ISO/TS 23151:2021
(Main)Nanotechnologies — Particle size distribution for cellulose nanocrystals
Nanotechnologies — Particle size distribution for cellulose nanocrystals
This document describes methods for the measurement of particle size distributions for cellulose nanocrystals using atomic force microscopy and transmission electron microscopy. The document provides a protocol for the reproducible dispersion of the material using ultrasonication, as assessed using dynamic light scattering. Sample preparation for microscopy, image acquisition and data analysis are included.
Nanotechnologies — Distribution en taille des particules pour les nanocristaux de cellulose
General Information
Standards Content (Sample)
TECHNICAL ISO/TS
SPECIFICATION 23151
First edition
2021-09
Nanotechnologies — Particle size
distribution for cellulose nanocrystals
Nanotechnologies — Distribution en taille des particules pour les
nanocristaux de cellulose
Reference number
© ISO 2021
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ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 2
5 Dispersion of CNCs .2
5.1 General considerations. 2
5.2 Dispersion of CNCs by sonication . 3
5.3 Dynamic light scattering assessment of dispersions . 4
5.4 Determination of optimal sonication energy . 5
6 Sample preparation for microscopy .5
6.1 General considerations. 5
6.2 AFM sample preparation . 6
6.3 TEM sample preparation . 6
7 Atomic force microscopy . 6
7.1 General . 6
7.2 Instrumentation and accessories . 7
7.3 Microscope calibration . 7
7.4 Data acquisition . 7
7.5 Image analysis. 8
8 Transmission electron microscopy . 8
8.1 General . 8
8.2 Instrumentation and accessories . 9
8.3 Microscope calibration . 9
8.4 Data acquisition . 9
8.5 Image analysis. 9
9 Data analysis .10
9.1 General . 10
9.2 Assessment of data quality . 10
9.3 Fitting distribution models to data . 10
9.4 Measurement uncertainty . 11
10 Test report .12
10.1 Atomic force microscopy . 12
10.1.1 General information .12
10.1.2 Sample. 12
10.1.3 Data acquisition . . .12
10.1.4 Image analysis . 13
10.2 Transmission electron microscopy . 13
10.2.1 General information .13
10.2.2 Sample. 13
10.2.3 Data acquisition . . .13
10.2.4 Image analysis . 14
Annex A (informative) Assessment of CNC dispersions .15
Annex B (informative) Assessment of applied imaging force .16
Annex C (informative) Interlaboratory comparison results: AFM .18
Annex D (informative) Interlaboratory comparison results: TEM .25
Bibliography .34
iii
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
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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
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www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
Any feedback or questions on this document should be directed to the user’s national standards body. A
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committees.
iv
Introduction
Cellulose nanomaterials, including cellulose nanocrystals (CNCs) and cellulose nanofibrils, are
anticipated to have significant commercial impact. Cellulose nanocrystals are produced from naturally
occurring cellulose, primarily from wood pulps and annual plants, by acid hydrolysis. Their production
from readily available cellulose sources makes them a candidate for use as a potentially non-toxic,
biodegradable and sustainable nanomaterial. The recent demonstration of the feasibility of large-scale
CNC production and the availability of infrastructure for harvesting raw materials will facilitate their
commercial development. CNCs and cellulose nanofibrils are produced in a number of countries on pilot,
pre-commercial or commercial scales. Estimates of the market potential for cellulosic nanomaterials
are as high as 35 million metric tons annually, depending on the predicted applications and the
[10],[11]
estimated market penetration . Standards for characterization of CNCs are required for material
certification to facilitate sustained commercial and applications development.
Cellulose nanocrystals have high crystallinity and are nanorods with high aspect ratio, surface area
and mechanical strength. They assemble to give a chiral nematic phase with unique optical properties
and their surface chemistry can be modified to ensure colloidal stability in water and to facilitate
dispersion in a variety of matrices. These properties, plus their biocompatibility, low cost and minimal
toxicity, enable many potential applications. Industrial producers are working with receptor industries
in various application areas, including nanocomposite materials, health and personal care products,
paints, adhesives and thin films, rheology modifiers and optical films and devices. Standardization
activities within ISO/TC 229 and ISO/TC 6 have focused on nomenclature and terminology,
characterization methods in general and specific methods for determining surface functional groups,
metal ion and dry ash content. Particle size distribution is also a key property for CNC characterization.
