ISO 19056-3:2022

INTERNATIONAL ISO
STANDARD 19056-3
First edition
2022-01
Microscopes — Definition and
measurement of illumination
properties —
Part 3:
Incident light fluorescence microscopy
with incoherent light sources
Microscopes — Définition et mesurage des propriétés d'éclairage —
Partie 3: Microscopie par fluorescence à lumière incidente avec
sources lumineuses incohérentes
Reference number
© ISO 2022
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Published in Switzerland
ii
Contents  Page
Foreword .iv
Introduction .v
1 Scope . 1
2  Normative references . 1
3  Terms and definitions . 1
4  Measurands . 1
4.1 General . 1
4.2 Illumination brightness . 1
4.3 Temporal stability . 2
4.3.1 General . 2
4.3.2 Short-term stability of radiant flux . 2
4.3.3 Long term stability of radiant flux . 2
4.4 Uniformity . 3
5  Measurement procedure . 3
5.1 General . 3
5.2 Microscope settings . 3
5.3 Illumination brightness . 4
5.4 Temporal stability . 4
5.5 Uniformity . 5
5.6 Spectral information . 6
6  Information provided to the user .6
Annex A (informative) Examples . 7
iii
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
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electrotechnical standardization.
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www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 172 Optics  and  photonics,
Subcommittee SC 5, Microscopes and endoscopes.
A list of all parts in the ISO 19056 series can be found on the ISO website.
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iv
Introduction
This document defines important quantities and methods to assess the quality of incident light
fluorescence illumination for microscopes as well as methods to measure them. Details covered will
enable users to compare different microscopes quantitatively with respect to their fluorescence
illumination prior to a purchase decision and chose an appropriate instrument according to their
application. Data obtained by following these methods also enables users to compare existing
instruments with each other or to track performance variations over time, assuring proper performance.
Additionally, what information is to be provided to the user is covered.
v
INTERNATIONAL STANDARD ISO 19056-3:2022(E)
Microscopes — Definition and measurement of
illumination properties —
Part 3:
Incident light fluorescence microscopy with incoherent
light sources
1 Scope
This document specifies procedures for the measurement of illumination brightness, temporal stability
and uniformity for incident light fluorescence microscopy. The measurement for uniformity is defined
in image planes or intermediate image planes only, when these planes are suitable for detection by
electronic imaging devices.
This document defines how illumination brightness, temporal stability and uniformity are measured,
and how this information is provided to the user.
NOTE The scope is intentionally limited to electronic imaging devices and (intermediate) image planes.
The visual observation by means of eyepieces would require a different measurement procedure and hence
result in ambiguities in the description of measurement procedures. Nevertheless, this document will give
useful estimates for the uniformity with visual observation as in this case an eyepiece is used to observe an
intermediate image plane (which is under the scope of this document).
2  Normative references
There are no normative references in this document.
3  Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
4  Measurands
4.1  General
For obtaining the desired image quality, when using a light microscope, the brightness, temporal
stability and uniformity of the image play an important role. This holds true for different applications
and various types of instruments. The measurement of illumination brightness and uniformity is
mandatory. The temporal stability is an optional measurement because it is only suitable for certain
types of light sources (e.g. arc lamps).
4.2  Illumination brightness
The illumination shall be bright enough to allow the detection of the details of the object under
investigation.
As this document is based on measurement procedures, the image brightness shall be expressed in
the corresponding SI unit. The radiometric unit irradiance shall be used because it is well suited for
the essentially monochromatic or narrow-band illumination spectral range used in fluorescence
microscopy.
The value is expressed as given by Formula (1):
P
E= (1)
A
where
E is the irradiance averaged over the area of the diaphragm used in W/mm ;
P is the radiant flux recorded by the power meter in W;
A is the area of diaphragm used in mm .
4.3  Temporal stability
4.3.1  General
The temporal stability of the radiant flux is an important factor when conducting experiments in a time
series. If experimental results are to be derived from variations in the image brightness, any variation
in illumination brightness due to temporal instability shall not contribute to these results.
The temporal stability shall be expressed for at least one of two different time scales.
4.3.2  Short-term stability of radiant flux
The radiant flu
...