DEPARTMENT OF HEALTH AND HUMAN SERVICES

Centers for Disease Control and Prevention

National Institute for Occupational Safety and Health

NIOSH Manual of Analytical Methods (NMAM), 5th Edition

 

 

 

Analysis of Carbon Nanotubes 

and Nanofibers on Mixed 

Cellulose Ester Filters by 

Transmission Electron 

Microscopy 

by M. Eileen Birch, Chen Wang, Joseph E. Fernback, H. Amy Feng, Quinn T. Birch, and Alan K. Dozier, 

NIOSH

 

 

 

1 Introduction CN- 2

2 Sample Preparation CN- 2

3 TEM Analysis and Counting Method CN- 6

4 Conclusions CN-18

5 References CN-18
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Analysis of Carbon Nanotubes and Nanofibers on MCE Filters by TEM 

1 Introduction

A transmission electron microscopy (TEM) method for the analysis of filter samples of 

carbon nanotubes and nanofibers (CNT and CNF) is described in this Chapter. The 

approach is a modification to NIOSH NMAM 7402, asbestos by TEM [NIOSH 1994], and 

was applied to personal breathing zone (PBZ) and area samples collected at CNT/CNF 

survey sites [Birch et al. 2011; Dahm et al. 2015]. Procedures specific to asbestos fiber 

identification and counting were eliminated from 7402. In contrast to asbestos, CNT 

materials used commercially are typically in the form of complex agglomerates, with 

highly entangled, curved tubes. This curvature can sometimes make even individual 

fibers difficult to size. As such, a modified method that considers these structural 

differences was needed for particle classification. 

TEM provides visualization of airborne CNT and CNF particles, indicating their size,

shape, and agglomeration state [Birch et al. 2016]. These materials have distinct features 

(e.g., graphene layers, cylindrical structures, and hollow cores) that distinguish them 

from other types of particles. Given the considerable differences between CNT/CNF 

particles and asbestos fibers, particle counting methods necessarily differ. In the case of 

CNT/CNF, all particles, agglomerates as well as single fibers, are counted as a CNT/CNF

‘structure.’ Unlike Method 7402, no minimum size cutoffs have been used to date 

because of a lack of knowledge on lung deposition and toxicological endpoints. 

Based on the number of CNT/CNF structures counted, and the collected air volume, a 

CNT/CNF concentration (i.e., structures/cm3) can be calculated. For samples collected at 

the survey sites, a significant positive correlation between structure counts and 

elemental carbon (EC) data was found, but there was considerable scatter in the data 

[Dahm et al. 2013, Dahm et al. 2015]. Given the method limitations discussed herein, the 

calculated values for structure counts are considered semi-quantitative indicators of air 

concentration.

2 Sample preparation 

For the field surveys on CNT/CNF, open-face, 25-mm, three-piece cassettes were used 

for air sampling, approximating the inhalable fraction [Dahm et al. 2013, Dahm et al. 

2015]. As specified in NMAM 7402, air samples were collected on mixed cellulose ester 

(MCE) filters having a nominal pore size of 0.8 μm (7402 specifies 0.45 to 1.2 μm), with 

sampling pumps operated at 5 L/min (0.5 to 16 L/min specified in 7402). Polycarbonate 

(PC) membrane filters also may be acceptable, but this media was not used in the field 

studies, and the filter preparation method differs from that described herein. 
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Analysis of Carbon Nanotubes and Nanofibers on MCE Filters by TEM 

The sampling period depends on the air concentration, and it is important to avoid filter 

overload. Some trial and error may be necessary. In general, if a filter deposit is clearly 

visible, the sample is likely overloaded. An estimate of structure counts may still be 

possible, but overloading results in particle deposition on previously deposited material 

and may preclude an accurate structure count. 

Three TEM grids per field sample, and one grid each for field and media blanks (when 

provided), were prepared using procedures outlined in NMAM 7402, with the following 

modifications: 

 A scalpel is used to remove a wedge-shaped portion from the filter. Do not use a cork 

borer to remove filter sections. 

