ISO/TS 23366:2023
(Main)Nanotechnologies — Performance evaluation requirements for quantifying biomolecules using fluorescent nanoparticles in immunohistochemistry
Nanotechnologies — Performance evaluation requirements for quantifying biomolecules using fluorescent nanoparticles in immunohistochemistry
This document describes minimum requirements for performance evaluation of applying fluorescent nanoparticles in quantitative immuno-histochemistry.
Nanotechnologies — Exigences d'évaluation des performances pour la quantification de biomolécules en immunohistochimie à l'aide de nanoparticules fluorescentes
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TECHNICAL ISO/TS
SPECIFICATION 23366
First edition
2023-05
Nanotechnologies — Performance
evaluation requirements for
quantifying biomolecules using
fluorescent nanoparticles in
immunohistochemistry
Nanotechnologies — Exigences d'évaluation des performances pour
la quantification de biomolécules en immunohistochimie à l'aide de
nanoparticules fluorescentes
Reference number
ISO/TS 23366:2023(E)
© ISO 2023
---------------------- Page: 1 ----------------------
ISO/TS 23366:2023(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
© ISO 2023 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 23366:2023(E)
Contents Page
Foreword .v
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Selection of fluorescent nanoparticles . 3
5.1 General . 3
5.2 Characteristics of nanoparticle . 3
6 Quantification system .4
6.1 Overall design . 4
6.2 Antibody . 5
6.2.1 General . 5
6.2.2 Antibody type . 5
6.2.3 Target antigen . 5
6.2.4 Applicability of antibody to method . 6
6.2.5 Animal species . 6
6.2.6 Immunoglobulin class . 6
6.2.7 Titre . 6
6.2.8 Specificity . 7
6.3 Staining procedure . 7
6.3.1 Staining conditions . . 7
6.3.2 Robustness of staining procedure . 7
6.4 Image processing . 8
6.4.1 Image quality and relevant factors . 8
6.4.2 Selection of image processing software . 8
7 Comparability of results . 9
7.1 Light source adjustment. 9
7.2 Reference material . 9
8 Performance characteristics . 9
8.1 Background . 9
8.1.1 General . 9
8.1.2 Autofluorescence . 10
8.1.3 Nonspecific absorption of nanoparticles . 10
8.1.4 Nonspecific absorption of antibodies . 10
8.1.5 Loss of intensity and interferences . 10
8.2 Reference material dependent indices . 11
8.2.1 General . 11
8.2.2 Limit of detection (LOD) . 11
8.2.3 Limit of quantification (LOQ) . 11
8.2.4 Linearity and dynamic range . 11
8.3 Robustness .12
9 Validation and verification .12
9.1 General .12
9.2 Single lab precision .12
9.3 Reproducibility . 13
10 Reporting .13
Annex A (informative) Example of the reference material .14
iii
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ISO/TS 23366:2023(E)
Annex B (informative) Example of the nanoparticle aggregation/agglomeration evaluation
method .16
Bibliography .18
iv
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ISO/TS 23366:2023(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
v
© ISO 2023 – All rights reserved
---------------------- Page: 5 ----------------------
ISO/TS 23366:2023(E)
Introduction
Fluorescent nanoparticles are expanding their market into bio-technological and research fields as a
labelling material to be used for immunohistochemical staining.
Conventionally, various fluorescent dyes, including FITC (fluorescent isothiocyanate), rhodamine-
isothiocyanate and sulforhodamine 101 acid chloride, have been used for immunohistochemical
staining. They are still powerful tools for identifying localization of target biomolecules, for example,
proteins and sugar chains, mainly for qualitative analyses. They are also applied to quantitative analysis
in combination with various algorithms for calculating signal intensity related to the quantity of the
target biomolecules. The quantification system generally consists of sample preparation, staining,
microscopic observation and photography, and image processing for obtaining quantification results
as shown in Figure 1. For reliable measurement results of quantification, fluorescent dyes that are
brighter and more photostable by exposure to the excitation light are more appropriate.
Large number of fluorescent nanoparticles are available in the market. Generally, they show higher
brightness and are more resistant to photobleaching, compared to the conventional fluorescent dyes.
The characteristics of fluorescent nanoparticles can be an advantage for the quantification of target
biomolecules by immunohistochemical methods also combining with the same algorithm employed for
[1][2][3][4]
the quantification with conventional fluorescent dyes.
In this context, various staining kits with fluorescent nanoparticles and various quantification systems
[5]
have been developed and are available in the market. Thus, the needs to realise the compatibility of
various systems are expanding in the research and industrial fields.
In this document the minimum requirements for performance evaluation of products and application
using fluorescent nanoparticles is addressed. This document provides information ensuring the
comparability of the results of relative quantification by using fluorescent nanoparticles.
This document does not provide industry segment specific performance criteria for the workflow of
measuring biomolecules. When applicable, users can also additionally consult existing industry specific
standards.
Figure 1 — Target quantification process by using of fluorescent nanoparticles
vi
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---------------------- Page: 6 ----------------------
TECHNICAL SPECIFICATION ISO/TS 23366:2023(E)
Nanotechnologies — Performance evaluation
requirements for quantifying biomolecules using
fluorescent nanoparticles in immunohistochemistry
1 Scope
This document describes minimum requirements for performance evaluation of applying fluorescent
nanoparticles in quantitative immuno-histochemistry.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology 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/
3.1
fluorescent nanoparticle
nanoparticle emitting fluorescence excited by light of specific wavelength
3.2
cell block array
paraffin block re-embedded with plural cylinders gouged out from paraffin embedded cell suspension
for histopathology
3.3
cell block array section
thin slice of cell block array obtained by cutting cell block array using microtome, and mounted onto a
glass slide
3.4
agglomerate
collection of weakly or medium strongly bound particles where the resulting external surface area is
similar to the sum of the surface areas of the individual components
Note 1 to entry: The forces holding an agglomerate together are weak forces, for example van der Waals forces or
simple physical entanglement.
