ISO 19430:2016

INTERNATIONAL ISO
STANDARD 19430
First edition
2016-12-15
Particle size analysis — Particle
tracking analysis (PTA) method
Analyse granulométrique — Méthode d’analyse de suivi de
particule (PTA)
Reference number
©
ISO 2016
© ISO 2016, Published in Switzerland
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ii © ISO 2016 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Symbols and abbreviated terms . 4
5 Principles . 4
5.1 General . 4
5.2 Key physical parameters. 5
5.3 Detection limits . 5
5.3.1 Lower size limit . . 5
5.3.2 Upper size limit . 6
5.3.3 Sample and sampling volume . 6
5.3.4 Maximum particle number concentration . 6
5.3.5 Minimum particle number concentration . 7
5.4 Measurement precision and uncertainties . 7
5.4.1 General. 7
5.4.2 Measurement precision . 7
5.4.3 Size range . 8
5.4.4 Counting efficiency . 8
5.4.5 Sizing accuracy . 9
5.4.6 Size resolution . . 9
6 Apparatus .10
7 Procedure.11
7.1 General .11
7.2 Sample preparation .12
7.3 Instrument set-up and initialisation .12
7.4 Measurement .13
7.4.1 Sample delivery . .13
7.4.2 Sample illumination .13
7.4.3 Particle imaging and tracking .14
7.4.4 Track analysis .14
7.5 Results evaluation .14
7.5.1 General.14
7.5.2 Particle size evaluation .14
7.5.3 Distribution analysis.14
7.5.4 Data analysis and reporting .14
8 System qualification and quality control .15
8.1 General .15
8.2 System installation requirements .15
8.3 System maintenance .15
8.4 System operation .15
8.5 System qualification .16
9 Data recording .17
10 Test report .17
Annex A (informative) Theory.20
Annex B (informative) Apparatus settings and best practice .23
Bibliography .25
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
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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
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The committee responsible for this document is Technical Committee ISO/TC 24, Particle
characterization including sieving, Subcommittee SC 4, Particle characterization.
iv © ISO 2016 – All rights reserved

Introduction
Regulatory, scientific and commercial requirements for nanomaterial characterization or
characterization of particulate suspensions where particle sizing and counting is required provide a
strong case for further development of techniques such as Particle Tracking Analysis (PTA), also known
[14]
as Nanoparticle Tracking Analysis (NTA) . Due to the fact that the term PTA covers a larger size
1)
range and is more generic , the term PTA is used throughout this document to refer to NTA and PTA.
For all aims and purposes, the term PTA also means NTA in this document.
PTA is based on measuring the diffusion movement of particles in a suspension by means of laser
illumination, imaging of scattered light, particle identification and localization, and individual particle
2)
tracking . In this case, suspension is an even dispersion of particles, gas bubbles or other liquid
droplets. The hydrodynamic diameter of the individual particles, droplets or bubbles is related to
Brownian motion parameters via the Stokes–Einstein equation.
In recent years the academic community working in fields such as liposomes and other drug
delivery vehicles, nanotoxicology, viruses, exosomes, protein aggregation, inkjet inks, pigment
particles, cosmetics, foodstuffs, fuel additives and fine bubbles began using the PTA technology for
[10]
characterization. An ASTM standard guide (E2834–12) was developed to give guidance to the
measurement of particle size distribution by means of Nanoparticle Tracking Analysis. The present
document aims to broaden the scope of the specification and to introduce system tests for PTA
operation.
This document outlines the theory and basic principles of the particle tracking analysis method along
with its limitations and advantages. It also describes commonly used instrument configurations and
measurement procedures as well as system qualifications and data reporting. One of the key aspects
is the meaning of the data and its interpretation. It should be noted that the key measurand obtained
from PTA measurement is the number-based particle size distribution where the size is taken to mean
the hydrodynamic diameter (3.11) of the particles in the sample. This size can be different from other
[6] [4].
sizes obtained with different techniques such as dynamic light scattering or electron microscopy
1) NTA is the most recognised abbreviation for the technique described in this document. However the Particle
Tracking Analysis (PTA) includes NTA in its size range of measurements.