 Only one sample wedge should be placed on each slide. Do not affix the section to the 

slide with any type or adhesive, as specified in 7402. (Note: After carbon coating the 

wedge and removing small portions for transfer to TEM grids, the remaining portion 

can be secured [e.g., with tape] to the slide for long term storage, if desired.)

 The “hot block” technique [Baron and Pickford 1986] is used to clear (collapse) the 

filter wedge. Other methods also may be suitable.

A description of the procedures used for sample and grid preparation is provided in the 

following three sections. 

a. Filter 

Carefully open the sampling cassette with a cassette opener, with the particle 

collection side facing upwards (Figure 1, step 1). Remove about one fourth of the 25-

mm MCE filter with a clean scalpel. Place the wedge, particle side up, on a frosted 

glass microscope slide labelled with the sequence and sample numbers (Figure 1, 

step 2). Include a media (filter) blank with each set of slides to be coated. Use the 

“hot block” technique [Baron and Pickford 1986] to clear (collapse) the filter wedge 

(Figure 1, step 3). After clearing the filter, place the slides in a carbon coater to apply 

a thin carbon film (Figure 1, step 4); the filter surface should be evenly coated. After 

coating the filter wedges, the samples are ready for transfer to TEM grids. A Jaffe 

wick washer [Jaffe 1948] is used for final processing of the sample grids. Preparation 

of the washer and transfer of the sample sections to grids therein, for final 

processing, are described in the next two sections. 
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Analysis of Carbon Nanotubes and Nanofibers on MCE Filters by TEM 

Figure 1. Filter preparation steps prior to loading sample portions on TEM grids: (1) 

remove 25-mm MCE filter from air sampling cassette; (2) remove about one fourth of 

the filter and place the filter wedge on a glass slide; (3) clear the MCE filter with 

acetone vapor in a hot block; (4) apply carbon coating; (5) cut section from coated 

filter and divide into three portions, one per grid. Each filter portion has particles 

sandwiched between the carbon film and collapsed filter. 

 

 

b. Jaffe Wick washer

Prepare a Jaffe wick washer using a petri dish (15-mm depth recommended, but 

other sizes may be suitable), stainless steel screen (20 mesh), and lens tissue (Figure

2, a) or a stainless steel screen disk (40 mesh) (Figure 2, b) as a wicking substrate 

(one disk per sample). Bend two edges of the 20-mesh screen to form a platform 

(Figure 2) that fits inside the petri dish and is no higher than half the dish height. The 

platform supports the tissue (Figure 2, a) or stainless disk (Figure 2, b) onto which 

the sample grids are placed and keeps them above the solvent level during sample 

processing (Figure 2). 
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Analysis of Carbon Nanotubes and Nanofibers on MCE Filters by TEM 

Figure 2. Sample transfer and final processing: place a piece of lens tissue (a) or a 

stainless steel screen disk (b) on a stainless screen platform in a Jaffe wick washer 

filled with dimethylformamide (DMF). Sides of the platform are bent to keep sample 

grids above the solvent level. Depending on size, multiple disks can be used to hold 

grids. 

Use a pencil to label the lens tissue with the sample identifiers and place the tissue 

on the 20-mesh screen platform (Figure 2, a). The samples placed on the tissue 

should be the same set that was coated (together) in the carbon coater. Place the 

petri dish in a fume hood and carefully add dimethylformamide (DMF) to the dish 

until the lens tissue is saturated or screen disks are wetted, but ensure that no 

puddles form on surfaces of the tissue or disk. A combination of DMF and acetone 

also may be used for filter dissolution. (Note!: DMF [CAS 68-12-2] is flammable and 

hazardous in case of skin contact, eye contact, ingestion, and inhalation. Ensure 

adequate ventilation, proper personal protective equipment, and engineering 

controls in handling and storage. Consult SDS prior to use.) In a row, place three TEM 

grids per sample in the designated areas of the tissue or on a disk (Figure 2). If 

screen disks are used, place three TEM grids per sample on each (Figure 2, b). 

