ISO 21073:2019

Overview

ISO 21073:2019 - Microscopes - Confocal microscopes - Optical data of fluorescence confocal microscopes for biological imaging - defines standardized optical performance quantities for fluorescence confocal laser scanning microscopy (CLSM). The standard applies specifically to single-point confocal scanners using single-photon excitation and provides measurement and declaration rules so manufacturers and users can compare imaging performance reliably for biological specimens.

Key topics and technical requirements

• Resolution (lateral and axial): Defined as the full width at half maximum (FWHM) of the confocal point spread function (cPSF) measured at the centre of the field. Resolution measurements must use unprocessed data and a Gaussian fit.
• Strength of optical sectioning: Measured as FWHM of the response when scanning a thin fluorescent layer or reflective planar object through focus - a key metric for optical sectioning capability.
• Measurement conditions and test objects:
  • Use point-like fluorescent microspheres smaller than half the expected resolution.
  • Thin fluorescent layers for axial tests must be significantly thinner than the expected sectioning strength.
  • Detection pinhole sizing specified in Airy units (AU); 1 AU is generally recommended.
• Sampling and field requirements:
  • Pixel size should be at least ten times smaller than the theoretical resolution.
  • Scanned field should be at least ten times larger than the theoretical resolution.
• Data integrity and signal path:
  • Recordings must be linear (no detector/excitation saturation).
  • Include full cPSF range (max and min) to avoid clipped FWHM estimates.
• Additional performance quantities covered in the standard (as listed in the document):
  • Field uniformity and centring accuracy
  • Co-registration accuracy (multi-channel alignment)
  • Stability of illumination power
  • Field number of the confocal scan optic
  • Scanning frequency
• Declaration requirements: Measurements must be reported with test conditions (excitation/detection wavelengths, objective designation, pinhole size and reference wavelength, polarization, etc.).

Applications and who uses it

• Enables manufacturers to publish comparable optical data for confocal microscopes.
• Helps core facilities, imaging scientists, and life-science researchers to:
  • Select instruments that meet required resolution and optical sectioning for fluorescence biological imaging.
  • Monitor instrument performance and perform acceptance testing.
  • Ensure reproducible quantitative imaging and multi-channel co-registration.
• Useful for procurement, method validation, and quality assurance in research labs, microscopy cores, and imaging-enabled diagnostics.

Related standards

• ISO 10934-1 and ISO 10934-2 (vocabulary and advanced microscopy terms) are normative references for terminology used in ISO 21073:2019.

Keywords: ISO 21073:2019, confocal microscope, confocal laser scanning microscopy, fluorescence imaging, resolution, FWHM, cPSF, Airy unit, optical sectioning, co-registration, imaging performance.