Note 2 to entry: Agglomerates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO 26824:2022, 3.1.2]
3.5
agglomeration
process through which agglomerates form
1
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ISO/TS 23366:2023(E)
3.6
aggregate
particle comprising strongly bonded or fused particles where the resulting external surface area is
significantly smaller than the sum of surface areas of the individual components
Note 1 to entry: The forces holding an aggregate together are strong forces, for example, covalent or ionic bonds,
or those resulting from sintering or complex physical entanglement.
Note 2 to entry: Aggregates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO 26824:2022, 3.1.3, modified — Note 1 to entry has been adapted.]
3.7
aggregation
process through which aggregates form
3.8
quantum yield
number of quanta emitted per quantum absorbed
[SOURCE: ISO 22493:2014, 6.6.9]
3.9
molar extinction coefficient
optical density of 1 M fluorescent substance per 1 cm of optical path of absorption cell
3.10
photobleaching
destruction of fluorescing properties of molecules by light, resulting in reduced fluorescence of the
sample
[SOURCE: ISO 10934:2020, 3.2.31]
4 Principle
Immunofluorescence methods are based on staining of thin tissue sections with specific antibodies
recognizing intended molecules, e.g. proteins and sugars. The specific antibodies are prepared by
immunizing animals, e.g. goat, rabbit or mouse, with target molecules. While monoclonal and polyclonal
antibodies are used for immunofluorescence methods, antibodies need to be labelled with fluorescent
substances, e.g. dyes or nanoparticles. For investigating localization and quantity of the target molecules,
tissue sections are stained with fluorescence labelled antibodies. There are two basic methods for
staining with labelled antibodies. One is the direct immunofluorescence method, the other is an indirect
immunofluorescence method. The direct immunofluorescence method is performed by using a single
fluorescence-labelled antibody recognizing the target molecules. The indirect immunofluorescence
method is performed with a non-labelled antibody recognizing the target molecules (first antibody)
and antibody recognizing the first antibody (second antibody). When the first antibody (e.g. IgG) is
produced in mouse, anti-mouse IgG antibody should be selected for the second antibody. In addition,
other molecular systems enhancing fluorescence signal are also available in market. For example, the
avidin-biotin system is well known for this application. In the avidin-biotin system, the second antibody
can be labelled with biotin and reacted with avidin conjugated with fluorescent materials including
nanoparticles, e.g. quantum dots (QD), i.e. semiconductor particles with a few nanometres in size. The
first antibody-second antibody-avidin-QD complex emits a strong fluorescence signal with less photo
bleaching. It is preferred for the quantification of the target molecules by using nanoparticles including
QD.
Fluorescence signal from the stained specimen is measured by fluorescence microscopy, which is
an optical imaging technique that detects simultaneously the emitted fluorescence from the field of
view using a camera. Fluorescence intensity, namely the emitted fluorescence, can be measured as
an intensity value in fluorescence microscopy, which is computed by summing together the intensity
2
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ISO/TS 23366:2023(E)
values from a group of individual pixels in a digital image acquired using a digital camera. In addition
to the measurement of integrated fluorescence intensity, counting bright spots is used to quantify the
target biomolecules.
Quantitative comparison of the intensity data or the numbers of the bright spots requires sound
experimental design and appropriate operation of the whole quantitative photometric system
including a digital camera e.g. a charge coupled device (CCD) or a scientific complementary metal
oxide semiconductor (sCMOS). Issues of the quantitative comparison of intensity data involving the
digital camera 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 described in Reference [15].
For quantitative analysis by immunofluorescence microscopy, fluorescence intensity or the number of
bright spots can be compared to the measured value by enzyme-linked immuno-sorbent assay (ELISA)
or fluorescence activated cell sorter (FACS) and a calibration curve drawn (see A.3). Fluorescence
microscopy measures the fluorescence intensity or the number of bright spots from immunostained
thin slice of tissues and cells, however, it does not measure the biomolecule number per cell. It measures
the fluorescence intensity or the number of bright spots from a slice of a population of the cells although
it is corelated to the number of biomolecules in cells when fluorescence intensity or the number of the
bright spots is measured from a sufficient number of the cells in a field of view. In this sense, microscopic
measurement of fluorescence intensity or the number of bright spots is a relative measurement.
Fluorescence intensity does not in itself have an associated SI unit, because it is a relative measurement.
The number of the bright spots has “unit one” but it is also a relative measurement in principle. A relative
intensity measurement (RIM) is determined as the ratio of one intensity measurement or bright spot
count to another. The fluorescence intensity measurement is an accurate estimate of the ratio of the
irradiance from part or all of a specimen, to the irradiance from part or all of the same or another
specimen. For the counting of bright spots, the results can be interpreted in a similar way with the
integrated fluorescence intensity measurements.
Indices for performance evaluation of quantitative values and application of CBA (cell block array) for
realizing comparability among values that form various quantification systems are described in the
following clauses.
5 Selection of fluorescent nanoparticles
5.1 General
Nanoparticles shall be selected to fit the purpose of the quantification system. Required performance of
nanoparticles varies with respect to quantity of target biomolecules, performance of imaging systems
including sensitivity, available excitation wavelengths, colours for staining including multicolour
staining. For the selection of nanoparticle-labelled antibodies, including commercially available labelled
antibodies, the performance of nanoparticles and the titre of the antibodies should be evaluated (see
5.2).
5.2 Characteristics of nanoparticle
When nanoparticle labelling is performed in the laboratory, the nanoparticle shall be selected based on
the characteristics of nanoparticle. The characteristics of nanoparticles to be evaluated shall include
but not limited to the following.
a) Brightness.