Depending on disk size, multiple disks can be used. Use one TEM grid each for the 

blanks (field and media). 

c. Sample transfer and final processing 

Using a scalpel and tweezers, cut and remove three small sections of the coated filter 

wedge as shown in Figure 1, step 5, starting at the center area (bottom left of wedge) 

of the filter and moving outward, across the bottom, towards the filter edge. The area 

of the cut sections should be similar and enough to cover a grid. Carbon side up, 

carefully place the excised filter sections on the three grids. If lens tissue is used, 
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Analysis of Carbon Nanotubes and Nanofibers on MCE Filters by TEM 

place the section from the center of the filter (Figure1, Step 5, A) on the leftmost grid, 

the middle section (Figure1, Step 5, B) on the center grid, and the outermost section 

(Figure1, Step 5, C) on the rightmost grid. The locations are labeled as shown in 

Figure 1, step 5. When all filter sections have been transferred to the grids, slowly 

add more solvent to raise the level as high as possible without disturbing the sample 

preparations. Cover the petri dish, if lens tissue is used, and elevate one side of the 

dish by placing a microscope slide under it (allowing drops of condensed solvent to 

form near the edge of the cover rather than in the center, where they can drip on the 

grid preparation). To dissolve the filter, the tissue method requires the grids to sit 

overnight, while the screen disk method requires only about 30 minutes. If screen 

disks are used, leave the petri dish uncovered, in a fume hood or other well 

ventilated area. Once the residual MCE polymer is completely dissolved in the 

solvent, only particles embedded in the carbon film remain on the grid, as illustrated 

in Figure 3. Ensure that the processed grids are solvent free prior to the TEM 

analysis.

Figure 3. Removal of residual MCE polymer from carbon-coated sample by 

dimethylformamide (DMF). After polymer removal, only particles embedded in the 

carbon film remain on the grid. 

3

a. Sample quality

.TEM analysis and counting method 

Align the microscope for TEM applications following the manufacturer’s instructions. 

Load the sample into a TEM grid holder and insert the holder into the TEM column. 

First, examine the grid at low magnification (500-1,000X) to determine the filter 

loading and preparation quality. In general, to be acceptable for analysis, the grid 

should have at least 75% intact grid openings and a particle loading less than about 

25% (area coverage). 
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Analysis of Carbon Nanotubes and Nanofibers on MCE Filters by TEM 

If these criteria are met, randomly select at least four openings (with intact carbon 

film) from four quadrants of the grid and estimate the number of particles per 

opening to determine the distribution variability. If the particle loading is uneven or 

heavy, the sample might not be useful. Discuss any uneven distributions or loading 

issues with the requestor of the analysis. 

b. Definition of a CNT/CNF structure 

Count all particles that meet the following definition of a CNT structure:

 Any CNT or CNF individual fiber (Figure 4) with an aspect ratio (i.e., length to 

width) greater than 3:1. There are no minimum length or diameter cutoffs. If a 

fiber structure has the morphological characteristics of CNT/CNF and meets the 

3:1 aspect ratio criterion, it is counted. In our work, isolated CNT fiber or bundle 

structures were relatively rare. CNT agglomerates were far more common. 

Figure 4. Examples of CNT fiber structures with various lengths and widths. 
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Analysis of Carbon Nanotubes and Nanofibers on MCE Filters by TEM 

 Any particle agglomerate with associated CNT/CNF: There are many types of 

structures that fit this criterion. Some structures, classified as matrix particles, 

contain relatively few CNT/CNF, being mainly amorphous carbon or other 

components. In contrast, structures that consist mainly of CNT/CNF are classified 

as cluster particles. 

In this chapter, ‘agglomerate’ refers to both cluster (Figure 5) and matrix (Figure 6) 

particles. Although matrix particles have associated CNT, they are often observed as 

fiber structures protruding from the particle edge. The structure of the CNT, 

presumably within the matrix, cannot be determined in the TEM analysis. Another 

classification problem occurs when agglomerates have cluster and matrix regions 

(Figure 7) of similar size. Structure counts are discussed in the next section. 

 Figure 5. Examples of CNT/CNF clusters: (a)-(f) collected from workplaces. 
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Analysis of Carbon Nanotubes and Nanofibers on MCE Filters by TEM 

Figure 6. Examples of CNT matrix particles: (a)-(d) collected from workplaces. 

