SIST ISO 13322-1:2015
Particle size analysis - Image analysis methods - Part 1: Static image analysis methods
Particle size analysis - Image analysis methods - Part 1: Static image analysis methods
ISO 13322-1:2014 is applicable to the analysis of images for the purpose of determining particle size distributions where the velocity of the particles against the axis of the optical system of the imaging device is zero. The particles are appropriately dispersed and fixed in the object plane of the instrument. The field of view may sample the object plane dynamically either by moving the sample support or the camera provided this can be accomplished without any motion effects on the image. Captured images can be analysed subsequently.
ISO 13322-1:2014 concentrates upon the analysis of digital images created from either light or electron detection systems. It considers only image evaluation methods using complete pixel counts.
Analyse granulométrique - Méthodes par analyse d'images - Partie 1: Méthodes par analyse d'images statiques
Granulometrijska analiza - Metode analize slike - 1. del: Statične metode analize slike
Ta del standarda ISO 13322 velja za analize slik za določanje razporeditve velikosti delcev, kadar je hitrost delcev glede na os optičnega sistema naprave za slikanje enaka nič. Delci so ustrezno razpršeni in fiksni v predmetni ravnini instrumenta. Vidno polje lahko vzorči predmetno ravnino dinamično s premikanjem podpore vzorca ali predložene kamere. To je mogoče doseči brez kakršnih koli učinkov gibanja na sliko. Posnete slike je mogoče analizirati pozneje.
Ta del standarda ISO 13322 se osredotoča na analizo digitalnih slik, ustvarjenih s sistemi za zaznavanje svetlobe ali elektronov. Ne zajema metode ustvarjanja slike, čeprav je nastavitev zaznavanja, ki je izbrana skupaj s kalibracijo, pomembna za natančnost določanja velikosti delcev. Ta del standarda ISO 13322 zajema samo metode ocenjevanja slik z uporabo popolnega štetja pikslov. Vrsta porazdelitve (glede na število ali prostornino) skupaj s širino porazdelitve velikosti delcev ima bistven vpliv na število delcev za merjenje, da se zagotovi natančnost v okviru določenih mejah zaupanja. Primer je prikazan v prilogi A. Avtomatizacija analize je mogoča, da se izmeri zadostno število delcev za zahtevano stopnjo natančnosti.
Ta del standarda ISO 13322 ne obravnava priprave vzorca. Kljub temu so podvzorčenje, razpršitev in predstavitev delcev za merjenje ključni deli operativne verige dejanj, ki so potrebna za zagotovitev pravilnosti in natančnosti končnih rezultatov.
General Information
Relations
Overview
ISO 13322-1:2014 - Particle size analysis - Image analysis methods - Part 1: Static image analysis methods defines requirements and guidance for using digital images to determine particle size distributions when particles are stationary (zero velocity relative to the optical axis). The standard covers digital images from light or electron detection systems and image evaluation methods based on complete pixel counts. It focuses on image acquisition factors that affect sizing accuracy, image analysis workflows, calibration, traceability and uncertainty estimation.
Key topics and requirements
The standard addresses practical and technical elements essential to robust particle size analysis by image:
- Scope and definitions: terms such as area equivalent diameter, Feret diameters, measurement frame and segmentation methods.
- Preparation for image capture: procedures to ensure particles are appropriately dispersed and fixed in the object plane; guidance on sampling the measurement field without motion effects.
- Sample presentation considerations: comments on sample splitting and the influence of touching particles, particle distribution and the number of particles required to meet precision targets.
- Quality of captured images: criteria such as contrast, pixels per particle, and imaging conditions that affect segmentation and measurement accuracy.
- Image analysis and segmentation: recommendations for edge detection, thresholding and complete-pixel-count evaluation methods for deriving particle metrics.
- Counting procedures: rules for handling particles cut by frame edges, touching particles and measurement conventions.
- Calibration and traceability: requirements for calibration verification, use of reference scales and estimation of systematic uncertainties.
- Accuracy and qualification: guidance on qualification tests, use of reference materials and estimating measurement uncertainty.
- Reporting: essential content for the test report and annexes (e.g., sample size estimation, common segmentation methods, process flow chart).
Applications and users
ISO 13322-1:2014 is applicable wherever static image-based particle sizing is used to produce particle size distributions. Typical applications include:
- Quality control and R&D for powders, suspensions and particulate materials
- Materials science and microscopy labs using light or electron imaging
- Instrument manufacturers, service labs and metrology teams establishing traceable measurements
Primary users:
- Analytical scientists and laboratory technicians performing image-based particle size analysis
- QA/QC managers specifying test methods and acceptance criteria
- Instrument vendors developing or verifying imaging-based particle sizing systems
Related standards
- ISO 13322-2 (Dynamic image analysis methods)
- ISO 9276-1, ISO 9276-2 (representation and calculations for particle size results)
- ISO 14488, ISO 14887 (sampling and sample preparation guidance)
ISO 13322-1:2014 is essential reading for anyone implementing or validating static image analysis methods for particle size distribution with an emphasis on calibration, traceability and reproducible measurement practice.
Standards Content (Sample)
SLOVENSKI STANDARD
01-februar-2015
1DGRPHãþD
SIST ISO 13322-1:2006
*UDQXORPHWULMVNDDQDOL]D0HWRGHDQDOL]HVOLNHGHO 6WDWLþQHPHWRGHDQDOL]H
VOLNH
Particle size analysis - Image analysis methods - Part 1: Static image analysis methods
Analyse granulométrique - Méthodes par analyse d'images - Partie 1: Méthodes par
analyse d'images statiques
Ta slovenski standard je istoveten z: ISO 13322-1:2014
ICS:
19.120 Analiza velikosti delcev. Particle size analysis. Sieving
Sejanje
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
INTERNATIONAL ISO
STANDARD 13322-1
Second edition
2014-05-15
Particle size analysis — Image analysis
methods —
Part 1:
Static image analysis methods
Analyse granulométrique — Méthodes par analyse d’images —
Partie 1: Méthodes par analyse d’images statiques
Reference number
©
ISO 2014
© ISO 2014
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
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Published in Switzerland
ii © ISO 2014 – All rights reserved
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions and list of symbols . 1
3.1 Terms and definitions . 1
3.2 Symbols . 4
4 Preparation for image capture . 5
4.1 Introduction . 5
4.2 Procedures . 5
5 Sample preparation demands for method description . 6
5.1 Sample splitting and reduction . 6
5.2 Touching particles . 6
5.3 Particle distribution. 6
5.4 Number of particles to be counted . 6
5.5 Particle suspending fluid . 7
6 Quality of captured images . 7
6.1 General . 7
6.2 Pixels per particle . 7
7 Image analysis . 8
7.1 General . 8
7.2 Size classes and magnification . 8
8 Counting procedure . 9
8.1 General . 9
8.2 Particle image edges . 9
8.3 Particles cut by the edge of the measurement frame.10
8.4 Touching particles .11
8.5 Measurements .12
9 Calculation of the particle size results .12
10 Calibration and traceability .12
10.1 General .12
10.2 Recommendations and requirements .13
11 Accuracy .14
11.1 General .14
11.2 Reference materials .14
11.3 Instrument preparation .14
11.4 Qualification test .15
11.5 Qualification acceptance .15
12 Test report .15
Annex A (informative) Estimation of the number of particles to be counted for a given accuracy .17
Annex B (informative) Common segmentation methods for particle edge detection.22
Annex C (informative) Flow chart showing a typical image analysis method.23
Bibliography .24
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 on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical Barriers
to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 24, Particle characterization including sieving,
Subcommittee SC 4, Particle characterization.