2) For the purpose of this document “tracking” will mean “following in terms of particle x and y position” and the
“track” will mean “the path of that particle defined by such x and y coordinates of each step”
INTERNATIONAL STANDARD ISO 19430:2016(E)
Particle size analysis — Particle tracking analysis (PTA)
method
1 Scope
This document describes the evaluation of the number–based particle size distribution in liquid
dispersions (solid, liquid or gaseous particles suspended in liquids) using the particle tracking analysis
method for diffusion velocity measurements.
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 terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at http://www.iso.org/obp
— IEC Electropedia: available at http://www.electropedia.org/
3.1
nanoscale
length range approximately from 1 nm to 100 nm
Note 1 to entry: Properties that are not extrapolations from larger sizes are predominantly exhibited in this
length range.
[SOURCE: ISO/TS 80004-1:2015, 2.1]
3.2
nano-object
material with one, two or three external dimensions in the nanoscale (3.1)
Note 1 to entry: The second and third external dimensions are orthogonal to the first dimension and to each other.
[SOURCE: ISO/TS 80004-1:2015, 2.5]
3.3
nanoparticle
nano-object (3.2) with all three external dimensions in the nanoscale (3.1)
Note 1 to entry: If the lengths of the longest to the shortest axes of the nano-object differ significantly (typically
by more than three times), the terms nanofibre or nanoplate are intended to be used instead of the term
nanoparticle.
[SOURCE: ISO/TS 80004-4:2011, 2.4]
3.4
particle
minute piece of matter with defined physical boundaries
Note 1 to entry: A physical boundary can also be described as an interface.
Note 2 to entry: A particle can move as a unit.
Note 3 to entry: This general particle definition applies to nano-objects (3.2).
[SOURCE: ISO/TS 80004-6:2013, 2.9]
3.5
agglomerate
collection of weakly bound particles or aggregates or mixtures of the two 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/TS 80004-4:2011, 2.8]
3.6
aggregate
particle comprising strongly bonded or fused particles where the resulting external surface area may
be significantly smaller than the sum of calculated surface areas of the individual components
Note 1 to entry: The forces holding an aggregate together are strong forces, for example covalent 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/TS 80004-4:2011, 2.7]
3.7
particle size
linear dimension of a particle (3.4) determined by a specified measurement method and under specified
measurement conditions
Note 1 to entry: Different methods of analysis are based on the measurement of different physical properties.
Independent of the particle property actually measured, the particle size can be reported as a linear dimension,
e.g. as an equivalent spherical diameter.
[SOURCE: ISO/TS 80004-6:2013, 3.1.1]
3.8
particle size distribution
distribution of particles (3.4) as a function of particle size (3.7)
Note 1 to entry: Particle size distribution may be expressed as cumulative distribution or a distribution density
(distribution of the fraction of material in a size class, divided by the width of that class).
[SOURCE: ISO/TS 80004-6:2013, 3.1.2]
3.9
equivalent diameter
diameter of a sphere that produces a response by a given particle-sizing method, that is equivalent to
the response produced by the particle being measured
Note 1 to entry: The physical property to which the equivalent diameter refers is indicated using a suitable
subscript [ISO 9276-1:1998].
Note 2 to entry: For discrete-particle-counting, light-scattering instruments, an equivalent optical diameter is used.
2 © ISO 2016 – All rights reserved

Note 3 to entry: Other material constants like density of the particle are used for the calculation of the equivalent
diameter like Stokes diameter or sedimentation equivalent diameter. The material constants, used for the
calculation, should be reported additionally.
Note 4 to entry: For inertial instruments, the aerodynamic diameter is used. Aerodynamic diameter is the
−3
diameter of a sphere of density 1 000 kg m that has the same settling velocity as the irregular particle.
