1. ARTICLE IN PRESS
Ultramicroscopy 110 (2010) 751–757
Contents lists available at ScienceDirect
Ultramicroscopy
journal homepage: www.elsevier.com/locate/ultramic
Cryo-staining techniques in cryo-TEM studies of dispersed nanotubes
Eran Edri, Oren Regev n
Department of Chemical Engineering, Ben-Gurion University of the Negev, 84105 Beer Sheva, Israel
a r t i c l e in fo abstract
Article history: The combination of cryo-TEM and staining is employed for studying protein-enabled dispersion of
Received 20 August 2009 carbon nanotubes in aqueous solution. The same staining agent is used for both positive- and negative-
Received in revised form staining. We are able to image the adsorbed layer of protein or polysaccharide on the nanotube but not
1 March 2010
the individual molecule. The process is not artifact-free due to change in ionic strength of the solution.
Accepted 26 March 2010
However, our results are in line with other, not related, experimental techniques. The obtained
information could be used to update models suggested based on, e.g., scattering data.
Keywords: & 2010 Elsevier B.V. All rights reserved.
Staining
Positive
Negative
Cryo-TEM
Nanotube
Dispersion
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751
2. Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752
2.1. Materials and sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752
2.2. Staining procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752
2.3. Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753
3. Results and discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753
3.1. Contrast enhancement by negative staining (NS). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753
3.2. Contrast enhancement by positive staining (PS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754
3.3. Artifacts and interpretations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 756
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 756
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 756
1. Introduction is an advantageous quality. Efficient dispersion of nanotubes is
obtained by high frequency sonication in the presence of an
The wide applicative range of single walled carbon nanotubes amphiphilic dispersing agent (‘a dispersant’). The sonication
(SWNT) provokes a worldwide research effort on the paths exfoliates tubes from the bundle while the amphiphilic agent
towards their application [1–3]. Strong van-der Waals attraction covers their surface by adsorption [7,8]. However, not all SWNT
forces acting between single walled carbon nanotubes [4,5] are exfoliated [8], and the large bundles are usually removed by,
induce bundling and prevent SWNT dispersion in most organic e.g., centrifugation. The adsorbed amphiphilic molecule stabilizes
and aqueous media. Obtaining dispersed nanotubes is a desired the recovered SWNT in solution, by electrostatic repulsion, by
step in the course towards application of SWNT in, e.g., materials steric hindrance or by other mechanisms [9]. With the intention
engineering [3] or drug delivery [6], where their high aspect ratio of making carbon nanotubes (CNT) biocompatible and employable
as drug delivery agents, the use of biopolymers, such as proteins,
as dispersants has recently become popular [10–13]. However,
n
Corresponding author. Tel.: + 972 86472145; fax: + 972 86472916. due to lack of appropriate research tools a clear physico-chemical
E-mail address: oregev@bgu.ac.il (O. Regev). picture at supramolecular level has not yet been attained. Such
0304-3991/$ - see front matter & 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ultramic.2010.03.010

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752 E. Edri, O. Regev / Ultramicroscopy 110 (2010) 751–757
Fig. 1B at four times higher magnification, still with no observable
features over the nanotube. In most carbon-based materials
such as proteins, surfactants and polymers similar phenomenon
is reported: the individual molecules cannot be imaged by
cryo-TEM.
In this work two strategies are adopted to enhance the contrast
of the dispersant molecules: (1) electron density enhancement of
the objects’ environment (indirect contrast enhancement), i.e.,
negative staining, and (2) direct enhancement of the objects’
electron density, i.e., positive staining.
Neither staining [25] nor cryo-TEM [22] is new to the TEM
community. Yet, the combination of both has become popular
only in the last decade [26]. Major contributions come from the
Fig. 1. Cryo-TEM images (not stained) of (A) BSA- and (B) GA-dispersed SWNT.
molecular biology community. The cryo-negative staining (cryo-
Although the SWNTs are easily discerned (white arrows), BSA or GA molecules
cannot be identified without the help of a staining agent. The black arrow indicates NS) method was established by Adrian et al. [26] and has been
attached Ni and Y catalyst particles. used to study the (hydrated) structure of complex proteins [27–
29]. Proteins with molecular weight down to $ 120 kDa were
imaged [30]. However, a cryo-positive staining (cryo-PS) approach
picture is required in applications as drug delivery (for instance has not yet been reported to the best of our knowledge. In this
control of drug dosage, targeting), or in materials science (e.g. paper (cryo-) positive staining is obtained simply by reducing the
wetting of SWNT surface) [14]. In an effort to obtain a mechanistic concentration of the staining agent (ammonium molybdate, AM)
model at the molecular level of the dispersed system we propose from a saturated solution used for negative staining (16 wt%,
a combination of microscopic methods. Although not artifact-free 0.8 M) to 0.5–1 wt%. The two approaches, i.e. cryo-positive and
(on which we expand later), the methods withhold unique cryo-negative staining, are used to study the BSA-SWNT system at
advantages and reveal interesting properties with regard to two pH values (pH5 and pH10), representing two different BSA
CNT–dispersant interactions. conformations (normal and basic, respectively) [17]. We note that
Previously, the protein bovine serum albumin (BSA) was used either method is not free of artifacts induced by the high ionic
to disperse SWNT [15,16]. It was found that due to the pH- strength of the staining agent. Nonetheless, imaging at two
sensitive conformation of BSA [17] different SWNT recoveries extreme ionic strengths reveals similar results and corresponds
could be obtained [16]. Generally speaking, changing the BSA with previous reports [31] obtained by completely different
conformation from a bulky ‘heart shaped’ normal conformation (at experimental methods.