This second edition cancels and replaces the first edition (ISO 13322-1:2004), which has been technically
revised.
ISO 13322 consists of the following parts, under the general title Particle size analysis — Image analysis
methods:
— Part 1: Static image analysis methods
— Part 2: Dynamic image analysis methods
iv © ISO 2014 – All rights reserved
Introduction
The purpose of this part of ISO 13322 is to give guidance when using images for particle size analysis.
Image analysis is a technique that has gained popularity in different applications. The aim of this part
of ISO 13322 is to give a standardized description of the technique used and its validation. This part of
ISO 13322 does not describe specific instruments and is restricted to those parts of the acquisition of
images that are relevant to the accuracy of the particle size analysis.
This part of ISO 13322 includes methods of calibration verification and recommends using a certified
standard as a reference scale. However it is sensible to make some measurements on particles under
study, or other reference objects, of known size so that the likely systematic uncertainties introduced by
the equipment can be assessed.
Errors introduced at all stages of the analysis from sub-division of the sample to generation of the final
result add to the total uncertainty of measurements and it is important to obtain estimates for the
uncertainty arising from each stage.
Essential operations are identified to ensure that measurements made conform to this part of ISO 13322
and are traceable.
INTERNATIONAL STANDARD ISO 13322-1:2014(E)
Particle size analysis — Image analysis methods —
Part 1:
Static image analysis methods
1 Scope
This part of ISO 13322 is applicable to the analysis of images for the purpose of determining particle
size distributions where the velocity of the particles against the axis of the optical system of the imaging
device is zero. The particles are appropriately dispersed and fixed in the object plane of the instrument.
The field of view may sample the object plane dynamically either by moving the sample support or the
camera provided this can be accomplished without any motion effects on the image. Captured images
can be analysed subsequently.
This part of ISO 13322 concentrates upon the analysis of digital images created from either light or
electron detection systems. It does not address the method of creating the image although the detection
settings chosen together with its calibration are important to particle sizing accuracy. This part of
ISO 13322 considers only image evaluation methods using complete pixel counts.
Both the type of distribution, (by number or by volume) together with the width of the particle size
distribution has a very material influence upon the number of particles to be measured to secure the
desired accuracy within the specified confidence limits. An example is shown in Annex A.
Automation of the analysis is possible in order to measure sufficient particle numbers for a required
degree of precision.
This part of ISO 13322 does not address the sample preparation. However, the sub sampling, dispersion
and presentation of particles to be measured are a vital part of the operational chain of actions necessary
to ensure accuracy and precision of any final result.
NOTE Further details about sampling and sample preparation can be found in ISO 14887 and ISO 14488.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 9276-1, Representation of results of particle size analysis — Part 1: Graphical representation
ISO 9276-2, Representation of results of particle size analysis — Part 2: The calculations of average particle
sizes/diameters and moments from particle size distributions
ISO 14488, Particulate materials — Sampling and sample splitting for the determination of particulate
properties
3 Terms and definitions and list of symbols
3.1 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1.1
area equivalent diameter
diameter of a circle having the same area as the projected image of the particle
Note 1 to entry: It is also known as the Heywood diameter or as the equivalent circular diameter.
3.1.2
binary image
digitized image consisting of an array of pixels, each of which has a value of 0 or 1, whose values are
normally represented by dark and bright regions on the display screen or by the use of two distinct
colours
3.1.3
contrast (of an image)
difference between the intensity of the particle image with respect to the
background near to the particle
3.1.4
edge detection
methods used to detect transition between objects and background
Note 1 to entry: See segmentation method (3.1.13).
3.1.5
Feret diameter
distance between two parallel tangents on opposite sides of the image of a particle
3.1.6
field of view
field which is viewed by the viewing device
Note 1 to entry: The full image frame of a digital imaging device corresponds to its field of view.
SEE: Figure 1.
3.1.7
grey image
image in which multiple grey level values are permitted for each pixel
3.1.8
image analysis
processing and data reduction operation which yields a numerical or logical result from an image
3.1.9
measurement field
field which is composed by the set of all measurement frames
SEE: Figure 1.
3.1.10
measurement frame
selected area from the field of view in which particles are sized and counted for image analysis
SEE: Figure 1.
3.1.11
pixel
picture element
individual sample in a digital image that has been formed by uniform sampling in both the horizontal
and vertical directions
2 © ISO 2014 – All rights reserved
3.1.12
raster pattern
scanning order of measurement frames in the total measurement field
SEE: Figure 1.
3.1.13
segmentation method
strategy employed to separate the objects of interest from their surroundings
Note 1 to entry: Method of dividing the particle image from the background.
Note 2 to entry: See edge detection (3.1.4).
3.1.14
threshold
grey level value which is set to discriminate objects of interest from background
Key
1 measurement frame
2 field of view
3 raster pattern of measurement frames
4 measurement field
X enlarged view of a field of view
Figure 1 — Relationship between the terms “field of view”, “measurement frame”, “raster
pattern” and “measurement field”
3.2 Symbols
A projected area of particle i
i
α horizontal calibration factor
α vertical calibration factor
d minimum feature length
d diameter of a circle
c
N number of particles to be measured
n measured number of pixels within a circle
c
n numbers of particles in size interval Δx
j j
P probability that particle i exists in the measuring frame (also called Miles-Lantuéjoul factor)
i
φ shape descriptor
i
σ standard deviation
V volume of particle i
i
x area equivalent diameter of particle i
A,i
x horizontal Feret diameter of object
F1
x vertical Feret diameter of object
F2
x dimension of particle i
i
x longest dimension of particle i, also called maximum Feret diameter
Fmax,i
x shortest dimension of particle i, also called minimum Feret diameter
Fmin,i
x horizontal dimension of object
x horizontal dimension of object in SI unit
1,m
x horizontal dimension of object in pixel
1,p
x vertical dimension of object
x vertical dimension of object in SI unit
2,m
x vertical dimension of object in pixel
2,p
x particle size corresponding to 10 % of the cumulative undersize distribution by volume
10,3
x particle size corresponding to 90 % of the cumulative undersize distribution by volume
90,3
Z horizontal side length of the rectangular measurement frame
Z vertical side length of the rectangular measurement frame
4 © ISO 2014 – All rights reserved
4 Preparation for image capture
4.1 Introduction
A pre-requisite for accurate particle size measurement using this method requires a full understanding
of the settings and calibration applied within the image capture device as well as a consideration of the
purpose for conducting the measurement.
The final settings and calibration of the image capture device need to be established via an iterative
approach. The size range of the particles within an unknown test sample has an influence upon the
settings required within the image capturing device. These remain unknown until the first image has
been taken, the result observed and the necessary adjustments to the image capture device to achieve
the desired accuracy of particle size measurement required. A fully trained operator shall conduct the
assessment.
The imaging instrument should be set up and operated in accordance with the manufacturer’s
recommendations considering the conditions prevailing.