[SOURCE: ISO/TS 80004-6:2013, 3.1.5]
3.10
light scattering
change in propagation of light at the interface of two media having different optical properties
[SOURCE: ISO 13320:2009, 3.1.17]
3.11
hydrodynamic diameter
equivalent spherical diameter of a particle in a liquid having the same diffusion coefficient as the real
particle in that liquid
[SOURCE: ISO/TS 80004-6:2013, 3.2.6]
3.12
particle tracking analysis
PTA
method where particles undergoing Brownian motion in a liquid suspension are illuminated by a laser
and the change in position of individual particles is used to determine particle size
Note 1 to entry: Analysis of the time-dependent particle position yields translational diffusion coefficient and
hence the particle size as hydrodynamic diameter using the Stokes-Einstein relationship.
Note 2 to entry: Nanoparticle Tracking Analysis (NTA) is often used to describe PTA. NTA is a subset of PTA since
PTA covers larger range of particle sizes than nanoscale (3.1).
[SOURCE: ISO/TS 80004-6:2013, 3.2.8, modified — Nanoparticle tracking analysis has been removed
from the term, and Notes 1 and 2 have been modified.]
3.13
nanomaterial
material with any external dimension in the nanoscale (3.1) or having internal structure or surface
structure in the nanoscale
[SOURCE: ISO/TS 80004-1:2015, 2.4]
3.14
diluent
non-volatile homogeneous liquid which is used to decrease the number concentration of particles
(3.4) in a suspension without any deleterious effects such as changing particle total number, state of
aggregation, particle size (3.7) or surface chemistry
3.15
viscosity
measure of the resistance to flow or deformation of a liquid
[SOURCE: ISO 3104:1994]
3.16
percentile
value of a variable below which a certain percentage of observations fall
[SOURCE: ISO 11064-4:2013, 3.7]
4 Symbols and abbreviated terms
For the purposes of this document, the following symbols and abbreviated terms apply.
CCD Charge Coupled Device
CMOS Complementary Metal Oxide Semiconductor
CV Coefficient of Variation (standard deviation divided by arithmetic
average)(ISO 27448:2009, 3.11)
CCD Charge Coupled Device
d hydrodynamic diameter metre m
translational diffusion coefficient in 1 dimension m /s
D
x
translational diffusion coefficient in 2 dimensions m /s
D
xy
translational diffusion coefficient in 3 dimensions m /s
D
xyz
η viscosity of the suspension medium pascal second Pa·s
2 −2 −1
k Boltzmann’s constant m kg s K
B
RSD Relative Standard Deviation (ISO/TR 13843:2000, 2.34) %
T absolute temperature kelvin °K
t time second s
mean square displacement in 1 dimension metre squared m
x
()
mean square displacement in 2 dimensions metre squared m
xy,
()
mean square displacement in 3 dimensions metre squared m
xy,,z
()
5 Principles
5.1 General
Determination of particle size distribution by PTA makes use of the Brownian motion and light
scattering properties of particles suspended in liquids. Irradiation of the sample (typically by means
of a laser beam of wavelength in the visible region) leads to light scattering by objects with a refractive
index that is different from that of the surrounding medium. Light scattered from each particle is
collected by magnifying optics and visualized by way of a suitable detector, such as a Charge Coupled
Device (CCD) or Complementary Metal Oxide Semiconductor (CMOS) camera. By recording a series of
sequential images, the instrument’s software tracks positions of particles as a function of time, allowing
analysis of their movement.
3)
[6] [13],
By tracking individual particles, undergoing random Brownian motion from frame to frame,
the average spatial displacement of the particles per unit time can be calculated, and this displacement
3) For the purpose of this document, “frame” will mean a still image obtained from video capturing of the moving
objects in PTA measurement equipment”.
4 © ISO 2016 – All rights reserved

[13]
can be related to the hydrodynamic diameter of the particles through the Stokes-Einstein equation .