Particle morphology and size distribution control some properties of individual CNCs and contribute
in part to their organization in suspensions, dry films and matrices. These properties and chemical
characteristics determine CNC colloidal stability, viscosity and self-assembly, as well as performance
in applications (e.g. reinforcement of nanocomposites). Length distribution may also be used to
differentiate among cellulose nanocrystal grades or products.
This document describes a method for reproducibly dispersing dry CNCs for preparation of microscopy
samples, provides image acquisition protocols for atomic force and transmission electron microscopy
and summarizes image analysis procedures for determining particle size distributions. The methods
are compatible with analysis of CNCs as produced by several processes and can be extended to surface
modified CNCs with adjustment of dispersion and sample deposition methods. The two microscopy
methods provide complementary information, and both have been widely used for size analysis of CNCs.
v
TECHNICAL SPECIFICATION ISO/TS 23151:2021(E)
Nanotechnologies — Particle size distribution for cellulose
nanocrystals
1 Scope
This document describes methods for the measurement of particle size distributions for cellulose
nanocrystals using atomic force microscopy and transmission electron microscopy. The document
provides a protocol for the reproducible dispersion of the material using ultrasonication, as assessed
using dynamic light scattering. Sample preparation for microscopy, image acquisition and data analysis
are included.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO/TS 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects
ISO 21363:2020, Nanotechnologies — Measurements of particle size and shape distributions by
transmission electron microscopy
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO/TS 80004-2 and the following
apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
cellulose nanocrystal
nanocrystal predominantly composed of cellulose with at least one elementary fibril (3.3), containing
predominantly crystalline and paracrystalline regions, with an aspect ratio of usually less than 50 but
usually greater than 5, not exhibiting longitudinal splits, inter-particle entanglement, or network-like
structures
Note 1 to entry: The dimensions are typically 3 nm to 50 nm in cross-section and 100 nm to several μm in length
depending on the source of the cellulose nanocrystal.
Note 2 to entry: The aspect ratio refers to the ratio of the longest to the shortest dimension.
Note 3 to entry: Historically cellulose nanocrystals have been called nanocrystalline cellulose (NCC), whiskers
such as cellulose nanowhiskers (CNW), and microfibrils such as cellulose microfibrils; they have also been called
spheres, needles or nanowires based on their shape, dimensions and morphology; other names have included
cellulose micelles, cellulose crystallites and cellulose microcrystals.
[SOURCE: ISO/TS 20477:2017]
3.2
cellulose nanofibril
cellulose nanofibre composed of at least one elementary fibril (3.3), containing crystalline,
paracrystalline and amorphous regions, with aspect ratio usually greater than 10, which may contain
longitudinal splits, entanglement between particles, or network-like structures
Note 1 to entry: The dimensions are typically 3 nm to 100 nm in cross-section and typically up to 100 μm in
length.
Note 2 to entry: The aspect ratio refers to the ratio of the longest to the shortest dimensions.
Note 3 to entry: The terms “nanofibrillated cellulose”, “nanofibrillar cellulose”, “microfibrillated cellulose”,
“microfibrillar cellulose”, “cellulose microfibril” and “cellulose nanofibre” have been used to describe cellulose
nanofibrils produced by mechanical treatment of plant materials often combined with chemical or enzymatic
pre-treatment steps.
Note 4 to entry: Cellulose nanofibrils produced from plant sources by mechanical processes usually contain
hemicellulose and in some cases lignin.
Note 5 to entry: Some cellulose nanofibrils might have functional groups on their surface as a result of the
manufacturing process.
[SOURCE: ISO/TS 20477:2017, 3.3.6, modified — Note 6 to entry has been deleted.]