There are specific definitions of CNT and CNF; however, field samples have contained 

complex mixtures of structures that often do not fit these narrow definitions. For 

example, products described as CNT can contain CNF and vice versa, and a variety of 

irregularly shaped, nanoscale carbon particles have been observed. In practice, if the 

analytical request specifies a ‘CNT’ or ‘CNF’ facility, these terms are used in the 

analytical report, along with the corresponding TEM images. In addition, images and 

information on other types of particles are provided.
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Analysis of Carbon Nanotubes and Nanofibers on MCE Filters by TEM 

c. Structure counts 

The counting method for individual fibrous structures generally follows that for 

asbestos, as in NMAM 7402 and EPA’s AHERA (Asbestos Hazard Emergency 

Response Act) method [40 CFR 763; NIOSH 1994]. That is, each fiber with an aspect 

ratio > 3:1 is counted and the diameter and length are recorded. At a minimum, the 

diameter and length ranges are recorded, and averages are determined, if 

appropriate. This procedure is negotiable with the requestor. 

As mentioned, in the field samples, isolated CNT fibers were rare relative to particle 

agglomerates. The classification of CNT/CNF agglomerates is accomplished by 

considering both particle size and structure. The size bin depends on the maximum 

crosswise dimension. At least one dimension, the maximum crosswise length, is 

recorded for each structure. Other characteristics also can be examined to meet 

investigator needs. Some agglomerates, especially larger ones, may contain a nonCNT/CNF component. In these instances, if the agglomerate looks to be mostly 

CNT/CNF, it is classified as a cluster. If non-CNT/CNF components appear more 

abundant, the matrix classification is used. However, as discussed, some 

agglomerates may have roughly equal cluster and matrix portions. For these, a 

matrix category is usually assigned but this classification is somewhat arbitrary. 

Given the difficulties of particle classification, it is important to provide 

representative images of all particles in the sample. 

If the particle loading and distribution are acceptable, an initial particle count can be 

performed at low magnification. Select the lowest magnification that shows the 

majority of the particulate of interest: 5,000-15,000X is usually sufficient. Smaller 

particles may not be visible at low magnification, but the purpose of this step is to 

assess the number of grid openings likely to be counted. If the sample composition is 

known (e.g., a laboratory generated CNT or CNF sample), an initial count can be 

made to count larger (micrometer sized) CNT (CNF) particles, which dominate the 

particle mass. After examination of the sample at low magnification, a switch to high 

magnification (e.g., 40,000X) is made to image particles/features not observable at 

low magnification. The magnification should be adjusted as required for counting 

and confirming the presence of CNT. 

As discussed, for the analysis, three 3-mm copper TEM grids are usually prepared 

from each sample. A total of 40 openings (stop counting at 100 structures) are 

examined at high magnification (13-15 openings for each grid). The number of grid 

openings counted per grid should be approximately equal for the three grid 

preparations, and the openings counted on each grid should be selected as randomly 

as possible. Only grid openings that have the carbon film intact should be counted. 

Any particles containing CNT/CNF are counted as CNT or CNF structures, which can 

range from single fibers (Figure 4) to structures of varying size and composition 

(Figs. 5-9). Counted CNT/CNF structures are categorized by size-bins based on the 
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Analysis of Carbon Nanotubes and Nanofibers on MCE Filters by TEM 

maximum crosswise dimension. In our studies [Dahm et al. 2015; Birch et al. 2016], 

most of the CNT agglomerates had a roughly spherical shape (i.e., the longest 

dimension was no more than twice the length of the orthogonal dimension). 

Therefore, the maximum crosswise dimension was used to categorize structure size. 

The counted structures were placed into five discrete size-bins, based on healthrelevant size fractions and observations from field studies [Birch 2011; Dahm et al. 

