ASTM F 3294 : 2018 : R2024
Current
The latest, up-to-date edition.
Standard Guide for Performing Quantitative Fluorescence Intensity Measurements in Cell-based Assays with Widefield Epifluorescence Microscopy
Hardcopy , PDF
English
01-10-2024
Committee |
F 04
|
DocumentType |
Guide
|
Pages |
13
|
PublisherName |
American Society for Testing and Materials
|
Status |
Current
|
Supersedes |
1.1This guidance document has been developed to facilitate the collection of microscopy images with an epifluorescence microscope that allows 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.
1.2This 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.
1.3Fluorescence 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.
1.4This 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.
1.5This 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.
ASTM F 3088 : 2022 | Standard Practice for Use of a Centrifugation Method to Quantify/Study Cell-Material Adhesive Interactions |
ASTM F 2997 : 2021 | Standard Practice for Quantification of Calcium Deposits in Osteogenic Culture of Progenitor Cells Using Fluorescent Image Analysis |
ASTM F 3510 : 2021 | Standard Guide for Characterizing Fiber-Based Constructs for Tissue-Engineered Medical Products |
ASTM F 3504 : 2021 | Standard Practice for Quantifying Cell Proliferation in 3D Scaffolds by a Nondestructive Method |
ASTM F 2944 : 2020 | Standard Practice for Automated Colony Forming Unit (CFU) Assays—Image Acquisition and Analysis Method for Enumerating and Characterizing Cells and Colonies in Culture |
Access your standards online with a subscription
Features
-
Simple online access to standards, technical information and regulations.
-
Critical updates of standards and customisable alerts and notifications.
-
Multi-user online standards collection: secure, flexible and cost effective.