3.3
elementary fibril
structure, originating from a single terminal enzyme complex, having a configuration of cellulose
chains specific to each cellulose-producing plant, animal, algal and bacteria species
[SOURCE: ISO/TS 20477:2017, 3.2.5]
4 Abbreviated terms
AFM atomic force microscopy
CNC(s) cellulose nanocrystal(s)
DLS dynamic light scattering
ILC interlaboratory comparison
PLL poly-L-lysine
PSD particle size distribution
PI polydispersity index
PVDF polyvinylidene difluoride
TEM transmission electron microscopy
VAMAS Versailles project on advanced materials and standards
5 Dispersion of CNCs
5.1 General considerations
Dry CNCs are aggregated and require energy input, typically by ultrasonication, for dispersion.
Previous studies have examined the sonication efficiency for CNCs derived from wood pulp by sulfuric
- +
acid hydrolysis and neutralization with sodium hydroxide which generates –SO Na groups on the
[12]
surface . The average CNC size and size distribution varied with the sample concentration even when
the sonication energy divided by mass of CNC was kept constant; CNC suspensions with a mass fraction
of 2 % were shown to be optimal for efficient dispersion by sonication. The protocol below has been
developed using spray-dried sodium exchanged sulfated CNCs. The protocol may require optimization
[12],[13]
for freeze-dried CNCs , CNCs produced from other cellulose biomass sources and CNCs with a
different loading of sulfate half esters or other negatively charged surface groups.
A procedure for sample preparation and sonication (probe sonicator) to generate a well-dispersed
CNC suspension is provided in 5.2. Bath sonication has been shown to be inadequate for dispersion of
[12][14]
CNCs . A protocol for analysis of CNC suspensions by DLS is provided in 5.3; general details on the
[7]
use of DLS for particle size determination are available in ISO 22412 .
Representative results illustrating changes in size (Z-average) and PI as a function of sonication energy
are provided in Annex A. The Z-average is the intensity-weighted harmonic mean diameter derived
[7]
from a cumulants analysis of DLS data, as described in ISO 22412 .
The Z-average provides the equivalent hydrodynamic diameter, the diameter of a sphere that will
diffuse at the same rate as the acicular CNC particle.
Although the Z-average determined by DLS is not a direct measure of CNC particle size, it provides a
useful and rapid means of assessing changes in relative size for a large number of CNC suspensions.
Recent developments in the use of field flow fractionation coupled with multiple detection systems for
[15]
CNC analysis may provide an alternative to DLS analysis . The protocols for dispersion by sonication
[12]
and DLS assessment have been used by three laboratories with repeatable and reproducible results
[15][16]
.
Plots of Z-average and PI as a function of sonication energy can be used to select an appropriate
sonication energy for specific samples, see 5.4. This selection is a compromise between applying
sufficient sonication to disperse most aggregates while ensuring that the applied sonication energy
does not damage the sample.
5.2 Dispersion of CNCs by sonication
Remove dry CNC from low temperature storage and keep unopened until the sample reaches room
temperature (typically several hours).
Use an analytical balance to weigh the desired amount of CNC in a polypropylene centrifuge tube.
Amounts of CNC in the 50 mg to 300 mg range have been used with either 15 ml or 50 ml centrifuge
tubes in this protocol for preparation of 2 % mass fraction CNC suspensions. Glass tubes can be used,
although some optimization of the protocol may be required since the sonication efficiency is sensitive
to a number of factors, including the probe depth and placement and the container material and
[17]
geometry .
Add deionized water to the tube in the amount required to obtain a 2 % mass fraction suspension
of CNC, close the tube cap, and shake the tube vigorously by hand for a few seconds to promote CNC
dispersion. Freshly obtained deionized water (18,2 MΩ cm) filtered with a 0,22 μm filter (typically part
of the purification system) is used throughout.
The optimal concentration of CNC for dispersion by ultrasonication is 2 % mass fraction; disruption
of aggregates and agglomerates by sonication is less effective at lower concentrations. If suspensions
of lower concentration are required, dilute the sonicated 2 % mass fraction CNC suspension with
deionized water to the desired concentration.
Leave the mixture at room temperature for 24 h for the CNC to disperse. The mixture can be shaken by
hand periodically to accelerate dispersion; a tube shaker may also be used.
Check the condition of the ultrasonic probe (a 6-mm probe is recommended for the volumes used here)
and clean if pitting or roughness of the surface is observed.