2013]. The structure categories also included a separate bin for single fibers. Size 

bins for the CNT/CNF structures have the following maximum crosswise dimensions: 

< 1 μm, 1-2 μm, 2-5 μm, 5-10 μm, and > 10 μm. This sizing scheme was used to 

analyze CNT/CNF samples collected during field surveys [Dahm et al. 2015]. A 

statistical analysis of the TEM counts for a subset of the field samples is provided in 

the following section. 

Figure 7. CNT particle with both matrix (right side) and cluster (left) features. 

Structure categorized as a CNT matrix particle. 
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Analysis of Carbon Nanotubes and Nanofibers on MCE Filters by TEM 

Figure 8. CNT particles in personal breathing zone samples collected from various 

sites: (a) MWCNT fiber (polymer composite site), b) SWCNT agglomerate from 

aerosolized, aqueous suspension (electronics site), c) MWCNT agglomerate 

(composites/thermoplastics site), and d) MWCNT agglomerate (primary 

manufacturer of CNT). 
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Analysis of Carbon Nanotubes and Nanofibers on MCE Filters by TEM 

Figure 9. Examples of CNT particles embedded in polymer composite: (a)-(d). 

d. Statistics 

A total of 3000 observations (structures/opening) from 75 field samples with light 

loading (<5 structures per grid opening) were analyzed. The mean, standard 

deviation, and confidence intervals (95% confidence level) were calculated. Factors 

considered included grid (1-3), opening (1-14), and loading level (1-3). Grids were 

counted in the order of grid 1, 2, and then 3, going from the center to the edge of the 

filter (wedge portion). From Figure 10 and Table 1, the means and variabilities of 

counts from different loading levels follow the same trend. The variability of grid 1 

(from center area of filter) was slightly higher than the other 2 grids. The variability 

becomes smaller when the loading level is increased. The box plot in Figure 11 shows 

the statistics of counts by openings. The means and variabilities of counts from 

opening 1 and 2 were slightly higher than counts from opening 12, 13 and 14. 

Because opening 1 was the first opening counted, and 14 the last, it is possible that 
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Analysis of Carbon Nanotubes and Nanofibers on MCE Filters by TEM 

the increased variability is due to higher analyst error during the initial and final 

counts. The test of half data (including openings 1-7) shows similar behavior as that 

for all data (Figure 12). Based on the results for this sample set, counts from one or 

two grids, with 7 openings per grid, may be adequate if the deposit is even. Larger 

data sets from samples with different loadings and collected from various sites are 

required to develop a statistical model for further analysis. 

Figure 10. Boxplot of CNT counts by categorized loading (1: mean ≤ 1, 2: 1 < mean ≤ 

2, and 3: mean > 2) from three grids. The trend of mean counts from grid 1 to 3 is 

consistent for different loading levels. Mean count of grid 1 is slightly higher than 

those of grid 2 and 3. 

 
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Analysis of Carbon Nanotubes and Nanofibers on MCE Filters by TEM 

 

Table 1. Statistics of sample counts obtained from different grids and loading levels 

(M=Mean, SD=Standard Deviation, CI=Confidence Intervals).

Loadinga

 Grid 1b Grid 2 b Grid 3 b

M (SD) 95% CI M (SD) 95% CI M (SD) 95% CI

Level 1 0.51 (0.84) [0.44, 0.58] 0.44 (0.64) [0.38, 0.49] 0.43 (0.66) [0.37, 0.49] 

Level 2 1.45 (1.12) [1.29, 1.60] 1.33 (0.91) [1.21, 1.46] 1.35 (0.90) [1.23, 1.48] 

Level 3 2.65 (1.32) [2.50, 2.80] 2.64 (1.10) [2.51, 2.77] 2.55 (1.07) [2.43, 2.68] 

All 1.33 (1.41) [1.24, 1.41] 1.26 (1.28) [1.18, 1.34] 1.24 (1.25) [1.16, 1.32] 

a Loading levels are defined by M, where Level 1 (M ≤ 1), Level 2 (1 < M ≤ 2) and Level 3 (M > 2). 

b Grids collected from different locations on a filter wedge (center to edge).