Brightness is important for the quantification of biomolecules by immunohistochemistry. Initial
brightness can be used as a characteristic for the selection of nanoparticles. It is a relative
brightness that is proportional to molar extinction coefficient and quantum yield. For the selection
of nanoparticles, these parameters, i.e. molar extinction coefficient and quantum yield, should be
evaluated.
3
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ISO/TS 23366:2023(E)
Molar extinction coefficient is defined as the optical density of 1 M fluorescent substance per 1 cm
of optical path of absorption cell, measured by absorption photometry. Quantum yield is defined as
the number of quanta emitted per quantum absorbed and can be measured with a fluorospectro-
photometer.
b) Photobleaching time.
When fluorescent dyes or nanoparticles are continuously irradiated by excitation light, their
emission output decreases and is eventually bleached. It can be characterized by the half-time of
bleaching when the number of emitted photons per hour is halved.
c) Particle size and size distribution.
Average particle size should be analysed with coefficient of variation (CV). Dynamic light scattering
(DLS) and scanning electron microscopy (SEM) can be used for this analysis. For SEM analysis
[16]
some standards are helpful.
d) Aggregation and agglomeration.
Agglomeration is the process or degree to form agglomerates that are a collection of weakly or
medium strongly bound particles. In agglomeration, the resulting external surface area is similar
to the sum of the surface areas of the individual particles. Aggregation is the process or degree to
form aggregates that are particles comprising strongly bonded or fused particles. In aggregation,
the resulting external surface area is significantly smaller than the sum of the surface areas of the
individual particles (see Clause 3).
An example of an evaluation method of aggregation is shown in Annex B.
[6]
NOTE That includes QD aggregate and agglomerate in biological media as well as the other types of
[7][8][9][10] [11]
nanoparticles. The excess aggregated QDs can be separated and removed.
e) Non-specific absorption of nanoparticles.
Nonspecific absorption of fluorescent nanoparticles can be a part of the background noise,
diminish the quality of fluoromicroscopic images, and hinder the relative-quantification analysis.
Nonspecific absorption of nanoparticles should be evaluated (see 7.1).
f) Uniformity of nanoparticle(s).
When nanoparticles are used as fluorescent substances, variation in particle size can reduce the
level of correlation between quantity of biomolecules and fluorescence intensity. Particle size shall
be evaluated along with CV and reported.
6 Quantification system
6.1 Overall design
When designing a quantification system for selected fluorescent nanoparticles, the intended use shall
be defined and documented. The quantification system described in this document uses technology
of immunohistochemistry that is an application staining thin sections of tissues with an antibody
specifically recognizing a target biomolecule. The thin section can be prepared from formalin fixed
paraffin embedded and frozen tissues. They are subsequently stained with antibodies linking to
nanoparticles. In some cases, another molecular system including biotin-avidin conjugation can be used
to detect the antibody binding to the target molecules (see 6.2.1). For the quantification, stained slides
are observed with fluorescence microscope and images of tissues are captured with a digital camera,
followed by quantification of the fluorescence signals from the tissue image with the image analysis
software. The design of the quantification system should cover all the steps of the process, from the
prepared tissue sections and the experimentally measured values, to the image processing and final
quantification of the data with the processing software.
4
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ISO/TS 23366:2023(E)
The design description shall contain but not be limited to the following specifications: the fluorescent
nanoparticle to be used and its performance, antibodies including nanoparticle labelled and non-
labelled antibodies when used, staining conditions, microscope system with imaging equipment,
spectral characteristics of the light source used, and image analysis software. The design shall be
reviewed to ensure conformity to the requirements of the system.
For selecting image processing software, requirements of the software shall be described. Minimum
exposure time for imaging is critical for the quality of image processing by the software as well as other
instrument configurations stated in 6.4. Staining procedure, imaging and image processing software
are not independent of each other. When quantification analysis method is newly developed or changed,
the design description shall be reviewed.
6.2 Antibody
6.2.1 General
Various antibodies are used in the immunohistochemistry. For examp
...
TECHNICAL ISO/TS
SPECIFICATION 23366
First edition
Nanotechnologies — Performance
evaluation requirements for
quantifying biomolecules using
fluorescent nanoparticles in
immunohistochemistry
Nanotechnologies — Exigences d'évaluation des performances pour
la quantification de biomolécules en immunohistochimie à l'aide de
nanoparticules fluorescentes
PROOF/ÉPREUVE
Reference number
ISO/TS 23366:2023(E)
© ISO 2023
---------------------- Page: 1 ----------------------
ISO/TS 23366:2023(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
PROOF/ÉPREUVE © ISO 2023 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 23366:2023(E)
Contents Page
Foreword .v
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Selection of fluorescent nanoparticles . 3
5.1 General . 3
5.2 Characteristics of nanoparticle . 3
6 Quantification system .4
6.1 Overall design . 4
6.2 Antibody . 5
6.2.1 General . 5
6.2.2 Antibody type . 5
6.2.3 Target antigen . 5
6.2.4 Applicability of antibody to method . 6
6.2.5 Animal species . 6
6.2.6 Immunoglobulin class . 6
6.2.7 Titre . 6
6.2.8 Specificity . 7
6.3 Staining procedure . 7
6.3.1 Staining Conditions . 7
6.3.2 Robustness of staining procedure . 7
6.4 Image processing . 8
6.4.1 Image quality and relevant factors . 8
6.4.2 Selection of image processing software . 8
7 Comparability of results . 9
7.1 Light source adjustment. 9
7.2 Reference material . 9
8 Performance Characteristics . .9
8.1 Background . 9
8.1.1 General . 9
8.1.2 Autofluorescence . 10
8.1.3 Nonspecific absorption of nanoparticles . 10
8.1.4 Nonspecific absorption of antibodies . 10
8.1.5 Loss of intensity and interferences . 10
8.2 Reference material dependent indices . 11
8.2.1 General . 11
8.2.2 Limit of detection (LOD) . 11
8.2.3 Limit of quantification (LOQ) . 11
8.2.4 Linearity and dynamic range . 11
8.3 Robustness .12
9 Validation and verification .12
9.1 General .12
9.2 Single lab precision .12
9.3 Reproducibility . 13
10 Reporting .13
Annex A (informative) Example of the reference material .14
iii
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ISO/TS 23366:2023(E)
Annex B (informative) Example of the nanoparticle aggregation/agglomeration evaluation
method .16
Bibliography .18
iv
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ISO/TS 23366:2023(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
v
© ISO 2023 – All rights reserved PROOF/ÉPREUVE
---------------------- Page: 5 ----------------------
ISO/TS 23366:2023(E)
Introduction
Fluorescent nanoparticles are expanding their market into bio-technological and research fields as a
labelling material to be used for immunohistochemical staining.