Sonication is most effective at low temperatures. Therefore, heating of the suspension during prolonged
sonication should be avoided. The temperature increase should not be more than 2 °C to 3 °C for the
amounts of dry CNC and processing energy recommended in this protocol, if the probe is in good working
condition, properly installed in the processor, and immersed in the suspension as recommended above.
During sonication, the tube may be placed in a room temperature water bath cooled when necessary
with a few ice cubes. Use of an ice bath is not recommended.
Immerse the ultrasonic probe in the suspension ensuring that the tip is centered in the tube and at least
1,3 cm both below the suspension surface and above the bottom of the tube.
Sonicate the suspension with the required energy (J/g dry CNC) at room temperature and an average
power of approximately 10 W. Ensure that the suspension surface remains as flat as possible and no
excessive aerosoling or bubbling is observed. If excessive aerosoling, bubbling, or suspension surface
fluctuation is observed, adjust the probe position immediately. Cover the tube to minimize loss of
suspension due to aerosoling.
[17]
The energy transfer efficiency may be measured calorimetrically to ensure that the applied
energy is reliable and does not change with time. Knowledge of the sonication energy is necessary for
comparisons between laboratories.
Remove the sample from the ultrasonic processor, and store for a short period of time at room
temperature (≈ 21 °C to 22 °C) or refrigerate (≈ 5 °C) for longer term storage.
NOTE This protocol has been tested with 50 mg to 300 mg dry CNC; preparation of suspensions with larger
amounts of CNC can require optimization of sonication conditions.
5.3 Dynamic light scattering assessment of dispersions
Set up the instrument as recommended in the manual.
Information on the importance of cell cleanliness and handling and proper technique for preparing and
[8]
transferring suspensions for DLS measurements is available in ISO/TR 22814 .
It is good laboratory practice to verify the operability of a DLS instrument by measuring a reference
nanomaterial (for which DLS data is available) to obtain Z-average and PI. Gold, silica and polystyrene
nanoparticles with diameter <100 nm are in the same size range as most CNC samples. For larger CNCs,
a reference material with diameter above 100 nm may be used. The use of a reference material from
[1]
a source qualified under ISO guidelines is recommended. The measured Z-average and PI should
be within the quoted uncertainty for the reference material. It is important to note that instrument
operability as verified using a reference material does not mean that a Z-average value obtained for
acicular CNCs is a quantitative or accurate measurement of diameter.
Dilute the 2 % mass fraction CNC suspensions to 0,1 % using deionized water, and then add 1 ml of
10 mmol/l NaCl solution to 1 ml of 0,1 % mass fraction CNC suspension to obtain 2 ml of 0,05 % mass
fraction suspension in 5 mmol/L NaCl. The 0,05 % suspension shall be analyzed within several hours of
preparation and shaken vigorously before transfer to the DLS cell. Filter the sample through a 0,45 μm
PVDF membrane syringe filter and discard the first several drops before adding the required volume to
the DLS cuvette. Ensure that there are no bubbles in the cell.
Place the cuvette in the instrument and equilibrate at the desired temperature. The time required for
equilibration will vary depending on the difference between the target temperature and the ambient
temperature. The equilibration time can be verified by measuring the temperature for an equivalent
volume of water under the same conditions. Adjust the scattering intensity using the instrument
software. Measure each sample three times with each measurement consisting of the average of a
number of runs (e.g. 10 runs of 10 s each).
Use the cumulants method to obtain the three-measurement average value and standard deviation for
Z-average and PI for the sample.
NOTE Different instrument optical configurations are available. The use of forward/backward scattering
and the scattering angle will affect the measured Z-average.
5.4 Determination of optimal sonication energy
Sonicate CNC suspensions with varying energies and measure the DLS Z-average and PI for each sample
as described in 5.3. Plot Z-average and PI against sonication energy.
Select the optimal sonication energy for production of a well-dispersed suspension from a region of the
curve where the measured Z-average and PI change slowly with increasing energy. An example plot is
shown in Annex A.
To ensure reproducibility, measure a minimum of three replicate, independently prepared samples
sonicated with the selected optimal sonication energy.
