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
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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 flux shall be recorded for a time interval of 5 min. During this time interval the detector of
the power meter is illuminated continuously and a power reading is recorded every second. The short-
term stability of radiant flux, S , is expressed as a percentage by Formula (2):
short
PP−
 
maxmin
S =×100 1− (2)
short  
PP+
 
maxmin
where
S is the short term stability in percent;
short
P is the maximum radiant flux recorded during the time interval of 5 min;
max
P is the minimum radiant flux recorded during the time interval of 5 min.
min
Care should be taken that the light source is permanently on during the complete time interval of 5 min,
because some light sources are only switched on during actual image acquisition.
4.3.3  Long term stability of radiant flux
The radiant flux shall be recorded for a time interval of 120 min. During this time interval the detector
of the power meter is illuminated every 30 s for one second. During the one second illumination of the
detector a power reading is recorded. The long term stability of radiant flux, S , is expressed as a
long
percentage by the Formula (3):
PP−
 
maxmin
S =×100 1− (3)
long  
PP+
 maxmin 
where
S is the long term stability in percent;
long
P is the maximum radiant flux recorded during the time interval of 120 min;
max
P is the minimum radiant flux recorded during the time interval of 120 min.
min
4.4  Uniformity
The microscope’s optical system shall achieve a certain degree of image uniformity in order to allow
the detection of the details of the object under investigation. A severe drop of the image brightness
towards the edge of the image field can result in brightness values that are not sufficient in the above
mentioned condition.
Furthermore, spatial variations of the image brightness over the image field may not always be
distinguished from spatial variations of the properties of the object under investigation.
The uniformity of the brightness, U, in the image field is expressed as a percentage by Formula (4):
B
IF,min
U=×100 (4)
B
IF,max
Where
U is the uniformity of brightness in the image field, in percent;
B is the minimum brightness in image field;
IF,min
B , is the maximum brightness in image field.
IF max
NOTE Depending on the size of the image field, the objective magnification and the size of the sensor pixels,
it might be necessary to apply a suitable averaging method in the computation of the uniformity.
5  Measurement procedure
5.
...