An ISO about Optical data of fluorescence confocal microscopes for biological imaging has been released in December 2019. 15 pages dedicated to image performance in confocal laser scanning microscopy.
The ISO 21073:2019 has recently been published and consists in standards on performance of microscopes for biological imaging. These standards are intended to provide comparable specifications of confocal microscopes by microscope manufacturers and to allow users to compare and monitor the imaging performance of their confocal microscopes.
1. What’s the scope of these standards?
The ISO 21073:2019 aims at specifying the metrics commonly used to assess image performance in confocal laser scanning microscopy, for the field of fluorescent biological specimens imaging. This document applies to confocal single point scanners using single photon excitation.
It has been thought and written by a committee of 12 participating members of several countries, specialized in Optics and photonics.
More specifically, it clarifies the topics of:
- resolution and strength of optical sectioning,
- uniformity of field and centering accuracy,
- co-registration accuracy,
- stability of illumination power,
- field number of the confocal scan optic,
- scanning frequency,
- theoretical resolution.
The ISO document can be purchased (CHF 88) and is protected by copyright, it cannot be copy/pasted.
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2. Are there other existing standards?
For more than a decade, the microscopy community has been discussing the need of shared references for fluorescence measurements, supported by the National Metrology Institutes1.
“In the 20 years or more since introduction of laser scanning confocal microscopes, it has not been feasible to assess how these systems compare.”
To quote A. Dixon and al, in their article titled “Need for Standardization of Fluorescence Measurements from the Instrument Manufacturer’s View”2: “In the 20 years or more since introduction of laser scanning confocal microscopes, it has not been feasible to assess how these systems compare. No one can assert with confident that instrument A in use in 1990 has better or worse sensitivity than instrument B operating in 2006. There is, therefore, a paramount need for standardized test samples and procedures for their use.”
As a first step towards standardization, the standard vocabulary had already been defined in ISO 10934-1:2002 (Vocabulary for microscopy — Part 1: Light microscopy) and ISO 10934-2:2007 (Vocabulary for microscopy — Part 2: Advanced techniques in light microscopy).
Both specify terms and definitions to be used in the field of light microscopy.
“Just think about the number of parameters or procedures involved in determining imaging resolution. If researchers use different characterization approaches, or even different imaging targets, there can be confusion.” 3
Encouraged by global trends to harmonize measurements, traceability, and accreditation, the ASTM (American Society for Testing and Materials, an international standards organization) has released in 2014 a guide covering the major topics and concerns involved in fluorescence detection and calibration of fluorescence instruments, under standard ASTM E2719-09(2014).
This new ISO 21073:2019 standard is another step towards internationally standardized results, that can be shared and compared between users, as well as used as a common ground with equipment manufacturers. On the way to tackle the reproducibility crisis! (Read article on reproducibility crisis)
To go further, you can browse at the following publications:
- Keeping up standards. Nature Photon 12, 117 (2018). doi.org/10.1038/s41566-018-0131-6 https://www.nature.com/articles/s41566-018-0131-6.
- Waters JC. Accuracy and precision in quantitative fluorescence microscopy. J Cell Biol. 2009;185(7): 1135–1148. doi:10.1083/jcb.20090309 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2712964/.
- Resch-Genger U., et al., How to Improve Quality Assurance in Fluorometry: Fluorescence-Inherent Sources of Error and Suited Fluorescence Standards. J Fluoresc 15, 337–362 (2005). doi.org/10.1007/s10895-005-2630-3 https://link.springer.com/article/10.1007/s10895-005-2630-3.
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(1) P. C. De Rose, L. Wang, A. K. Gaigalas, G. W. Kramer, U. Resch-Genger, U. Panne, Resch-Genger U. (eds) Standardization and Quality Assurance in Fluorescence Measurements I. Springer Series on Fluorescence, vol 5. Springer, DOI: 10.1007/4243_2008_049 (April 2008) https://link.springer.com/chapter/10.1007/4243_2008_049
(2) A. Dixon, et al., Resch-Genger U. (eds) Standardization and Quality Assurance in Fluorescence Measurements II. Springer Series on Fluorescence, vol 6. Springer, doi: 10.1007/4243_2008_026 (March 2008) https://link.springer.com/chapter/10.1007%2F4243_2008_026
(3) Keeping up standards. Nature Photon 12, 117 (2018). doi.org/10.1038/s41566-018-0131-6 https://www.nature.com/articles/s41566-018-0131-6
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