6 Sample preparation for microscopy
6.1 General considerations
There are a number of general considerations that apply to the preparation of CNCs deposited on a
suitable support for either AFM or TEM. The first consideration is the importance of ensuring that a
representative sample is used. When starting with dry CNCs, it is important to verify that the sample
is well-mixed prior to weighing a sub-sample for dispersion. It is recommended to prepare dispersions
from three sub-samples in order to confirm that the preparation of the dispersion by sonication is
reproducible. Sonicate each of the three samples with the required sonication energy and then measure
the DLS Z-average and PI as described in 5.3. Changes in Z-average of less than 5 % indicate reproducible
dispersion of the sample. Alternatively, the entire sonication curve can be measured for each of the sub-
samples.
A second consideration is the agglomeration of the CNCs in the initial suspension. Reduction (but not
complete removal) of aggregates and agglomerates in solution can be accomplished by sonication and
filtration.
The third consideration is the selection of an appropriate support or TEM grid and a deposition method
that minimizes agglomeration of particles, while maximizing the number of individual particles that
can be analyzed per image. The use of a positively charged support or grid is in principle useful for
immobilization of negatively charged CNCs. Further details for AFM and TEM are noted in 6.2 and 6.3,
respectively.
A final factor is the number of independently prepared samples that should be imaged and the number of
particles that must be analyzed. Imaging multiple samples will provide information on reproducibility
of the sample deposition process and its possible impact on the CNC size distribution. The number
of individual particles (n) analyzed for each sample must be sufficiently large that the parameters
which define the size distribution (e.g. mean and standard deviation for a normal distribution) can be
determined with the desired level of uncertainty. As a general guideline the uncertainty is inversely
proportional to the square root of n for normal distributions; an analysis of the effects of sample
size on measurement uncertainty for log normal distributions can be found in ISO 13322-1:2014,
[4]
Annex A . The number of particles required will increase with increasing polydispersity of the sample.
Recommended starting points in a number of studies range from 200 particles to 1 000 particles.
ISO 21363 recommends analysis of 500 particles as a starting point and this has been adopted for the
ILCs for AFM and TEM of CNCs that are summarized in Annexes C and D.
Although automation of AFM and TEM image analysis can be used successfully for a number of spherical
and high contrast nanomaterials (see ISO 21363 and references cited therein for TEM examples), there
are currently no reliable methods for automation of image analysis for CNCs.
Representative methods for sample deposition are outlined below.
NOTE Some optimization of sample concentrations and amounts can be required for specific CNC samples.
6.2 AFM sample preparation
Most AFM imaging of CNCs has employed mica as the support, typically coated with a thin layer of
[14],[18]
poly-L-lysine (PLL) . This surface coating is preferable to bare mica since electrostatic effects
help to immobilize the CNCs thereby minimizing particle agglomeration and possible artifacts due
to movement of particles during imaging. Other substrates have been used occasionally; see ISO/TR
[5]
19716 for additional details. The procedure below employs positively charged PLL coated mica and
uses spin coating for deposition. This deposition procedure provides more reproducible samples (area
to area particle density) than incubation methods and is designed to maximize the number of particles
[16],[19]
per image while minimizing agglomeration and aggregation .
Prepare a suspension of CNCs in water as described in Clause 5. Dilute 500-fold with deionized water.
Prepare a PLL-coated slide by adding an aliquot of 0,01 % mass fraction PLL solution to a freshly cleaved
mica substrate (e.g. 40 µl for 12 mm diameter mica and 200 ul for 2,54 cm × 2,54 cm mica). Place the
mica with PLL solution in a covered petri dish for 10 min. Rinse the mica substrate with deionized
water five times and dry in a nitrogen stream.
Pipette the freshly diluted CNC suspension onto the center of a freshly prepared PLL-mica substrate
that is mounted in the spin coater; volumes of 40 µl and 200 µl are adequate for 12 mm diameter and
2,54 cm × 2,54 cm mica, respectively. Ensure that the suspension covers most of the substrate area.