Although translational Brownian motion is a three-dimensional process, it is possible to use a one-,
two-, or three-dimensional diffusion coefficient to determine particle hydrodynamic diameter. The
relevant formulae are derived in Annex A and can be summarized with three formulae below:
2kTt
B
xD==t (1)
()
x
3πηd
4kTt
B
xy, ==Dt (2)
()
xy
3πηd
2kTt
B
xy,,zD==t (3)
()
xyz
πηd
Mean square displacement x can be measured in x and y directions independently to give two
()
independent values for particle size [Formula (1)]. In most PTA instruments, xy, is evaluated as
()
shown in Formula (2). It should be noted that in all three cases there is no assumption of two
dimensional movement of particles. All particles are assumed to be moving freely in all three
dimensions while the measurement is sampling the projection of each x, y and z component of that
movement onto the xy observation plane. As described in Annex A, these components (observables)
are independent variables
5.2 Key physical parameters
Formulae (1) to (3) show that as well as the diffusion coefficient, the temperature and the viscosity of
the sample shall be known in order to calculate the hydrodynamic diameter.
5.3 Detection limits
Like any measurement technique, PTA has detection limits in terms of the particle size and the particle
number concentration. These limits are heavily dependent on the particle material, diluent and
polydispersity of the sample.
Depending on the physical properties of the particles, the typical working range of the PTA can be from
about 10 nm to about 2 μm in diameter.
5.3.1 Lower size limit
The lower limit of detection in terms of the particle hydrodynamic diameter is determined (apart
from sensitivity and dynamic range of the camera) by the light scattering from the particles. It is the
combination of refractive indexes of the particle material and the diluent that affect the amount of light
scattering the detection and tracking system. A large difference in refractive indexes results in higher
scattering and therefore lower detection limit for all other parameters being the same.
Better tracking of highly scattering particles results in preferential counting of particles. The accuracy
of counting is covered in 5.4.4.
Sample polydispersity affects the ability to track and therefore analyse different size fractions in the
particle number-size distribution. The underlying effect is linked to the dynamic range of the video
capture and image analysis. In a polydisperse sample large particles scatter a lot more than small
particles making it difficult to detect or track small size particles. All the values in Table 1 are given
for monodisperse samples. In the case of a monodisperse gold spheres in suspension, the lower limit of
detection is typically 15 nm but can range from approximately 10 nm to 20 nm.
Below is the table of detection limits for commonly used dispersions (particle-diluent combinations).
Table 1 — Lower limit of detection for monodisperse suspensions of nanoparticles
Particle material Approximate lower detection limit
(Hydrodynamic diameter in nm)
Gold 15
Polystyrene 45
Silica 75
Biological materials 60
Other metals or metal oxides 25
General effects of samples and measurement parameters on the detection limits are described in the
subclauses below. The typical values quoted for room temperature water dispersion are provided in
Table 1. These values are approximate and could vary (as much as 30 %, for example leading to low
detection limit for gold ranging from approximately 10 nm to 20 nm) depending on factors such as
porosity of silica or the type of biological material.
5.3.2 Upper size limit
The upper particle size limit is limited by slowing Brownian motion at larger particle sizes. The motion
of such particles is very slow and long observation periods may be required. Very large particles can
also produce so much scattering that the detection system may not track much smaller particles in the
same polydisperse sample.
In the limit of very large particles (or gas bubbles) the sample may separate with heavy particles
sedimenting (or large bubble creaming). These effects shall be considered at all times for PTA
measurement.
5.3.3 Sample and sampling volume
In a number of applications the knowledge of sample volume and sampling volume involved in a PTA
measurement can be important. Typically used equipment requires approximately 1 ml of sample to be
used for measurement.