Conventionally, various fluorescent dyes, including FITC (fluorescent isothiocyanate), rhodamine-
isothiocyanate and sulforhodamine 101 acid chloride, have been used for immunohistochemical
staining. They are still powerful tools for identifying localization of target biomolecules, for example,
proteins and sugar chains, mainly for qualitative analyses. They are also applied to quantitative analysis
in combination with various algorithms for calculating signal intensity related to the quantity of the
target biomolecules. The quantification system generally consists of sample preparation, staining,
microscopic observation and photography, and image processing for obtaining quantification results
as shown in Figure 1. For reliable measurement results of quantification, fluorescent dyes that are
brighter and more photostable by exposure to the excitation light are more appropriate.
Large number of fluorescent nanoparticles are available in the market. Generally, they show higher
brightness and are more resistant to photobleaching, compared to the conventional fluorescent dyes.
The characteristics of fluorescent nanoparticles can be an advantage for the quantification of target
biomolecules by immunohistochemical methods also combining with the same algorithm employed for
[1][2][3][4]
the quantification with conventional fluorescent dyes.
In this context, various staining kits with fluorescent nanoparticles and various quantification systems
[5]
have been developed and are available in the market. Thus, the needs to realise the compatibility of
various systems are expanding in the research and industrial fields.
In this document the minimum requirements for performance evaluation of products and application
using fluorescent nanoparticles is addressed. This document provides information ensuring the
comparability of the results of relative quantification by using fluorescent nanoparticles.
This document does not provide industry segment specific performance criteria for the workflow of
measuring biomolecules. When applicable, users can also additionally consult existing industry specific
standards.
Figure 1 — Target quantification process by using of fluorescent nanoparticles
vi
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---------------------- Page: 6 ----------------------
TECHNICAL SPECIFICATION ISO/TS 23366:2023(E)
Nanotechnologies — Performance evaluation
requirements for quantifying biomolecules using
fluorescent nanoparticles in immunohistochemistry
1 Scope
This document describes minimum requirements for performance evaluation of applying fluorescent
nanoparticles in quantitative immuno-histochemistry.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology 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/
3.1
fluorescent nanoparticle
nanoparticle emitting fluorescence excited by light of specific wavelength
3.2
cell block array
paraffin block re-embedded with plural cylinders gouged out from paraffin embedded cell suspension
for histopathology
3.3
cell block array section
thin slice of cell block array obtained by cutting cell block array using microtome, and mounted onto a
glass slide
3.4
agglomerate
collection of weakly or medium strongly bound particles where the resulting external surface area is
similar to the sum of the surface areas of the individual components
Note 1 to entry: The forces holding an agglomerate together are weak forces, for example van der Waals forces or
simple physical entanglement.
Note 2 to entry: Agglomerates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO 26824:2022, 3.1.2]
3.5
agglomeration
process through which agglomerates form
1
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---------------------- Page: 7 ----------------------
ISO/TS 23366:2023(E)
3.6
aggregate
particle comprising strongly bonded or fused particles where the resulting external surface area is
significantly smaller than the sum of surface areas of the individual components
Note 1 to entry: The forces holding an aggregate together are strong forces, for example, covalent or ionic bonds,
or those resulting from sintering or complex physical entanglement.
Note 2 to entry: Aggregates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO 26824:2022, 3.1.3, modified — Note 1 to entry has been adapted.]
3.7
aggregation
process through which aggregates form
3.8
quantum yield
number of quanta emitted per quantum absorbed
[SOURCE: ISO 22493:2014, 6.6.9]
3.9
molar extinction coefficient
optical density of 1 M fluorescent substance per 1 cm of optical path of absorption cell
3.10
photobleaching
destruction of fluorescing properties of molecules by light, resulting in reduced fluorescence of the
sample
[SOURCE: ISO 10934:2020, 3.2.31]
4 Principle
Immunofluorescence methods are based on staining of thin tissue sections with specific antibodies
recognizing intended molecules, e.g. proteins and sugars. The specific antibodies are prepared by
immunizing animals, e.g. goat, rabbit or mouse, with target molecules. While monoclonal and polyclonal
antibodies are used for immunofluorescence methods, antibodies need to be labelled with fluorescent
substances, e.g. dyes or nanoparticles. For investigating localization and quantity of the target molecules,
tissue sections are stained with fluorescence labelled antibodies. There are two basic methods for
staining with labelled antibodies. One is the direct immunofluorescence method, the other is an indirect
immunofluorescence method. The direct immunofluorescence method is performed by using a single
fluorescence-labelled antibody recognizing the target molecules. The indirect immunofluorescence
method is performed with a non-labelled antibody recognizing the target molecules (first antibody)
and antibody recognizing the first antibody (second antibody). When the first antibody (e.g. IgG) is
produced in mouse, anti-mouse IgG antibody should be selected for the second antibody. In addition,
other molecular systems enhancing fluorescence signal are also available in market. For example, the
avidin-biotin system is well known for this application. In the avidin-biotin system, the second antibody
can be labelled with biotin and reacted with avidin conjugated with fluorescent materials including
nanoparticles, e.g. quantum dots (QD), i.e. semiconductor particles with a few nanometres in size. The
first antibody-second antibody-avidin-QD complex emits a strong fluorescence signal with less photo
bleaching. It is preferred for the quantification of the target molecules by using nanoparticles including
QD.