In order to achieve accurate particle size measurements it is preferred that the illumination be uniform
over the total field of view and of a type designed to create images of high contrast. The magnification
should be such as to provide a minimum number of pixels for the smallest particle consistent with the
accuracy demanded and set to achieve a sharp focus. The number of pixels for the smallest dimension of
a particle is relevant for cases where linear dimensions or combinations thereof are measured.
Distortion in the image might arise from a number of causes, but its presence and effect on the image
may be determined by selecting known sized particles or other reference objects of similar optical
properties at a number of points and orientations in the field of view. It is important to note that the
measurements made provide only two-dimensional, X and Y, information.
4.2 Procedures
The operator should decide why the result of the image analysis is required. Is a size distribution
by the number of particles in each size class required or is the volume of particles in each size class
the requirement? What accuracy and precision is required for the final result? These decisions will
have a significant influence upon the choice of settings and the method employed in conducting the
measurement.
For each material to be analysed and for each instrument employed the person conducting the analysis
shall ensure that the following procedures are followed.
a) Ensure an adequate calibration for both the X and Y-axis of the measurement frame exists for the
imaging instrument being employed, preferably by using a certified graticule or equivalent reference
of equal standing.
b) Ensure that the optical magnification employed is suitable and such as the image of the smallest
particle to be measured covers a sufficient number of pixels to support the required accuracy of
measurement.
c) Ensure that the illumination method and setting of any focus is correctly established to give a good
contrast and uniformity of illumination of any image gathered.
d) Ensure that the number of particles within the measurement frame is such as to minimize the
number of touching particles.
e) Ensure that a sufficient number of images of separate aliquot samples are gathered to provide a
suitable total number of particles with respect to the type of distribution, number or volume based,
and the width of the particle size distribution (see ISO 14488) and that they contain an adequate
statistical number of the largest particle of the target material (see Annex A).
f) Some implementations of the image analysis technique employ a large area X, Y servo or manually
controlled sample slide assembly. Such large slides enable many measurement frames of the particles
deposited to be examined. Should the method of fully separate measurement frames be employed
then any frame overlap shall be avoided. If the method of overlapping measurement frames, or
other methods of analysing particles that interact with the measurement frame edge is used, then
procedures shall be employed to ensure that each particle is only included into the total count for
the appropriate size class, once. For more information, see 8.3.
g) If appropriate, ensure that the image quality consisting of illumination, focus and magnification has
not changed at the end of the measurement. The requirement for this step depends on the variability
of the instrument employed.
h) For the case when image analysis is to be used for certified reference material measurement at the
end of the image gathering procedure, the calibration outlined in a) should be repeated and any
measured deviation recorded.
i) All the conditions, set up or established, for the target material shall be fully documented.
5 Sample preparation demands for method description
5.1 Sample splitting and reduction
As only a small amount of material is needed to prepare a test sample, this should be sub-divided from
the whole sample in a manner that ensures that the test sample is representative of the whole as specified
in ISO 14488.
5.2 Touching particles
In order to assess the degree of touching particles, a suitable optical resolution setting of the imaging
system should be chosen. The optical resolution should also meet the criteria set out in 4.2 b).
The number of particles touching each other should be minimized. It is a prime requirement of the
method that measurements shall be made on isolated particles. Touching particles measured as one
particle without a proper separation will introduce error.
It is often not possible to reliably detect touching particles by image analysis alone, but the influence
of touching particles on the result can be investigated experimentally by increasing or decreasing the
number of particles per image. If the number of particles cannot be changed, the influence on the results
can be investigated using a reference material with similar size and shape.
5.3 Particle distribution
There should be an adequate distribution of particles in the field of view. It may be necessary to examine
several fields of view if a large total particle count is required. The whole area of the measurement
field should be examined to ascertain whether there is noticeable segregation of particles (by size). The
requirements set out in 4.2 f) should be followed.
5.4 Number of particles to be counted
The number of particles to be analysed depends upon whether the final result is a particle size
distribution by the number or by the volume of particles. Considerable care has to be exercised in order
to ensure that the analysis is representative of the bulk sample as described in ISO 14488. This can be
demonstrated by splitting the bulk sample into at least three test samples. Each test sample should
contain sufficient particle numbers for a full measurement. Statistical analysis of the data will reveal the
repeatability of the method including sampling and dispersion.
NOTE See Annex A for more information.
6 © ISO 2014 – All rights reserved
5.5 Particle suspending fluid
It is likely that a large number of particle measurements will be of particles presented in a gas where an
adequate image contrast should be ensured. Should particle presentation require a liquid suspension
then it is preferred that such liquids be clean, particle-free, transparent and have a refractive index
as different as possible from the refractive index of particles to enhance the image contrast. Particles
presented in a mixed optical background such as in biological specimens, may require dynamic particle
by particle threshold selection.
WARNING — Automated particle by particle parameter selection applied for segmentation as
envisaged when mixed optical backgrounds are required cannot be validated for true particle
size and may result in particle size bias and reduced accuracy.
CAUTION — Particle systems of mixed optical properties may have inaccurate particle sizes
attributed to them due to possible threshold setting errors introducing particle size bias.
6 Quality of captured images
6.1 General
It is important for the analysis that the particles in the captured images are well dispersed. The number
of overlapping or touching particles reduces with the concentration of the particles in the frame (see
5.2). This is in conflict with the requirements of having a large particle number to obtain a high degree
of precision. A compromise should be established.
The contrast achieved should be consistent with the level of accuracy required. The difference between
the brightness of the particle and its background shall be a few times of the resolution of the grey level
image from zero to maximum brightness signal.
The accuracy of the results is strongly affected by the number of pixels for each particle image. When only
the projected area of the particles is measured as an average value, a few pixels may provide acceptable
results for the smallest particles. Higher pixel numbers are required for accurate information about
each individual particle.
6.2 Pixels per particle
Both the number of pixels forming the image of the particle and the relative position of the centring of
the image with respect to the fixed pixel pattern can have a material influence upon the final particle
size assessed from each particle image.
Image analysis can be a method of choice for the certification of reference materials. It also can be the
method of choice for general measurements. The conditions required to achieve a defined accuracy and
precision may be quite different for these two cases and warrant separate approaches.
6.2.1 Characterization of reference materials
Materials selected for reference materials are often spherical and having limited or mono dispersed
size distributions. In order to characterize such materials to a high order of accuracy requires that each
particle cover a substantial number of pixels. Errors in particle size assignment from digitized images
[9]
arise from two sources .
a) The number of pixels covered by the particle image in combination with the relative position of the
centring of the particle image with respect to the fixed, pixel pattern. A limited number of pixels
per image results in a variation in the reported size as a result of image centring with respect to
the fixed pixel array and due to the finite number of pixels. This results in a finite broadening of the
particle size distribution even from mono-sized particles.
b) The setting and control of the threshold of pixel detection. Any error in the setting of the threshold
level to decide whether a pixel is included or excluded, results in a bias to the size reported. The
influence of the threshold setting shall be carefully examined as demonstrated in Reference [12].
Any automated threshold setting algorithm which cannot demonstrate not inducing any error or
bias shall not be used for the purpose of characterization of reference materials.
