1 ASTM Standards:
images is only possible if the images are quantitative based on
sound experimental design and appropriate operation of the E131Terminology Relating to Molecular Spectroscopy
E284Terminology of Appearance
digital array detector, such as a charge coupled device (CCD)
E2186Guide for Determining DNA Single-Strand Damage
or a scientific complementary metal oxide semiconductor
in Eukaryotic Cells Using the Comet Assay
(sCMOS)orsimilarcamera.Issuesinvolvingthearraydetector
E2642Terminology for Scientific Charge-Coupled Device
and controller software settings including collection of dark
(CCD) Detectors
count images to estimate the offset, flat-field correction,
E2719Guide for Fluorescence—Instrument Calibration and
background correction, benchmarking of the excitation lamp
Qualification
and the fluorescent collection optics are considered.
E2825Guide for Forensic Digital Image Processing
1.2 This document is developed around epifluorescence
F2944Test Method for Automated Colony Forming Unit
microscopy, but it is likely that many of the issues discussed
(CFU) Assays—Image Acquisition and Analysis Method
here are applicable to quantitative imaging in other fluores-
forEnumeratingandCharacterizingCellsandColoniesin
cence microscopy systems such as fluorescence confocal
Culture
microscopy. This guide is developed around single-color fluo-
F2997Practice for Quantification of Calcium Deposits in
rescencemicroscopyimagingormulti-colorimagingwherethe
Osteogenic Culture of Progenitor Cells Using Fluorescent
measured fluorescence is spectrally well separated.
Image Analysis
1.3 Fluorescence intensity is a relative measurement and
F2998Guide for Using Fluorescence Microscopy to Quan-
does not in itself have an associated SI unit. This document
tify the Spread Area of Fixed Cells
doesdiscussmetrologyissuesrelatedtorelativemeasurements
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2.2 ISO Standards:
and experimental designs that may be required to ensure
ISO 13653Measurement of relative irradiance in the image
quantitative fluorescence measurements are comparable after
field
changing microscope, sample, and lamp configurations.
ISO/IEC 10918-1:1994Digital compression and coding of
1.4 This standard does not purport to address all of the
continuous-tonestillimages:Requirementsandguidelines
safety concerns, if any, associated with its use. It is the
ISO/TR12033:2009Guidancefortheselectionofdocument
responsibility of the user of this standard to establish appro-
image compression methods
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
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This guide is under the jurisdiction ofASTM Committee F04 on Medical and contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Surgical Materials and Devices and is the direct responsibility of Subcommittee Standards volume information, refer to the standard’s Document Summary page on
F04.46 on Cell Signaling. the ASTM website.
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Current edition approved Oct. 1, 2018. Published October 2018. DOI: 10.1520/ Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
F3294-18. 4th Floor, New York, NY 10036, http://www.ansi.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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