The subsampling methods can vary between manufacturers, yet the sampling volume of liquid that is
4)
being investigated within the PTA microscope field of view is often limited to a range of approximately
0,1 nl to 1 nl volume. The sampling volume, for the PTA measurement is limited laterally by the optical
field of view of the system to (typically of the order of) 100 μm by 100 μm area. The particles in that
area are tracked using imaging power of the optics with an approximate focus depth of (the order of)
10 μm which is taken as the sampling volume depth. This results in a sampling volume of 0,1 nl. Larger
sampling volumes may be obtained for optical systems with larger field of view or lower magnification.
In order to obtain a representative measurement of a sample (especially for low particle number
concentrations), the sampling volume should be increased. This is often achieved by sampling multiple
parts of the sample and performing a new measurement as described in Clause 7.
5.3.4 Maximum particle number concentration
When preparing samples or evaluating the applicability of PTA to existing samples, the limits of particle
number concentration shall be considered. This subclause addresses the limitations of the PTA method
in terms of the particle number concentration. All references in this document to concentration are made
to particle number concentration and not molar concentration or mass concentration due to the nature
of the measurement. Appropriate conversions can be made from (for example) mass concentration to
particle number concentrations at sample preparation stages. PTA requires highly diluted samples and
the optimal particle concentration is sample-dependent. The optimal particle concentration should be
4) As defined in ISO 10360-7:2011, 3.3, “field of view” is the area viewed by the imaging probing system.
6 © ISO 2016 – All rights reserved

assessed by preliminary tests on a series of dilutions. Depending on the level of prior knowledge about
the sample concentration, selecting an appropriate dilution can take several iterations.
The number of particles in the sampling volume of the instrument determines the number of tracks and
therefore the quality of the statistical result of the measurement. The more particles tracked, the more
representative the obtained particle distribution is. However, too large a number of particles affects the
ability to independently track particles in the field of view (see 5.3.3). If particle paths intersect (which
happens in highly concentrated samples), the tracks are rejected leading to less tracked data.
A typical value of maximum particle number concentration measurable with PTA is 10 particles per ml.
5.3.5 Minimum particle number concentration
Every PTA measurement shall contain enough particle tracks to reach a specified level of sampling
repeatability. In principle it is possible to track just one particle, but this may not be representative
or reproducible enough for a required measurement. So the minimum particle number concentration
is determined by the sampling level of the result the user is trying to achieve (see 5.4). In addition, if
the user is evaluating the particle size distribution over a wide range of sizes, the minimum particle
5)
number concentration required is larger as there are more size bins over which the tracking data
should be spread.
For instrument systems with a wide optical field of view/sampling volume (larger than that in 5.3.3),
the minimum measureable particle number concentration can be as low as 10 particle per ml.
5.4 Measurement precision and uncertainties
5.4.1 General
It is important to note that although PTA usually involves the simultaneous tracking and analysis of
multiple light-scattering particles, the diffusion coefficient and hence the hydrodynamic diameter of
each particle is determined individually before the data are integrated to produce a number-based
particle size distribution. It should be noted that the data processing algorithms can vary between
manufacturers.
5.4.2 Measurement precision
The number of tracks of different particles determines the level of representative sampling. Tracking
[2][4].
enough particles for an appropriate statistical representation of the sample is important
An example of an approximate relationship between the number of tracks, number of frames, video
length and the PTA measurement precision expressed in CV of modal particle size for a monodisperse
100 nm sample is reported in Table 2.
Table 2 — Approximate values showing dependence of PTA measurement on the number of
tracked particles, number of frames, video length
Number of tracks Number of frames Video length CV of modal particle size
%
s
400 130 5 < 10 %
700 230 8 < 8 %
1 000 300 10 < 5 %
2 000 600 20 < 3 %
NOTE Data were obtained for a 100 nm monodisperse sample of polysterene spheres.
5) For the purpose of this document, “size bin” refers to the particle diameter size range width which corresponds
to a given (size-number) histogram particle number count.
Table 2 should be used as a guidance in planning experiments.