For a circle of diameter, d , expressed in pixel units and covering a measured number of pixels, n , the
c c
standard deviation of n may be approximated by (see Reference [4]):
c
d 2
c
σ n =06, 8 (1)
()
c
The number of pixels, n , to form the image of a particle of diameter, d , can be estimated using Formula (1)
c c
for any standard deviation σ.
NOTE A larger number of pixels per particle also introduces a restriction upon the number of particles per
image frame that can be counted per slide.
6.2.2 General particle sizing
In this more general case, a wider particle size range is most likely. For this a balance shall be achieved,
whereby the largest particles are readily contained within the image frame restriction, while providing
sufficient pixels to describe the smallest particle to the desired accuracy. The threshold level may be set
as described in 8.2.
7 Image analysis
7.1 General
Modern image analyzers usually have algorithms available for enhancing the quality of the image prior to
analysis and for separating touching particles. It is permitted to use enhancement algorithms provided
that the measurements can be unambiguously associated with the particles in the original image and
the enhancement can be verified as not introducing additional errors of particle size or likely to bias
the final result. Irregularly shaped particles or particles with sharp corners should not be separated
since this would distort the shape of the particles. All touching irregular shaped particles should be
rejected from the measurement and a note should be made for the proportion of particles rejected from
each measurement frame, see 8.4. Touching spherical particles may be separated, as this gives only
minor distortion of the area of particles. A flow chart showing typical procedures used in carrying out
measurements by image analysis is given in Figure C.1.
7.2 Size classes and magnification
The theoretical limit for resolution of objects by size using image analysis is one pixel, although the
uncertainty of particle size increases with very low pixel numbers. Discretized representations should
be stored particle-by-particle with a resolution of one pixel. Note that any compression of images might
reduce the accuracy and the resolution. However, it is necessary to define the size classes for the final
reporting of results; the desire for maximum dynamic range of sizes covered in each frame should be
balanced by the necessity for accuracy, which is a function of the total number of particles counted,
the dynamic range and the number of pixels included in the smallest objects to be considered. It is
recommended that pixels be converted to SI length units prior to any reporting of size for quantitative
analysis.
The magnification used should be such that the smallest particles counted have a projected area
sufficient to cover an adequate number of pixels to meet the accuracy required. All particles measured
should be sized and stored with a resolution of one pixel. The final results are to be reported by grouping
the particles into size classes. For samples with a narrow size distribution, the grouping may be based
on a linear progression and for samples with a wide size distribution; the grouping may be based on a
logarithmic progression. The intervals for these progressions should be based on the dynamic range and
8 © ISO 2014 – All rights reserved
total number of particles counted. The particles assigned to a given class are those that have a diameter
that is equal to or greater than the lower limit of the class interval and less than the upper limit.
8 Counting procedure
8.1 General
The particle size distributions should be determined by counting those particle images that have been
accepted as passing the software selected criteria being employed for each measurement frame and
then summing these over all of the frames.
8.2 Particle image edges
Several segmentation methods exist for particle contour detection. These are for instance:
a) thresholding;
b) edge detection.
NOTE 1 See Annex B for more information.
These methods should be tested with reference materials in order to find out what method and parameter
set give the best approximation of the reference materials result and are suitable for the material that
has to be characterized.
NOTE 2 In the field of optical microscopy, the optical appearance of a real particle depends on the refractive
index of the particle, the refractive index of the surrounding medium, the surface structure, and the type of
illumination. Furthermore the optical particle image on the sensor may be slightly out of focus and is digitally
sampled on a discrete pixel grid. All these effects have influence on the particle image edges.
A threshold method can be established manually, if necessary.
EXAMPLE If a half-amplitude method is applicable, a small region of the background located a few pixels
away from the boundary of a typical particle is selected to establish the background value. The amplitude of
signal from pixels just fully responding to the particles presence is selected to establish the foreground value. The
threshold level is to be set at the average of these two values; see Reference [2].
NOTE 3 Manual threshold levels may be subjectively checked by direct comparison of the threshold image with
that of the original image. This subjective method does not constitute validation but readily detects incorrect
settings.
A second option is to “auto-threshold” the image. Such an auto-threshold procedure shall be validated
against a certified reference material having optical properties similar to that of the particles under
test. This can be a certified reference material or a certified reticule. Ensure that the threshold applied
is independent of particle size.
CAUTION — The use of a graticule or a reference material having different optical properties to
that of the material under test especially when the particles are suspended in a liquid can lead to
substantial bias in the reported particle size when this reference is used to establish a threshold
level.
CAUTION — Failure to set an appropriate threshold level can lead to significant bias in the
determination of the particles size. This bias depends on particle size. All particles are affected
but the relative value of influence to the particles size increases with decreasing particle size
under a given resolution.
8.3 Particles cut by the edge of the measurement frame
8.3.1 Method of counting all particles in a measurement frame
If all the objects that appear within the image frame (field of view) are accepted for measurement, the
accuracy of the final distribution will be impaired because some of the objects will be cut by the edge
of the image frame. To overcome this, a measurement frame is defined within the image frame. The
measurement frame can be used in the following two ways.
a) All the objects are allocated one pixel (e.g. the centroid) as the feature count point. Objects are
accepted only when their feature count point lies within the measurement frame; see Figure 2 a).
The measurement frame can be of any shape provided that there is enough space between the edges
of the two frames so that no accepted particle is cut by the edge of the image frame.
b) A rectangular frame is used with the bottom and right edges defined as reject sides. Objects lying
partially or wholly within the measurement frame and not touching the reject sides are accepted;
see Figure 2 b). There has to be sufficient space between the top and left edges of the two frames so
that no accepted objects are cut by the edge of the image frame. This covers all eventualities except
for particles intersecting two opposite sides of the frame; these would either be too large to be
measured at the magnification or would be so acicular that it is unsuitable for classification by area
anyway. Image analysis systems that reject all particles intersecting a frame edge use an effective
frame size that is different for each size class and also different for each particle shape.
a) The centroid of particles is in the measurement frame
b) Right and bottom sides are set to be the reject sides
NOTE Shaded particles are included in count; unshaded particles are excluded from count.
Figure 2 — Treatment of particles cut by the edge of the measurement frame
8.3.2 Method of neglecting particles cut by the edge of a measurement frame
All particles entirely inside the measurement frame are accepted for counting. All particles outside, or cut
by the edge, are neglected. This creates the situation where the probability for a particle to be included
in the measurement frame varies inversely with the size of the particle. This, therefore, introduces a
bias that is greater the larger the size of particle considered. The probability, P , of a particle i having
i
10 © ISO 2014 – All rights reserved
a horizontal Feret diameter of object x and a vertical Feret diameter of object x in a rectangular
F1 F2
measurement frame of size Z by Z is given by Formula (2) (see References [3][5]):
1 2
Zx− Zx−
()()
1 F1 2F2
P = (2)
i
ZZ
The population of particles in the measurement frame should, therefore, be divided by the probability,
P .
i
NOTE A very large number of frames may be required to minimize the error created by the edge effect
influence, especially when the particles are no longer very small compared to the frame size.
EXAMPLE A square frame of size 100 units times 100 units is used for counting a population of particles
of sizes ranging from two units to ten units. The count of the particles wholly in the measuring frame and the
correction factors are shown in Table 1.