Fluorescence signal from the stained specimen is measured by fluorescence microscopy, which is
an optical imaging technique that detects simultaneously the emitted fluorescence from the field of
view using a camera. Fluorescence intensity, namely the emitted fluorescence, can be measured as
an intensity value in fluorescence microscopy, which is computed by summing together the intensity
2
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values from a group of individual pixels in a digital image acquired using a digital camera. In addition
to the measurement of integrated fluorescence intensity, counting bright spots is used to quantify the
target biomolecules.
Quantitative comparison of the intensity data or the numbers of the bright spots requires sound
experimental design and appropriate operation of the whole quantitative photometric system
including a digital camera e.g. a charge coupled device (CCD) or a scientific complementary metal
oxide semiconductor (sCMOS). Issues of the quantitative comparison of intensity data involving the
digital camera 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 described in Reference [15].
For quantitative analysis by immune-fluorescence microscopy, fluorescence intensity or the number of
bright spots can be compared to the measured value by enzyme-linked immuno-sorbent assay (ELISA)
or fluorescence activated cell sorter (FACS) and a calibration curve drawn (see A.3). Fluorescence
microscopy measures the fluorescence intensity or the number of bright spots from immune-stained
thin slice of tissues and cells, however, it does not measure the biomolecule number per cell. It measures
the fluorescence intensity or the number of bright spots from a slice of a population of the cells although
it is corelated to the number of biomolecules in cells when fluorescence intensity or the number of the
bright spots is measured from a sufficient number of the cells in a field of view. In this sense, microscopic
measurement of fluorescence intensity or the number of bright spots is a relative measurement.
Fluorescence intensity does not in itself have an associated SI unit, because it is a relative measurement.
The number of the bright spots has “unit one” but it is also a relative measurement in principle. A relative
intensity measurement (RIM) is determined as the ratio of one intensity measurement or bright spot
count to another. The fluorescence intensity measurement is an accurate estimate of the ratio of the
irradiance from part or all of a specimen, to the irradiance from part or all of the same or another
specimen. For the counting of bright spots, the results can be interpreted in a similar way with the
integrated fluorescence intensity measurements.
Indices for performance evaluation of quantitative values and application of CBA (cell block array) for
realizing comparability among values that form various quantification systems are described in the
following clauses.
5 Selection of fluorescent nanoparticles
5.1 General
Nanoparticles shall be selected to fit the purpose of the quantification system. Required performance of
nanoparticles varies with respect to quantity of target biomolecules, performance of imaging systems
including sensitivity, available excitation wavelengths, colours for staining including multicolour
staining. For the selection of nanoparticle-labelled antibodies, including commercially available labelled
antibodies, the performance of nanoparticles and the titre of the antibodies should be evaluated (see
5.2).
5.2 Characteristics of nanoparticle
When nanoparticle labelling is performed in the laboratory, the nanoparticle shall be selected based on
the characteristics of nanoparticle. The characteristics of nanoparticles to be evaluated shall include
but not limited to the following.
a) Brightness.
Brightness is important for the quantification of biomolecules by immunohistochemistry. Initial
brightness can be used as a characteristic for the selection of nanoparticles. It is a relative
brightness that is proportional to molar extinction coefficient and quantum yield. For the selection
of nanoparticles, these parameters, i.e. molar extinction coefficient and quantum yield, should be
evaluated.
3
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Molar extinction coefficient is defined as the optical density of 1 M fluorescent substance per 1 cm
of optical path of absorption cell, measured by absorption photometry. Quantum yield is defined as
the number of quanta emitted per quantum absorbed and can be measured with a fluorospectro-
photometer.
b) Photobleaching time.
When fluorescent dyes or nanoparticles are continuously irradiated by excitation light, their
emission output decreases and is eventually bleached. It can be characterized by the half-time of
bleaching when the number of emitted photons per hour is halved.
c) Particle size and size distribution.
Average particle size should be analysed with coefficient of variation (CV). Dynamic light scattering
(DLS) and scanning electron microscopy (SEM) can be used for this analysis. For SEM analysis
[16]
some standards are helpful.
d) Aggregation and agglomeration.
Agglomeration is the process or degree to form agglomerates that are a collection of weakly or
medium strongly bound particles. In agglomeration, the resulting external surface area is similar
to the sum of the surface areas of the individual particles. Aggregation is the process or degree to
form aggregates that are particles comprising strongly bonded or fused particles. In aggregation,
the resulting external surface area is significantly smaller than the sum of the surface areas of the
individual particles (see Clause 3).
An example of an evaluation method of aggregation is shown in Annex B.
[6]
NOTE That includes QD aggregate and agglomerate in biological media as well as the other types of
[7][8][9][10] [11]
nanoparticles. The excess aggregated QDs can be separated and removed.
e) Non-specific absorption of nanoparticles.
Nonspecific absorption of fluorescent nanoparticles can be a part of the background noise,
diminish the quality of fluoromicroscopic images, and hinder the relative-quantification analysis.
Nonspecific absorption of nanoparticles should be evaluated (see 7.1).
f) Uniformity of nanoparticle(s).
When nanoparticles are used as fluorescent substances, variation in particle size can reduce the
level of correlation between quantity of biomolecules and fluorescence intensity. Particle size shall
be evaluated along with CV and reported.