For all the data sets in Table 2, the size bins for the particle size distribution were kept at 5 nm. Other
size bins may be used in other measurements. Larger size bins allow better count statistics but
compromise size resolution.
For a given bin size, the number of tracks recorded can be used as an indication of the data precision.
For such a Poisson distribution, the square root of the number of tracks in a size bin represents the
precision estimate. An assessment of precision should be implemented by sampling three or more times
the same sample and a CV be obtained for each size bin.
The precision of PTA measurement is affected by a number of factors including polydispersity,
distribution of particle track lengths and size. Thus the values in Table 2 represent a relatively ideal case.
This means that, to achieve a desired precision, a higher number of tracks should be counted for more
[4]
polydisperse samples. Methodology described in ISO 13322-1:2014, Annex A ab-initio calculation of
the number of tracks required for a given precision. Table 2 indicates the minimum number of frames
or video length to achieve a given CV of modal particle size. Number of tracks, number of frames and
video length follow an approximate linear relationship (within the experimental fluctuations of Table
2 data).
5.4.3 Size range
Particle tracking size limits have already been described in 5.3.1 and 5.3.2 regarding the lower and
upper size limits. It was also mentioned that the particle size range can affect the measurement
accuracy due to the effect of larger particles on counting smaller ones in a polydisperse sample.
A larger particle size range also affects the number of tracks allocated to each size bin in the particle
size distribution. Increasing the size range without increasing the number of size bins results in a
coarser size granularity in the particle size distribution. The user of this document should be guided
by the accuracy required for each size bin and ensure enough tracks are sampled in each size bin. This
means that the total number of tracks required for a larger size range can be notably larger.
5.4.4 Counting efficiency
Most PTA systems give an indication of the number of particles in the field of view and this can be
used to estimate the total number concentration in the sampling volume (see 5.3.3). The uncertainties
involved in this calculation are related to the optical properties of the instrument and the polydispersity
of the sample. Due to a finite depth of focus (typically ~10 μm), the particles are tracked and counted in
that volume only.
Measurement of particles critically depends on the ability of the PTA system to detect them. Larger
particles can be detected with more ease than smaller ones. Samples that contain particles of very
different sizes may therefore oversample (or overcount) larger particles. Due to the statistical nature
of this measurement the particle track lengths vary. Tracks that are too short or intersecting tracks
are rejected by the processing software. Some instrument manufacturers employ an optimization
procedure that optimises the threshold automatically whereas some allow users to set the minimum
threshold of tracks manually.
Another effect of large particles is related to their dynamics, smaller particles on average move greater
distances than larger particles between frames. In some cases, these particles exit the field of view and
in that case may be disregarded from the calculation. Larger particles at the same edge of the field of
view are more likely to be tracked for longer thus contributing more significantly to the overall count.
Conversely, a small particle outside the field of view has a greater chance of more rapidly entering the
field of view than a large particle at that same position. Therefore, providing the number of particles of
a given size per frame are being reported on, this in itself does not lead to a bias in the measurement.
8 © ISO 2016 – All rights reserved

5.4.5 Sizing accuracy
Based on Formulae (1) to (3) in Clause 5, the particle diameter (hydrodynamic diameter, d) depends
on uncertainty in the temperature of the sample, in the tabulated value of viscosity (which is itself
temperature-dependent) and on the error involved in measuring the particle mean displacement while
tracking.
An uncertainty of ± 3 K in temperature results in approximately ± 1 % additional error in diameter
evaluation. The temperature of the sample shall therefore be measured to better or equal to ± 3 K for
this standard to be applicable. It is also required to bring the sample and instrument into thermal
equilibrium before starting any measurement. A freshly injected sample should be allowed to
equilibrate for 1 min to 2 min before measurement.
Values for viscosity will often be taken from tabulated values for a given diluent. Viscosity varies with
temperature. Such variation shall be less than ± 2 % over the operational range of temperatures used
in an experiment. In the case of water at approximately room temperature, the ± 2 % requirement
for the accuracy of the viscosity implies a more stringent requirement on knowledge of the absolute
temperature (than ± 3 K) as the tabulated viscosity data and temperature are interdependent.