Table 1 — Example of corrected count
Diameter
Raw count Probability Corrected count
x
i
n P n /P
i i i i
arbitrary unit
2 81 0,96 84
4 64 0,92 70
6 49 0,88 56
8 36 0,85 42
10 25 0,81 31
8.3.3 Analysis method of overlapping measurement frames
The analysis of overlapping measurement frames provides an alternative method of overcoming edge
effects in order to include the maximum number of particles into the total valid particle count. This
method requires that the stage movement to expose the next measurement frame be of sufficient
accuracy and precision such that each particle can be assigned a positional index. This is an essential
requirement to prevent duplication of particle counts.
During the image processing of a measurement frame, particles having interacted with the measurement
frame edge are identified and their position listed for analysis in subsequent frames. All other particles
wholly within the measurement frame are included into the count and listed as being counted. A degree
of frame overlap was previously decided from knowledge of the size distribution and the magnification
selected. The overlap should be such that each particle is counted in one of the measurement frames.
This method includes all of those particles in the measurement field which do not interact with the
extreme edges of the field.
8.4 Touching particles
The slide preparation method should be chosen to give a minimum number of touching particles.
Nevertheless it is inevitable that there will be touching particles in each measurement frame and some
method for dealing with them is necessary.
However, the first difficulty is to have an automatic method of identifying touching particles. This can
be done by:
a) following the number of particles “created” by numerical separation procedure;
b) some criterion such as the shape factor or even;
c) by manual intervention; the statistical procedure for evaluating slides may also give some indication.
Numerical separation proced
...
INTERNATIONAL ISO
STANDARD 13322-1
Second edition
2014-05-15
Particle size analysis — Image analysis
methods —
Part 1:
Static image analysis methods
Analyse granulométrique — Méthodes par analyse d’images —
Partie 1: Méthodes par analyse d’images statiques
Reference number
©
ISO 2014
© ISO 2014
All rights reserved. Unless otherwise specified, 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
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Published in Switzerland
ii © ISO 2014 – All rights reserved
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions and list of symbols . 1
3.1 Terms and definitions . 1
3.2 Symbols . 4
4 Preparation for image capture . 5
4.1 Introduction . 5
4.2 Procedures . 5
5 Sample preparation demands for method description . 6
5.1 Sample splitting and reduction . 6
5.2 Touching particles . 6
5.3 Particle distribution. 6
5.4 Number of particles to be counted . 6
5.5 Particle suspending fluid . 7
6 Quality of captured images . 7
6.1 General . 7
6.2 Pixels per particle . 7
7 Image analysis . 8
7.1 General . 8
7.2 Size classes and magnification . 8
8 Counting procedure . 9
8.1 General . 9
8.2 Particle image edges . 9
8.3 Particles cut by the edge of the measurement frame.10
8.4 Touching particles .11
8.5 Measurements .12
9 Calculation of the particle size results .12
10 Calibration and traceability .12
10.1 General .12
10.2 Recommendations and requirements .13
11 Accuracy .14
11.1 General .14
11.2 Reference materials .14
11.3 Instrument preparation .14
11.4 Qualification test .15
11.5 Qualification acceptance .15
12 Test report .15
Annex A (informative) Estimation of the number of particles to be counted for a given accuracy .17
Annex B (informative) Common segmentation methods for particle edge detection.22
Annex C (informative) Flow chart showing a typical image analysis method.23
Bibliography .24
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 on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical Barriers
to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 24, Particle characterization including sieving,
Subcommittee SC 4, Particle characterization.
This second edition cancels and replaces the first edition (ISO 13322-1:2004), which has been technically
revised.
ISO 13322 consists of the following parts, under the general title Particle size analysis — Image analysis
methods:
— Part 1: Static image analysis methods
— Part 2: Dynamic image analysis methods
iv © ISO 2014 – All rights reserved
Introduction
The purpose of this part of ISO 13322 is to give guidance when using images for particle size analysis.
Image analysis is a technique that has gained popularity in different applications. The aim of this part
of ISO 13322 is to give a standardized description of the technique used and its validation. This part of
ISO 13322 does not describe specific instruments and is restricted to those parts of the acquisition of
images that are relevant to the accuracy of the particle size analysis.
This part of ISO 13322 includes methods of calibration verification and recommends using a certified
standard as a reference scale. However it is sensible to make some measurements on particles under
study, or other reference objects, of known size so that the likely systematic uncertainties introduced by
the equipment can be assessed.
Errors introduced at all stages of the analysis from sub-division of the sample to generation of the final
result add to the total uncertainty of measurements and it is important to obtain estimates for the
uncertainty arising from each stage.
Essential operations are identified to ensure that measurements made conform to this part of ISO 13322
and are traceable.
INTERNATIONAL STANDARD ISO 13322-1:2014(E)
Particle size analysis — Image analysis methods —
Part 1:
Static image analysis methods
1 Scope
This part of ISO 13322 is applicable to the analysis of images for the purpose of determining particle
size distributions where the velocity of the particles against the axis of the optical system of the imaging
device is zero. The particles are appropriately dispersed and fixed in the object plane of the instrument.
The field of view may sample the object plane dynamically either by moving the sample support or the
camera provided this can be accomplished without any motion effects on the image. Captured images
can be analysed subsequently.
This part of ISO 13322 concentrates upon the analysis of digital images created from either light or
electron detection systems. It does not address the method of creating the image although the detection
settings chosen together with its calibration are important to particle sizing accuracy. This part of
ISO 13322 considers only image evaluation methods using complete pixel counts.
Both the type of distribution, (by number or by volume) together with the width of the particle size
distribution has a very material influence upon the number of particles to be measured to secure the
desired accuracy within the specified confidence limits. An example is shown in Annex A.
Automation of the analysis is possible in order to measure sufficient particle numbers for a required
degree of precision.
This part of ISO 13322 does not address the sample preparation. However, the sub sampling, dispersion
and presentation of particles to be measured are a vital part of the operational chain of actions necessary
to ensure accuracy and precision of any final result.
NOTE Further details about sampling and sample preparation can be found in ISO 14887 and ISO 14488.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 9276-1, Representation of results of particle size analysis — Part 1: Graphical representation
ISO 9276-2, Representation of results of particle size analysis — Part 2: The calculations of average particle
sizes/diameters and moments from particle size distributions
ISO 14488, Particulate materials — Sampling and sample splitting for the determination of particulate
properties
3 Terms and definitions and list of symbols
3.1 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1.1
area equivalent diameter
diameter of a circle having the same area as the projected image of the particle
Note 1 to entry: It is also known as the Heywood diameter or as the equivalent circular diameter.
3.1.2
binary image
digitized image consisting of an array of pixels, each of which has a value of 0 or 1, whose values are
normally represented by dark and bright regions on the display screen or by the use of two distinct
colours
3.1.3
contrast (of an image)
difference between the intensity of the particle image with respect to the
background near to the particle
3.1.4
edge detection
methods used to detect transition between objects and background
Note 1 to entry: See segmentation method (3.1.13).
3.1.5
Feret diameter
distance between two parallel tangents on opposite sides of the image of a particle
3.1.6
field of view
field which is viewed by the viewing device
Note 1 to entry: The full image frame of a digital imaging device corresponds to its field of view.
SEE: Figure 1.