Spin the mica substrate at 4 000 rpm for 25 s with an acceleration rate of 2 000 rpm/s. Air dry the
sample and store in a desiccator under a positive pressure of nitrogen prior to imaging.
Some optimization (amount and concentration of CNC suspension, spin coating speed and time) of the
above procedure can be required, depending on the sample dispersion and aggregation level of the
initial sample.
NOTE Samples can also be prepared by incubating an aliquot of CNC suspension (≈ 80 µl of 0,001 % mass
fraction CNC for 2,54 cm × 2,54 cm mica) on PLL-coated mica for 2 min, washing 5 times with deionized water and
drying under nitrogen. Typically the level of agglomeration will be higher and the area-to-area reproducibility
[16],[20]
lower for samples prepared by incubation than for those prepared by spin coating .
6.3 TEM sample preparation
[14],[21]-[23]
Sample preparation for TEM has been described in several recent reviews . The following
procedure is typical of many literature studies and has been employed to characterize a reference
material and samples for an interlaboratory comparison (see Annex D).
Prepare a suspension of CNCs in water as described in Clause 5. Dilute the suspension ≈100-fold with
deionized water and vortex-mix for 5 s.
Plasma clean (2 min) a carbon film covered copper grid (e.g. 200 mesh, Ted Pella 01840-F). Deposit
10 µl of CNC suspension on the grid, leave for 4 min and then wick away excess liquid with a filter paper.
Wash the sample by adding one drop of deionized water to the grid and wicking with a filter paper after
several seconds.
Stain the sample by depositing 10 µl of filtered (0,22 µm PVDF filter) 2 % mass fraction uranyl acetate
solution on the grid and leaving for 4 min. Immerse the grid in deionized water, remove the sample and
air dry for at least 1 h prior to installation in the microscope.
NOTE After uranyl acetate staining the grid can be washed by adding one drop of deionized water and
wicking with filter paper, rather than immersion in water.
7 Atomic force microscopy
7.1 General
Atomic force microscopy is used to measure the PSD for length and height for CNCs. Lateral dimensions
derived from AFM images are influenced by tip-particle convolution. Due to the high aspect ratios
of CNCs, the effect of convolution is proportionally smaller for length. However, the magnitude of
the broadening due to tip-particle convolution is comparable to the CNC width and therefore has a
significant effect on width measurements. Measurement of width by AFM is not recommended unless
[24]
a correction for convolution effects is applied . Imaging conditions shall be optimized to ensure that
the minimum possible imaging force is used to prevent compression of the particles. Size measurements
shall only be derived from areas that have not previously been scanned.
7.2 Instrumentation and accessories
The following instruments and accessories can be used to image CNCs by atomic force microscopy and
measure the particle size distribution:
─ AFM capable of high-resolution imaging in contact and intermittent contact mode;
─ AFM probes for both contact and intermittent contact imaging in air;
─ either calibration grids or nanoparticle reference materials, or both;
─ image analysis software.
7.3 Microscope calibration
Dimensional calibration of the microscope shall be verified prior to CNC imaging unless the calibration
records indicate that this is not necessary. The frequency of microscope calibration depends on the
type of instrument and its stability, the purpose of the measurements and potential changes in ambient
operating conditions. Calibration, if necessary, shall be carried out according to the manufacturer’s
instructions. General guidance for calibration of height and lateral dimensions for AFM is provided in
[3] [25]
ISO 11952 and a more practical guide for users is currently under development . The use of multiple
standards that cover the appropriate x-y and z-scales for CNC imaging and that have certified values
and uncertainty and metrological traceability are preferred. Typical calibration standards include step
height standards (z-scale) and 2D lateral measurement standards that have equidistant structures with
defined features with a fixed spacing (x-y scale).
7.4 Data acquisition
Select an appropriate tip for intermittent contact mode imaging and install in the AFM. CNCs have been
imaged with cantilevers varying in spring constant from k ≈ 40 N/m to k < 10 N/m and give comparable
results provided that care is taken to minimize the imaging force.
Select initial scan parameters and tune the cantilever resonance.
Install the sample and engage the tip and adjust parameters for intermittent contact mode imaging.