6 Quantification system
6.1 Overall design
When designing a quantification system for selected fluorescent nanoparticles, the intended use shall
be defined and documented. The quantification system described in this document uses technology
of immunohistochemistry that is an application staining thin sections of tissues with an antibody
specifically recognizing a target biomolecule. The thin section can be prepared from formalin fixed
paraffin embedded and frozen tissues. They are subsequently stained with antibodies linking to
nanoparticles. In some cases, another molecular system including biotin-avidin conjugation can be used
to detect the antibody binding to the target molecules (see 6.2.1). For the quantification, stained slides
are observed with fluorescence microscope and images of tissues are captured with a digital camera,
followed by quantification of the fluorescence signals from the tissue image with the image analysis
software. The design of the quantification system should cover all the steps of the process, from the
prepared tissue sections and the experimentally measured values, to the image processing and final
quantification of the data with the processing software.
4
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ISO/TS 23366:2023(E)
The design description shall contain but not be limited to the following specifications: the fluorescent
nanoparticle to be used and its performance, antibodies including nanoparticle labelled and non-
labelled antibodies when used, staining conditions, microscope system with imaging equipment,
spectral characteristics of the light source used, and image analysis software. The design shall be
reviewed to ensure conformity to the requirements of the system.
For selecting image processing software, requirements of the software shall be described. Minimum
exposure time for imaging is critical for the quality of image processing by the software as well as other
instrument configurations stated in 6.4. Staining procedure, imaging and image processing software
are not independent of each other. When quantification analysis method is newly developed or changed,
the design de
...
© ISO 2022 – All rights reserved
ISO/DTS TS 23366:2023(E)
ISO TC 229/WG 5
Date: 2023-02-20
Secretariat: BSI
Nanotechnologies — Performance evaluation requirements for quantifying biomolecules using
fluorescent nanoparticles in immunohistochemistry
Nanotechnologies — Exigences d'évaluation des performances pour la quantification de biomolécules en
immunohistochimie à l'aide de nanoparticules fluorescentes
To help you, this guide on writing standards was produced by the ISO/TMB and is available at
https://www.iso.org/iso/how-to-write-standards.pdf
A model manuscript of a draft International Standard (known as “The Rice Model”) is available at
https://www.iso.org/iso/model_document-rice_model.pdf
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ISO/DTS TS 23366 :2023(E)
© ISO 2023, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or
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ISO/DTS TS 23366 :2023(E)
Contents
Foreword . v
Introduction. vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Selection of fluorescent nanoparticles . 3
5.1 General . 3
5.2 Characteristics of nanoparticle . 3
6 Quantification system . 4
6.1 Overall design . 4
6.2 Antibody . 5
6.2.1 General . 5
6.2.2 Antibody type . 5
6.2.3 Target antigen . 6
6.2.4 Applicability of antibody to method . 6
6.2.5 Animal species . 6
6.2.6 Immunoglobulin class . 6
6.2.7 Titre . 7
6.2.8 Specificity . 7
6.3 Staining procedure . 7
6.3.1 Staining Conditions . 7
6.3.2 Robustness of staining procedure . 8
6.4 Image processing . 8
6.4.1 Image quality and relevant factors . 8
6.4.2 Selection of image processing software . 9
7 Comparability of results . 9
7.1 Light source adjustment . 9
7.2 Reference material . 9
8 Performance Characteristics . 10
8.1 Background . 10
8.1.1 General . 10
8.1.2 Autofluorescence . 10
8.1.3 Nonspecific absorption of nanoparticles . 10
8.1.4 Nonspecific absorption of antibodies . 11
8.1.5 Loss of intensity and interferences . 11
8.2 Reference material dependent indices . 11
8.2.1 General . 11
8.2.2 Limit of detection (LOD) . 11
8.2.3 Limit of quantification (LOQ) . 12
8.2.4 Linearity and dynamic range . 12
8.3 Robustness . 12
9 Validation and verification . 13
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ISO/DTS TS 23366 :2023(E)
9.1 General . 13
9.2 Single lab precision . 13
9.3 Reproducibility . 13
10 Reporting . 14
Annex A (informative) An example of the reference material . 15
Annex B (informative) An example nanoparticle aggregation/agglomeration evaluation
method . 17
Bibliography . 19
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ISO/DTS TS 23366 :2023(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO
collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directiveswww.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any
patent rights identified during the development of the document will be in the Introduction and/or on
the ISO list of patent declarations received (see www.iso.org/patentswww.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation onof the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the World
Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT)), see the following URL:
www.iso.org/iso/foreword.htmlwww.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
© ISO 2022 – All rights reserved 5
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ISO/DTS TS 23366 :2023(E)
Introduction
Fluorescent nanoparticles are expanding their market into bio-technological and research fields as a
labelling material to be used for immunohistochemical staining.
Conventionally, various fluorescent dyes, including FITC (fluorescent isothiocyanate), rhodamine-
isothiocyanate and sulforhodamine 101 acid chloride, have been used for immunohistochemical staining.
They are still powerful tools for identifying localization of target biomolecules, for example, proteins and
sugar chains, mainly for qualitative analyses. They are also applied to quantitative analysis in
combination with various algorithms for calculating signal intensity related to the quantity of the target
biomolecules. The quantification system generally consists of sample preparation, staining, microscopic
observation and photography, and image processing for obtaining quantification results as shown in
Figure 1. For reliable measurement results of quantification, fluorescent dyes that are brighter and more
photostable by exposure to the excitation light are more appropriate.
Large number of fluorescent nanoparticles are available in the market. Generally, they show higher
brightness and are more resistant to photobleaching, compared to the conventional fluorescent dyes. The
characteristics of fluorescent nanoparticles can be an advantage for the quantification of target
biomolecules by immunohistochemical methods also combining with the same algorithm employed for
[1 ][2 ][3 ][4 ]
the quantification with conventional fluorescent dyes [. ] [ ] [ ] [ ].