3.1.7
grey image
image in which multiple grey level values are permitted for each pixel
3.1.8
image analysis
processing and data reduction operation which yields a numerical or logical result from an image
3.1.9
measurement field
field which is composed by the set of all measurement frames
SEE: Figure 1.
3.1.10
measurement frame
selected area from the field of view in which particles are sized and counted for image analysis
SEE: Figure 1.
3.1.11
pixel
picture element
individual sample in a digital image that has been formed by uniform sampling in both the horizontal
and vertical directions
2 © ISO 2014 – All rights reserved
3.1.12
raster pattern
scanning order of measurement frames in the total measurement field
SEE: Figure 1.
3.1.13
segmentation method
strategy employed to separate the objects of interest from their surroundings
Note 1 to entry: Method of dividing the particle image from the background.
Note 2 to entry: See edge detection (3.1.4).
3.1.14
threshold
grey level value which is set to discriminate objects of interest from background
Key
1 measurement frame
2 field of view
3 raster pattern of measurement frames
4 measurement field
X enlarged view of a field of view
Figure 1 — Relationship between the terms “field of view”, “measurement frame”, “raster
pattern” and “measurement field”
3.2 Symbols
A projected area of particle i
i
α horizontal calibration factor
α vertical calibration factor
d minimum feature length
d diameter of a circle
c
N number of particles to be measured
n measured number of pixels within a circle
c
n numbers of particles in size interval Δx
j j
P probability that particle i exists in the measuring frame (also called Miles-Lantuéjoul factor)
i
φ shape descriptor
i
σ standard deviation
V volume of particle i
i
x area equivalent diameter of particle i
A,i
x horizontal Feret diameter of object
F1
x vertical Feret diameter of object
F2
x dimension of particle i
i
x longest dimension of particle i, also called maximum Feret diameter
Fmax,i
x shortest dimension of particle i, also called minimum Feret diameter
Fmin,i
x horizontal dimension of object
x horizontal dimension of object in SI unit
1,m
x horizontal dimension of object in pixel
1,p
x vertical dimension of object
x vertical dimension of object in SI unit
2,m
x vertical dimension of object in pixel
2,p
x particle size corresponding to 10 % of the cumulative undersize distribution by volume
10,3
x particle size corresponding to 90 % of the cumulative undersize distribution by volume
90,3
Z horizontal side length of the rectangular measurement frame
Z vertical side length of the rectangular measurement frame
4 © ISO 2014 – All rights reserved
4 Preparation for image capture
4.1 Introduction
A pre-requisite for accurate particle size measurement using this method requires a full understanding
of the settings and calibration applied within the image capture device as well as a consideration of the
purpose for conducting the measurement.
The final settings and calibration of the image capture device need to be established via an iterative
approach. The size range of the particles within an unknown test sample has an influence upon the
settings required within the image capturing device. These remain unknown until the first image has
been taken, the result observed and the necessary adjustments to the image capture device to achieve
the desired accuracy of particle size measurement required. A fully trained operator shall conduct the
assessment.
The imaging instrument should be set up and operated in accordance with the manufacturer’s
recommendations considering the conditions prevailing.
In order to achieve accurate particle size measurements it is preferred that the illumination be uniform
over the total field of view and of a type designed to create images of high contrast. The magnification
should be such as to provide a minimum number of pixels for the smallest particle consistent with the
accuracy demanded and set to achieve a sharp focus. The number of pixels for the smallest dimension of
a particle is relevant for cases where linear dimensions or combinations thereof are measured.
Distortion in the image might arise from a number of causes, but its presence and effect on the image
may be determined by selecting known sized particles or other reference objects of similar optical
properties at a number of points and orientations in the field of view. It is important to note that the
measurements made provide only two-dimensional, X and Y, information.
4.2 Procedures
The operator should decide why the result of the image analysis is required. Is a size distribution
by the number of particles in each size class required or is the volume of particles in each size class
the requirement? What accuracy and precision is required for the final result? These decisions will
have a significant influence upon the choice of settings and the method employed in conducting the
measurement.
For each material to be analysed and for each instrument employed the person conducting the analysis
shall ensure that the following procedures are followed.
a) Ensure an adequate calibration for both the X and Y-axis of the measurement frame exists for the
imaging instrument being employed, preferably by using a certified graticule or equivalent reference
of equal standing.
b) Ensure that the optical magnification employed is suitable and such as the image of the smallest
particle to be measured covers a sufficient number of pixels to support the required accuracy of
measurement.
c) Ensure that the illumination method and setting of any focus is correctly established to give a good
contrast and uniformity of illumination of any image gathered.
d) Ensure that the number of particles within the measurement frame is such as to minimize the
number of touching particles.
e) Ensure that a sufficient number of images of separate aliquot samples are gathered to provide a
suitable total number of particles with respect to the type of distribution, number or volume based,
and the width of the particle size distribution (see ISO 14488) and that they contain an adequate
statistical number of the largest particle of the target material (see Annex A).
f) Some implementations of the image analysis technique employ a large area X, Y servo or manually
controlled sample slide assembly. Such large slides enable many measurement frames of the particles
deposited to be examined. Should the method of fully separate measurement frames be employed
then any frame overlap shall be avoided. If the method of overlapping measurement frames, or
other methods of analysing particles that interact with the measurement frame edge is used, then
procedures shall be employed to ensure that each particle is only included into the total count for
the appropriate size class, once. For more information, see 8.3.
g) If appropriate, ensure that the image quality consisting of illumination, focus and magnification has
not changed at the end of the measurement. The requirement for this step depends on the variability
of the instrument employed.
h) For the case when image analysis is to be used for certified reference material measurement at the
end of the image gathering procedure, the calibration outlined in a) should be repeated and any
measured deviation recorded.
i) All the conditions, set up or established, for the target material shall be fully documented.
5 Sample preparation demands for method description
5.1 Sample splitting and reduction
As only a small amount of material is needed to prepare a test sample, this should be sub-divided from
the whole sample in a manner that ensures that the test sample is representative of the whole as specified
in ISO 14488.
5.2 Touching particles
In order to assess the degree of touching particles, a suitable optical resolution setting of the imaging
system should be chosen. The optical resolution should also meet the criteria set out in 4.2 b).
The number of particles touching each other should be minimized. It is a prime requirement of the
method that measurements shall be made on isolated particles. Touching particles measured as one
particle without a proper separation will introduce error.
It is often not possible to reliably detect touching particles by image analysis alone, but the influence
of touching particles on the result can be investigated experimentally by increasing or decreasing the
number of particles per image. If the number of particles cannot be changed, the influence on the results
can be investigated using a reference material with similar size and shape.
5.3 Particle distribution
There should be an adequate distribution of particles in the field of view. It may be necessary to examine
several fields of view if a large total particle count is required. The whole area of the measurement
field should be examined to ascertain whether there is noticeable segregation of particles (by size). The
requirements set out in 4.2 f) should be followed.
5.4 Number of particles to be counted
The number of particles to be analysed depends upon whether the final result is a particle size
distribution by the number or by the volume of particles. Considerable care has to be exercised in order
to ensure that the analysis is representative of the bulk sample as described in ISO 14488. This can be
demonstrated by splitting the bulk sample into at least three test samples. Each test sample should
contain sufficient particle numbers for a full measurement. Statistical analysis of the data will reveal the
repeatability of the method including sampling and dispersion.