Adjust scan rate, gains and setpoint as needed to obtain optimal trace and retrace tracking. Record
several large size images (5 μm × 5 μm or 10 μm × 10 μm) to verify the overall morphology and
homogeneity of the sample.
Prior to collecting images for analysis, image one or more sample areas (1 µm × 1 µm or smaller) with
multiple setpoint values in order to determine the minimum imaging force that can be used. Plots of
height for 10 or more individual CNCs as a function of the ratio between the amplitude setpoint (A )
sp
and the free amplitude (A ) can be used to determine the minimum imaging force that allows for stable
imaging and to estimate the uncertainty contribution in the height measurements due to variation of
applied force as a result of amplitude. Alternatively, plots of height versus applied force can be used.
Examples of both approaches are shown in Annex B.
Acquire a series of 1 µm × 1 µm AFM images with a minimum resolution of 512 pixels × 512 pixels,
0,8 Hz to 1,0 Hz scan rate, and Z-piezo range of 1 µm to 2 µm. Collect images in different regions close
to the centre of the substrate avoiding areas previously imaged. Collect a sufficient number of images to
provide the required number of individual CNCs for size measurement, considering the factors outlined
in 6.1. Typically, an average of ≈ 25 individual CNCs/image can be analyzed for 1 µm × 1 µm AFM images
using the sample preparation protocol provided in 6.2.
NOTE 1 The resolution is approximately 2 nm/pixel for 1 µm × 1 µm images with 512 pixels × 512 pixels.
Assuming a 1-pixel measurement error, this pixel size will give a relative uncertainty of approximately 1,3 % for
a 50 nm long CNC. For CNC suspensions with a large fraction of short (< 50 nm) particles, it can be desirable to
increase the resolution (although at the cost of added data acquisition time) by scanning smaller areas or using
1 024 pixels × 1 024 pixels.
NOTE 2 Optionally the cantilever can be calibrated by acquiring a thermal tune spectrum to determine the
[26]
resonance frequency and quality factor; the spring constant can be determined using the Sader method .
NOTE 3 The image quality can deteriorate after recording a number of images, either due to changes in tip
sharpness or contamination. In such cases it is necessary to use multiple tips to record a sufficient number of
images to analyze the required number of individual CNC particles.
[27]
The relative humidity has been reported to change the height of CNCs . It is recommended either to
record the relative humidity or to image in a humidity-controlled environment, or both.
7.5 Image analysis
Flatten images using a first-order polynomial fit using the AFM software, after excluding CNCs using
threshold masking. Save the flattened images for size analysis using appropriate software, such as
1) TM 2)
Gwyddion 2.35 , Scanning Probe Image Processor (SPIP by Image Metrology A/S) or microscope
software.
For each image, measure the length and height for all individual particles. Analyze adjacent particles
only if the separation between them is clearly established in the contact or near-contact areas. Exclude
aggregated particles, particles crossing or touching an edge of the image, particles <25 nm long,
particles crossing each other and particles with imaging artifacts.
Measure the particle length as the longest distance from one end of the CNC to the other. Use a standard
approach for measuring the particle height; for example, measure the maximum height along the long
axis used to measure the length. Ensure that random noise spikes are excluded when measuring the
maximum height.
Record height and length data for all particles and save the image with analyzed particles numbered,
which can be useful post-analysis if any anomalous data are detected.
An example of an AFM image analysis procedure using Gwyddion is provided in Annex C.
NOTE The identification of individual particles is challenging due to the irregular shape of some CNC
particles and the fact that the height can vary across the length of the particle.
8 Transmission electron microscopy
8.1 General
Transmission electron microscopy is used to measure the particle size distribution for CNC length and
width but does not provide information on the particle height. Adequate contrast requires staining,
[21]
typically with uranyl acetate; other staining methods have also been used .
1) Gwyddion 2.35 is the trade name of a product supplied by the Czech Metrology Institute. It is a free and open
source software, available at: http:// gwyddion .net/ .This information is given for the convenience of users of this
document and does not constitute an endorsement by ISO or IEC of the product named.
2) SPIP™ is an example of a suitable product available commercially. This information is given for the convenience
of users of this document and does not constitute an endorsement by ISO or IEC of this product.
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