In this context, various staining kits with fluorescent nanoparticles and various quantification systems
[5 ]
have been developed and are available in the market [. ]. Thus, the needs to realise the compatibility of
various systems are expanding in the research and industrial fields.
In this document the minimum requirements for performance evaluation of products and application
using fluorescent nanoparticles is addressed. This document provides information ensuring the
comparability of the results of relative quantification by using fluorescent nanoparticles.
This document does not provide industry segment specific performance criteria for the workflow of
measuring biomolecules. When applicable, users can also additionally consult existing industry specific
standards.
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ISO/DTS TS 23366 :2023(E)
Figure 1 — Target quantification process by using of fluorescent nanoparticles
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TECHNICAL SPECIFICATION ISO/TS 23366:2023(E)
Nanotechnologies — Performance evaluation requirements for
quantifying biomolecules using fluorescent nanoparticles in
immunohistochemistry
1 Scope
This document describes minimum requirements for performance evaluation of applying fluorescent
nanoparticles in quantitative immuno-histochemistry.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology 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/
3.1
fluorescent nanoparticle
nanoparticle emitting fluorescence excited by light of specific wavelength
3.2
cell block array
paraffin block re-embedded with plural cylinders gouged out from paraffin embedded cell suspension for
histopathology
3.3
cell block array section
thin slice of cell block array obtained by cutting cell block array using microtome, and mounted onto a
glass slide
3.4
agglomerate
collection of weakly or medium strongly bound particles where the resulting external surface area is
similar to the sum of the surface areas of the individual components
Note 1 to entry: The forces holding an agglomerate together are weak forces, for example van der Waals forces or
simple physical entanglement.
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ISO/TS 23366:2023(E)
Note 2 to entry: Agglomerates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO 26824:2013, 2022, 3.1.2]
3.5
agglomeration
process through which agglomerates form
3.6
aggregate
particle comprising strongly bonded or fused particles where the resulting external surface area is
significantly smaller than the sum of surface areas of the individual components
Note 1 to entry: The forces holding an aggregate together are strong forces, for example, covalent or ionic bonds, or
those resulting from sintering or complex physical entanglement.
Note 2 to entry: Aggregates are also termed secondary particles and the original source particles are termed primary
particles.
[SOURCE: ISO 26824:2013, 2022, 3.1.3, modified — Note 1 to entry has been adapted.]
3.7
aggregation
process through which aggregates form
3.8
quantum yield
number of quanta emitted per quantum absorbed
[SOURCE: ISO 22493:2014, 6.6.9]
3.9
molar extinction coefficient
molar absorption coefficient
optical density of 1M1 M fluorescent substance per 1 cm of optical path of absorption cell
3.10
photobleaching
destruction of fluorescing properties of molecules by light, resulting in reduced fluorescence of the
sample
[SOURCE: ISO 10934:2020, 3.2.31]
4 Principle
Immunofluorescence methods are based on staining of thin tissue sections with specific antibodies
recognizing intended molecules, e.g.,. proteins and sugars. The specific antibodies are prepared by
immunizing animals, e.g.,. goat, rabbit, or mouse etc.,, with target molecules. While monoclonal and
polyclonal antibodies are used for immunofluorescence methods, antibodies need to be labelled with
fluorescent substances, e.g.,. dyes or nanoparticles. For investigating localization and quantity of the
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ISO/TS 23366:2023(E)
target molecules, tissue sections are stained with fluorescence labelled antibodies. There are two basic
methods for staining with labelled antibodies. One is the direct immunofluorescence method, the other
is an indirect immunofluorescence method. The direct immunofluorescence method is performed by
using a single fluorescence-labelled antibody recognizing the target molecules. The indirect
immunofluorescence method is performed with a non-labelled antibody recognizing the target molecules
(first antibody) and antibody recognizing the first antibody (second antibody). When the first antibody
(e.g. IgG) is produced in mouse, anti-mouse IgG antibody should be selected for the second antibody. In
addition, other molecular systems enhancing fluorescence signal are also available in market. For
example, the avidin-biotin system is well known for this application. In the avidin-biotin system, the
second antibody can be labelled with biotin and reacted with avidin conjugated with fluorescent
materials including nanoparticles, e.g.,. quantum dots (QD), i.e.,. semiconductor particles with a few
nanometres in size. The first antibody-second antibody-avidin-QD complex emits a strong fluorescence
signal with less photo bleaching. It is preferred for the quantification of the target molecules by using
nanoparticles including QD.
Fluorescence signal from the stained specimen is measured by fluorescence microscopy, which is an
optical imaging technique that detects simultaneously the emitted fluorescence from the field of view
using a camera. Fluorescence intensity, namely the emitted fluorescence, can be measured as an intensity
value in fluorescence microscopy, which is computed by summing together the intensity values from a
group of individual pixels in a digital image acquired using a digital camera. In addition to the
measurement of integrated fluorescence intensity, counting bright spots is used to quantify the target
biomolecules.
Quantitative comparison of the intensity data or the numbers of the bright spots requires sound
experimental design and appropriate operation of the whole quantitative photometric system including
a digital camera e.g.,. a charge coupled device (CCD) or a scientific complementary metal oxide
semiconductor (sCMOS). Issues of the quantitative comparison of intensity data involving the digital
camera 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 described in the Reference [15].
For quantitative analysis by immune-fluorescence microscopy, fluorescence intensity or the number of
bright spots can be compared to the measured value by enzyme-linked immuno-sorbent assay (ELISA)
or fluorescence activated cell sorter (FACS) and a calibration curve drawn (See Annexsee A.3)).
Fluorescence microscopy measures the fluorescence intensity or the number of bright spots from
immune-stained thin slice of tissues and cells, however, it does not measure the biomolecule number per
cell. It measures the fluorescence intensity or the number of bright spots from a slice of a population of
the cells although it is corelated to the number of biomolecules in cells when fluorescence intensity or the
number of the bright spots is measured from a sufficient number of the cells in a field of view. In this
sense, microscopic measurement of fluorescence intensity or the number of bright spots is a relative
measurement.