NOTE See Annex A for more information.
6 © ISO 2014 – All rights reserved
5.5 Particle suspending fluid
It is likely that a large number of particle measurements will be of particles presented in a gas where an
adequate image contrast should be ensured. Should particle presentation require a liquid suspension
then it is preferred that such liquids be clean, particle-free, transparent and have a refractive index
as different as possible from the refractive index of particles to enhance the image contrast. Particles
presented in a mixed optical background such as in biological specimens, may require dynamic particle
by particle threshold selection.
WARNING — Automated particle by particle parameter selection applied for segmentation as
envisaged when mixed optical backgrounds are required cannot be validated for true particle
size and may result in particle size bias and reduced accuracy.
CAUTION — Particle systems of mixed optical properties may have inaccurate particle sizes
attributed to them due to possible threshold setting errors introducing particle size bias.
6 Quality of captured images
6.1 General
It is important for the analysis that the particles in the captured images are well dispersed. The number
of overlapping or touching particles reduces with the concentration of the particles in the frame (see
5.2). This is in conflict with the requirements of having a large particle number to obtain a high degree
of precision. A compromise should be established.
The contrast achieved should be consistent with the level of accuracy required. The difference between
the brightness of the particle and its background shall be a few times of the resolution of the grey level
image from zero to maximum brightness signal.
The accuracy of the results is strongly affected by the number of pixels for each particle image. When only
the projected area of the particles is measured as an average value, a few pixels may provide acceptable
results for the smallest particles. Higher pixel numbers are required for accurate information about
each individual particle.
6.2 Pixels per particle
Both the number of pixels forming the image of the particle and the relative position of the centring of
the image with respect to the fixed pixel pattern can have a material influence upon the final particle
size assessed from each particle image.
Image analysis can be a method of choice for the certification of reference materials. It also can be the
method of choice for general measurements. The conditions required to achieve a defined accuracy and
precision may be quite different for these two cases and warrant separate approaches.
6.2.1 Characterization of reference materials
Materials selected for reference materials are often spherical and having limited or mono dispersed
size distributions. In order to characterize such materials to a high order of accuracy requires that each
particle cover a substantial number of pixels. Errors in particle size assignment from digitized images
[9]
arise from two sources .
a) The number of pixels covered by the particle image in combination with the relative position of the
centring of the particle image with respect to the fixed, pixel pattern. A limited number of pixels
per image results in a variation in the reported size as a result of image centring with respect to
the fixed pixel array and due to the finite number of pixels. This results in a finite broadening of the
particle size distribution even from mono-sized particles.
b) The setting and control of the threshold of pixel detection. Any error in the setting of the threshold
level to decide whether a pixel is included or excluded, results in a bias to the size reported. The
influence of the threshold setting shall be carefully examined as demonstrated in Reference [12].
Any automated threshold setting algorithm which cannot demonstrate not inducing any error or
bias shall not be used for the purpose of characterization of reference materials.
For a circle of diameter, d , expressed in pixel units and covering a measured number of pixels, n , the
c c
standard deviation of n may be approximated by (see Reference [4]):
c
d 2
c
σ n =06, 8 (1)
()
c
The number of pixels, n , to form the image of a particle of diameter, d , can be estimated using Formula (1)
c c
for any standard deviation σ.
NOTE A larger number of pixels per particle also introduces a restriction upon the number of particles per
image frame that can be counted per slide.
6.2.2 General particle sizing
In this more general case, a wider particle size range is most likely. For this a balance shall be achieved,
whereby the largest particles are readily contained within the image frame restriction, while providing
sufficient pixels to describe the smallest particle to the desired accuracy. The threshold level may be set
as described in 8.2.
7 Image analysis
7.1 General
Modern image analyzers usually have algorithms available for enhancing the quality of the image prior to
analysis and for separating touching particles. It is permitted to use enhancement algorithms provided
that the measurements can be unambiguously associated with the particles in the original image and
the enhancement can be verified as not introducing additional errors of particle size or likely to bias
the final result. Irregularly shaped particles or particles with sharp corners should not be separated
since this would distort the shape of the particles. All touching irregular shaped particles should be
rejected from the measurement and a note should be made for the proportion of particles rejected from
each measurement frame, see 8.4. Touching spherical particles may be separated, as this gives only
minor distortion of the area of particles. A flow chart showing typical procedures used in carrying out
measurements by image analysis is given in Figure C.1.
7.2 Size classes and magnification
The theoretical limit for resolution of objects by size using image analysis is one pixel, although the
uncertainty of particle size increases with very low pixel numbers. Discretized representations should
be stored particle-by-particle with a resolution of one pixel. Note that any compression of images might
reduce the accuracy and the resolution. However, it is necessary to define the size classes for the final
reporting of results; the desire for maximum dynamic range of sizes covered in each frame should be
balanced by the necessity for accuracy, which is a function of the total number of particles counted,
the dynamic range and the number of pixels included in the smallest objects to be considered. It is
recommended that pixels be converted to SI length units prior to any reporting of size for quantitative
analysis.
The magnification used should be such that the smallest particles counted have a projected area
sufficient to cover an adequate number of pixels to meet the accuracy required. All particles measured
should be sized and stored with a resolution of one pixel. The final results are to be reported by grouping
the particles into size classes. For samples with a narrow size distribution, the grouping may be based
on a linear progression and for samples with a wide size distribution; the grouping may be based on a
logarithmic progression. The intervals for these progressions should be based on the dynamic range and
8 © ISO 2014 – All rights reserved
total number of particles counted. The particles assigned to a given class are those that have a diameter
that is equal to or greater than the lower limit of the class interval and less than the upper limit.
8 Counting procedure
8.1 General
The particle size distributions should be determined by counting those particle images that have been
accepted as passing the software selected criteria being employed for each measurement frame and
then summing these over all of the frames.
8.2 Particle image edges
Several segmentation methods exist for particle contour detection. These are for instance:
a) thresholding;
b) edge detection.
NOTE 1 See Annex B for more information.
These methods should be tested with reference materials in order to find out what method and parameter
set give the best approximation of the reference materials result and are suitable for the material that
has to be characterized.
NOTE 2 In the field of optical microscopy, the optical appearance of a real particle depends on the refractive
index of the particle, the refractive index of the surrounding medium, the surface structure, and the type of
illumination. Furthermore the optical particle image on the sensor may be slightly out of focus and is digitally
sampled on a discrete pixel grid. All these effects have influence on the particle image edges.
A threshold method can be established manually, if necessary.
EXAMPLE If a half-amplitude method is applicable, a small region of the background located a few pixels
away from the boundary of a typical particle is selected to establish the background value. The amplitude of
signal from pixels just fully responding to the particles presence is selected to establish the foreground value. The
threshold level is to be set at the average of these two values; see Reference [2].
NOTE 3 Manual threshold levels may be subjectively checked by direct comparison of the threshold image with
that of the original image. This subjective method does not constitute validation but readily detects incorrect
settings.
A second option is to “auto-threshold” the image. Such an auto-threshold procedure shall be validated
against a certified reference material having optical properties similar to that of the particles under
test. This can be a certified reference material or a certified reticule. Ensure that the threshold applied
is independent of particle size.