 

Figure 11. Boxplot of CNT counts by openings (1-14). The variability in openings 1 

and 2 significantly higher than that of openings 13 and 14. The means of openings 1, 2 

and 3 were also higher than the means of openings 12, 13, and 14. 
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Analysis of Carbon Nanotubes and Nanofibers on MCE Filters by TEM 

Figure 12. Boxplot of CNT counts by grids (1-3) from all data (counts from openings1-

14) and half data (counts from openings 1-7). 

 
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Analysis of Carbon Nanotubes and Nanofibers on MCE Filters by TEM 

e. Limit of detection (LOD)

For the field samples [Dahm et al. 2015], the ‘no structures detected’ TEM samples

were those with counts below the LOD, defined as 1 CNT or CNF structure per

sample. The corresponding unit of structures per cm3 LOD (air concentration) was

then calculated (by the requestor) based on the total air volume sampled. 

f. CNT/CNF polymer composites

Dusts generated from CNT/CNF polymer composites that have been mechanically

worked (e.g., drilled, cut, sanded, blasted, etc.) contain particles with CNT/CNF

embedded in the polymer matrix. Our analyses of polymer composite dusts have 

shown little evidence of individual fiber release from the composite matrix, but 

respirable sized matrix particles with protruding CNT/CNF have been observed 

(Figure 9). These findings are consistent with other reports [Bello et al. 2012; 

Kingston et al. 2014] on potential CNT/CNF release from polymer matrices. Particle 

counts for these types of samples also can be performed, though only a limited 

number of surveys have been conducted. Because the CNT/CNF are embedded in a 

polymer matrix, the toxicity of these dusts may differ substantially from dusts with

free (unbound) agglomerates. Nevertheless, representative images of the particles

and information on particle shape and size can be documented.

g. Limitations

The composition of CNT powders varies substantially, especially those produced or

used in workplaces. These materials contain varying amounts of amorphous carbon 

and residual catalyst metals, and there are many types of structures, usually in

agglomerate form. As mentioned, in the case of composite dusts, CNT are embedded

in a polymer matrix, which complicates their measurement. This chapter concerns

the analysis of CNT powders.

In the case of ‘matrix’ particles, TEM images show mainly dark regions, with varying

amounts of CNT observed at the particle’s edge. The composition of material in the 

dark regions is unknown. For example, it may be mainly CNT, amorphous carbon, or

a combination of both. Thus, based on overall envelope size, a particle consisting

mainly of amorphous carbon is counted in the same bin as an agglomerate composed

mainly of CNT. Both matrix and cluster (structures with mainly CNT) type particles 

were common in field samples. A different counting method [Chen et al. 2012] was 

applied to a specific type of MWCNT (Mitsui MWCNT-7) used in an animal inhalation 

study but this particular product, having a more fibrous structure and more single 

fibers, differs from the CNT materials manufactured/used by U.S. companies. Images

(Figs. 4-9) of the different particle types found in our field surveys make obvious the 

difficulty in developing a standardized TEM structure count method for these 

materials.
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Analysis of Carbon Nanotubes and Nanofibers on MCE Filters by TEM 

4. Conclusions

The complexity and variety of structures makes CNT/CNF particle counting a challenge.

Unlike elemental carbon or other less selective measures, TEM can confirm the presence 

of CNT. However, with respect to quantification, it is important to recognize the 

limitations of a counting method. Because of the heterogeneity of CNT powders,

particles in the same size bin can vary greatly in their properties. Complex structures of

varying mass, density, shape, and composition (e.g., amorphous carbon, residual

catalyst) can fall into the same size bin. And dissimilar particles (e.g., cluster and matrix)

in a given bin may have different toxicological effects. Thus, even for a given material,

particle envelope size alone may not be an adequate risk indicator. Given the 

heterogeneity and variety of the CNT/CNF products to which workers are exposed, it is

important to apply several methods to assess worker exposure, through inhalation and

other routes.

Disclaimer 

Mention of any company or product does not constitute endorsement by NIOSH. In addition, 

citations to websites external to NIOSH do not constitute NIOSH endorsement of the

sponsoring organizations or their programs or products. Furthermore, NIOSH is not

responsible for the content of these websites. All web addresses referenced in this 

document were accessible as of the publication date.

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