Fluorescence intensity does not in itself have an associated SI unit, because it is a relative measurement.
The number of the bright spots has “unit one” but it is also a relative measurement in principle. A relative
intensity measurement (RIM) is determined as the ratio of one intensity measurement or bright spot
count to another. The fluorescence intensity measurement is an accurate estimate of the ratio of the
irradiance from part or all of a specimen, to the irradiance from part or all of the same or another
specimen. For the counting of bright spots, the results can be interpreted in a similar way with the
integrated fluorescence intensity measurements.
Indices for performance evaluation of quantitative values and application of CBA (cell block array) for
realizing comparability among values that form various quantification systems are described in the
following clauses.
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ISO/TS 23366:2023(E)
5 Selection of fluorescent nanoparticles
5.1 General
Nanoparticles shall be selected to fit the purpose of the quantification system. Required performance of
nanoparticles varies with respect to quantity of target biomolecules, performance of imaging systems
including sensitivity, available excitation wavelengths, colours for staining including multicolour
staining. For the selection of nanoparticle-labelled antibodies, including commercially available labelled
antibodies, the performance of nanoparticles and the titre of the antibodies should be evaluated. (See
(see 5.2)).
5.2 Characteristics of nanoparticle
When nanoparticle labelling is performed in the laboratory, the nanoparticle shall be selected based on
the characteristics of nanoparticle. The characteristics of nanoparticles to be evaluated shall include but
not limited to the following;.
1) brightness
a) Brightness.
Brightness is important for the quantification of biomolecules by immunohistochemistry. Initial
brightness can be used as a characteristic for the selection of nanoparticles. It is a relative brightness
that is proportional to molar extinction coefficient and quantum yield. For the selection of
nanoparticles, these parameters, i.e.,. molar extinction coefficient and quantum yield, should be
evaluated.
Molar extinction coefficient is defined as the optical density of 1M1 M fluorescent substance per 1 cm
of optical path of absorption cell, measured by absorption photometry. Quantum yield is defined as
the number of quanta emitted per quantum absorbed and can be measured with a fluorospectro-
photometer.
photobleachingb) Photobleaching time.
When fluorescent dyes or nanoparticles are continuously irradiated by excitation light, their
emission output decreases and is eventually bleached. It can be characterized by the half-time of
bleaching when the number of emitted photons per hour is halved.
particlec) Particle size and size distribution.
Average particle size should be analysed with coefficient of variation (CV). Dynamic light scattering
(DLS) and scanning electron microscopy (SEM) can be used for this analysis. For SEM analysis some
[16]
standards are helpful.
aggregationd) Aggregation and agglomeration.
Agglomeration is the process or degree to form agglomerates that are a collection of weakly or
medium strongly bound particles. In agglomeration, the resulting external surface area is similar to
the sum of the surface areas of the individual particles. Aggregation is the process or degree to form
aggregates that are particles comprising strongly bonded or fused particles. In aggregation, the
resulting external surface area is significantly smaller than the sum of the surface areas of the
individual particles (see clause Clause 3).
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ISO/TS 23366:2023(E)
An example of an evaluation method of aggregation is shown in Annex B.
[6]
NOTE That includes QD aggregate and agglomerate in biological media as well as the other types of
[7][8][9][10 ] [11 ]
nanoparticles [. ]. The excess aggregated QDs can be separated and removed [. ].
none) Non-specific absorption of nanoparticles.
Nonspecific absorption of fluorescent nanoparticles can be a part of the background noise, diminish
the quality of fluoromicroscopic images, and hinder the relative-quantification analysis. Nonspecific
absorption of nanoparticles should be evaluated (Seesee 7.1).
uniformityf) Uniformity of nanoparticle (s)).
When nanoparticles are used as fluorescent substances, variation in particle size can reduce the level
of correlation between quantity of biomolecules and fluorescence intensity. Particle size shall be
evaluated along with CV and reported.
6 Quantification system
6.1 Overall design
When designing a quantification system for selected fluorescent nanoparticles, the intended use shall be
defined and documented. The quantification system described in this document uses technology of
immunohistochemistry that is an application staining thin sections of tissues with an antibody
specifically recognizing a target biomolecule. The thin section can be prepared from formalin fixed
paraffin embedded and frozen tissues. They are subsequently stained with antibodies linking to
nanoparticles. In some cases, another molecular system including biotin-avidin conjugation can be used
to detect the antibody binding to the target molecules (see 6.2.1). For the quantification, stained slides
are observed with fluorescence microscope and images of tissues are captured with a digital camera,
followed by quantification of the fluorescence signals from the tissue image with the image analysis
software. The design of the quantification system should cover all the steps of the process, from the
prepared tissue sections and the experimentally measured values, to the image processing and final
quantification of the data with the processing software.
The design description shall contain but not be limited to the following specifications: the fluorescent
nanoparticle to be used and its performance, antibodies including nanoparticle labelled and non-labelled
antibodies when used, staining conditions, microscope system with imaging equipment, spectral
characteristics of the light source used, and image analysis software. The design shall be reviewed to
ensure conformity to the requirements of the system.
For selecting image processing software, requirements of the software shall be described. Minimum
exposure time for imaging is critical for the quality of image processing by the software as well as other
instrument configurations stated in the clause 6.4. Staining procedure, imaging, and image processing
software are not independent of each other. When quantification analysis method is newly developed or
changed, the design description shall be reviewed.
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ISO/TS 23366:2023(E)
6.2 Antibody
6.2.1 General
Various antibodies are used in the immunohistochemistry. For example, a single labelled antibody for
direct immunofluorescence methods and a set of two antibodies comprising primary antibody
recognizing a target biomolecule and labelled second antibo
...
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