CAUTION — The use of a graticule or a reference material having different optical properties to
that of the material under test especially when the particles are suspended in a liquid can lead to
substantial bias in the reported particle size when this reference is used to establish a threshold
level.
CAUTION — Failure to set an appropriate threshold level can lead to significant bias in the
determination of the particles size. This bias depends on particle size. All particles are affected
but the relative value of influence to the particles size increases with decreasing particle size
under a given resolution.
8.3 Particles cut by the edge of the measurement frame
8.3.1 Method of counting all particles in a measurement frame
If all the objects that appear within the image frame (field of view) are accepted for measurement, the
accuracy of the final distribution will be impaired because some of the objects will be cut by the edge
of the image frame. To overcome this, a measurement frame is defined within the image frame. The
measurement frame can be used in the following two ways.
a) All the objects are allocated one pixel (e.g. the centroid) as the feature count point. Objects are
accepted only when their feature count point lies within the measurement frame; see Figure 2 a).
The measurement frame can be of any shape provided that there is enough space between the edges
of the two frames so that no accepted particle is cut by the edge of the image frame.
b) A rectangular frame is used with the bottom and right edges defined as reject sides. Objects lying
partially or wholly within the measurement frame and not touching the reject sides are accepted;
see Figure 2 b). There has to be sufficient space between the top and left edges of the two frames so
that no accepted objects are cut by the edge of the image frame. This covers all eventualities except
for particles intersecting two opposite sides of the frame; these would either be too large to be
measured at the magnification or would be so acicular that it is unsuitable for classification by area
anyway. Image analysis systems that reject all particles intersecting a frame edge use an effective
frame size that is different for each size class and also different for each particle shape.
a) The centroid of particles is in the measurement frame
b) Right and bottom sides are set to be the reject sides
NOTE Shaded particles are included in count; unshaded particles are excluded from count.
Figure 2 — Treatment of particles cut by the edge of the measurement frame
8.3.2 Method of neglecting particles cut by the edge of a measurement frame
All particles entirely inside the measurement frame are accepted for counting. All particles outside, or cut
by the edge, are neglected. This creates the situation where the probability for a particle to be included
in the measurement frame varies inversely with the size of the particle. This, therefore, introduces a
bias that is greater the larger the size of particle considered. The probability, P , of a particle i having
i
10 © ISO 2014 – All rights reserved
a horizontal Feret diameter of object x and a vertical Feret diameter of object x in a rectangular
F1 F2
measurement frame of size Z by Z is given by Formula (2) (see References [3][5]):
1 2
Zx− Zx−
()()
1 F1 2F2
P = (2)
i
ZZ
The population of particles in the measurement frame should, therefore, be divided by the probability,
P .
i
NOTE A very large number of frames may be required to minimize the error created by the edge effect
influence, especially when the particles are no longer very small compared to the frame size.
EXAMPLE A square frame of size 100 units times 100 units is used for counting a population of particles
of sizes ranging from two units to ten units. The count of the particles wholly in the measuring frame and the
correction factors are shown in Table 1.
Table 1 — Example of corrected count
Diameter
Raw count Probability Corrected count
x
i
n P n /P
i i i i
arbitrary unit
2 81 0,96 84
4 64 0,92 70
6 49 0,88 56
8 36 0,85 42
10 25 0,81 31
8.3.3 Analysis method of overlapping measurement frames
The analysis of overlapping measurement frames provides an alternative method of overcoming edge
effects in order to include the maximum number of particles into the total valid particle count. This
method requires that the stage movement to expose the next measurement frame be of sufficient
accuracy and precision such that each particle can be assigned a positional index. This is an essential
requirement to prevent duplication of particle counts.
During the image processing of a measurement frame, particles having interacted with the measurement
frame edge are identified and their position listed for analysis in subsequent frames. All other particles
wholly within the measurement frame are included into the count and listed as being counted. A degree
of frame overlap was previously decided from knowledge of the size distribution and the magnification
selected. The overlap should be such that each particle is counted in one of the measurement frames.
This method includes all of those particles in the measurement field which do not interact with the
extreme edges of the field.
8.4 Touching particles
The slide preparation method should be chosen to give a minimum number of touching particles.
Nevertheless it is inevitable that there will be touching particles in each measurement frame and some
method for dealing with them is necessary.
However, the first difficulty is to have an automatic method of identifying touching particles. This can
be done by:
a) following the number of particles “created” by numerical separation procedure;
b) some criterion such as the shape factor or even;
c) by manual intervention; the statistical procedure for evaluating slides may also give some indication.
Numerical separation procedures are not recommended for separating particle aggregates into individual
particles as they can change the size of the particles in the image. Particle size distributions obtained
by such methods have no traceability. Such procedures for identifying aggregates can be investigated
by comparing the results with the size counting performed on the original untreated image. Touching
spherical particles may be separated, as this should give only minor distortion of the area of the particle.
Identification of touching particles on the basis of shape is not fool proof, in particular for compact
overlapping agglomerates, and will not distinguish real out-of-shape or oversized particles. In cases
where touching particles cannot be avoided, careful use of various techniques, e.g. fractal analysis to
identify aggregates or model-based separation techniques may be used to separate the particles. This
procedure should not be employed within certified reference material measurement procedures.
8.5 Measurements
The measurement of the perimeter of particles depends strongly on the image-analysis system used.
Accordingly, the primary measurement is the projected area of each particle, expressed in pixels, then
the longest and the shortest Feret diameters of ea
...
Frequently Asked Questions
SIST ISO 13322-1:2015 is a standard published by the Slovenian Institute for Standardization (SIST). Its full title is "Particle size analysis - Image analysis methods - Part 1: Static image analysis methods". This standard covers: ISO 13322-1:2014 is applicable to the analysis of images for the purpose of determining particle size distributions where the velocity of the particles against the axis of the optical system of the imaging device is zero. The particles are appropriately dispersed and fixed in the object plane of the instrument. The field of view may sample the object plane dynamically either by moving the sample support or the camera provided this can be accomplished without any motion effects on the image. Captured images can be analysed subsequently. ISO 13322-1:2014 concentrates upon the analysis of digital images created from either light or electron detection systems. It considers only image evaluation methods using complete pixel counts.
ISO 13322-1:2014 is applicable to the analysis of images for the purpose of determining particle size distributions where the velocity of the particles against the axis of the optical system of the imaging device is zero. The particles are appropriately dispersed and fixed in the object plane of the instrument. The field of view may sample the object plane dynamically either by moving the sample support or the camera provided this can be accomplished without any motion effects on the image. Captured images can be analysed subsequently. ISO 13322-1:2014 concentrates upon the analysis of digital images created from either light or electron detection systems. It considers only image evaluation methods using complete pixel counts.
SIST ISO 13322-1:2015 is classified under the following ICS (International Classification for Standards) categories: 19.120 - Particle size analysis. Sieving. The ICS classification helps identify the subject area and facilitates finding related standards.
SIST ISO 13322-1:2015 has the following relationships with other standards: It is inter standard links to SIST ISO 13322-1:2006. Understanding these relationships helps ensure you are using the most current and applicable version of the standard.
You can purchase SIST ISO 13322-1:2015 directly from iTeh Standards. The document is available in PDF format and is delivered instantly after payment. Add the standard to your cart and complete the secure checkout process. iTeh Standards is an authorized distributor of SIST standards.
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