Each particle track contains a number of steps over which the particle has been tracked. The longer the
particle is tracked, the greater the accuracy of the evaluation of its hydrodynamic diameter (providing
[12]
zero bias) . The uncertainty in evaluating the x [see Formula (1)] for each particle is therefore
()
proportional to 1 n , where n is the number of steps in each track.
steps steps
Any vibration or movement that is not a result of particle Brownian motion will lead to a decrease in
the reported size of particles, and as such shall be avoided. Therefore, steps shall be taken to prevent
vibration affecting the measurement. The tracking software shall be capable of detecting non-Brownian
motion and either correcting for it or at least alerting the operator to its presence.
5.4.6 Size resolution
Size resolution refers to the ability to distinguish two closely size-related particle populations.
This parameter is determined by the uncertainty in measurement accuracy of a single particle, the
reproducibility of the data in each size bin as well as the counting efficiency (see 5.4.4) in each size class
of the particle distribution.
6 Apparatus
PTA equipment will generally comprise a common collection of basic components, with the possibility of
additional peripherals that may be desirable or required for specific experiment types. The components
of the core apparatus are described below.
Figure 1 illustrates a common geometry of the PTA experimental setup. It should be noted that the
orientation shown implies the particle tracking in the xy plane. The arrangement of optical illumination
and detection may also be rotated about x axis with the CCD Camera pointing along the y direction.
The right angles between the illumination laser and the optical detection is not a requirement as other
angles allowing dark field imaging of the sample are commonly used.
See Annex B for information on apparatus settings and best practice.
6.1 Sample cell (with sample in dispersion). The sample to be analysed is held in the sample cell.
This cell shall be inert to the sample, and shall be able to hold the sample at a stable thermal equilibrium.
The temperature of the sample and cell shall be measureable, and as a minimum requirement the
temperature of the sample shall be measured with ± 3 K precision. The cell shall, at least in part, be
optically transparent to allow illumination of the sample by the radiation source and collection of
scattered light by the optical assembly.
6.2 Laser (or another light source).The laser source shall be such that the intensity and wavelength
provide appropriate scattering from particles without bleaching, destroying or modifying them in any
way. The wavelength and intensity shall also be appropriate for image collection by the digital camera.
The beam shall be focused to maximize illumination of in-focus particles, and to minimize optical noise
generated by the illumination of out-of-focus particles. The irradiation shall give rise to minimal localized
heating or photophoresis.
For certain experimental procedures, such as the visualization of fluorescently labelled particles
against a non-fluorescent scattering background, the radiation shall be monochromatic and the
wavelength matched to both the excitation wavelength of the fluorophore and the optical filters used in
signal collection.
6.3 Optical microscope (with optical magnification). Scattered (or emitted) light is collected
and delivered to the image capture apparatus through a series of lenses, filters and mirrors generally
resembling a conventional optical microscope. It is worth noting that magnification sufficient to image the
particles is unnecessary, the only requirement being sufficient resolution to allow suitable measurement
of the motion of the particles being measured.
6.4 Digital video camera. The image capture apparatus is a digital camera equipped with either a
CCD or CMOS detector sensitive enough to image the light scattered by the particles in the sample. The
camera frame rate (typically 10 to 60 frames per second) shall be such that sufficient displacement data
are collected for each particle, allowing accurate particle tracking and determination of particle size. It
should be noted that the camera frame rate is not typically adjustable by the user and instead is more
commonly set by the choice of camera by the manufacturer.
6.5 Tracking and data processing computer. A video of illuminated particles undergoing Brownian
motion shall be captured. The video shall be analysed using particle tracking software. The software
should be capable of processing each of the video images; identifying, localizing, and counting individual
particles; tracking discrete particles from frame to frame by recognizing the same particle in separate
10 © ISO
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