pH values 4–8) to more loose basic conformation (pH48) results
in reduced SWNT recoveries. Further expanding the BSA con-
formation at acidic pH values (expanded conformation) results in 2. Experiments
zero recovery of individual SWNTs expressed as demixing:
collapsed nanotubes and water. In this contribution we study 2.1. Materials and sample preparation
properties such as surface coverage and adsorbed protein layer
thickness (on dispersed SWNT) by transmission electron micro- As produced (AP)-SWNT prepared by arc-discharge method
scopy, since they were found to be important parameters in CNT were purchased from Carbolex (diameter $ 1.4 nm, catalyst Ni,
dispersion and stabilization [18]. and Y, purity 70%vol) and dispersed by BSA (Sigma-Aldrich A3803
Imaging of CNT is commonly conducted by various techniques and A7906, Cohn Fraction V, purity 498%) or gum arabic (Aldrich
such as AFM [19], SEM and TEM [20]. However, these techniques Acacia 26,077-0) in deionized water (18.2 MO cm). For BSA
are conducted on a dried sample, which does not represent the solutions the pH was calibrated with 1 M HCl and NaOH [16].
true state in solution, that is to say, in a dried state. Drying De-agglomeration of SWNT is carried out by sonication in an ice
artifacts result a misleading picture [21]. For example, the drying cold Bath-sonicator (Elma sonic model S10; 30 W, 37 kHz, Sonics
process significantly increases the concentrations and dehydrates & Materials Inc.) for $ 6 h followed by centrifugation ( Â 6240g for
the sample. The use of low temperature TEM (cryogenic-TEM or 30 min; Megafuge 1.0, Heraues) and extraction of $ 85% of the
cryo-TEM) bypasses the drying problem, as the imaging takes supernatant volume. All the solutions were stable for months.
place on a vitrified solution-phase sample [22]. However, as is Initial SWNT and dispersant concentrations are 2 and 4 mg mL À 1,
apparent in Fig. 1, while CNTs’ high electron density facilitates respectively, in all samples.
their imaging in TEM [23] the dispersant molecules (usually a
hydrocarbon-based material; here, BSA or the polysaccharide gum
arabic (GA)) have low electron density and cannot be imaged 2.2. Staining procedures
directly in the cryo-TEM. Previously, this stumbling block was
tackled by labeling the BSA molecules with gold nanoparticles A saturated solution of ammonium molybdate (AM, Fluka
[24]. Although the results were instructive, the method cannot be 09878) was prepared by dissolving 1.2–1.4 g of salt in 875 mL of
used to study a wider range of parameters, e.g. pH, due to water. 125 mL of 10 M NaOH was added to bring the pH to $ 4–7.
instability of the labeling agent (nanoparticles). After rigorous shaking, and leaving the solution to stand (for
Here, BSA-dispersed SWNT are imaged without the aid of a several minutes), the solution phase separates and a precipitate is
staining agent to enhance the image contrast. SWNT are easily topped with a clear solution of dissolved AM. Only the top phase
discerned (Fig. 1) in the cryo-TEM with a diameter of $ 1.4 nm. is used for staining, where the concentration is 16 wt% for
However, the dispersant molecules (BSA in this case) with negative staining [30].
Rg $2.7 nm are not observed in the TEM micrographs due to their For positive staining a 0.5–1 wt% AM solution was prepared by
low electron density (Fig. 1A), and subsequently low mass diluting the 16 wt% solution (corresponding to 0.8 M). For other
thickness contrast [23]. The polysaccharide GA with a diameter pH values, 1 M HCl or NaOH solutions were used for pH
larger than BSA (Rg $30 nm [37,38]) is shown for comparison in adjustments.

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2.3. Microscopy Table 1
Thickness of dispersant layer on dispersed SWNT.
TEM grids (300-mesh Cu Lacey substrate; Ted Pella, Ltd.) were SWNT imaged diameter (nm)
used throughout the study. The samples were examined using an
FEI Tecnai 12 G2 TWIN TEM operated at 120 kV at 3 mm under- Dispersant/method Unstaineda NS (r) SANS Rgb Ref.
focus. Images were recorded with a Gatan charge-coupled device
Gum arabic o2 11(3) 17 7 1 30 710 [37,38]
camera (model 794) and analyzed by Digital Micrograph software,
BSA pH5 o2 20(6) – 2.67 [17]
Version 3.1. Staining of the dispersed SWNT solutions was BSA pH10 o2 6(2) – 4 2.67 –
performed as described before [26,30] or through a modified
procedure (i.e. instead of floating the grid on the staining solution a
From cryo-TEM.
b
drop, the staining is applied ‘on-the-grid’) [32,33]. The samples Free in solution.
were prepared either in a controlled environment vitrification
system (CEVS) or in a vitrification robot system (VitrobotTM) at
498% humidity. Cryo-TEM samples were examined at low dose imaging individual BSA molecule could be below the reported
mode below À 175 1C, held by a cryo-TEM holder, Gatan model resolution.
626. Image acquisition, analysis and measurements were con- Imaging of individual BSA molecule has been indeed unattain-
ducted by Digital Micrograph software (Version 3.1). able; however, we claim that clustering of BSA molecules around
the SWNT surface results in increased imaged SWNT diameter,
due to depletion of the staining agent from the SWNT-BSA
complex. The diameter of unstained SWNT is $ 1.4 nm (Fig. 1),
3. Results and discussion
and in agreement with the manufacturer specifications corre-
sponding to individual SWNT [36]. However, the diameter of
We have previously found that for BSA-dispersed SWNT,
stained SWNT in Fig. 2 is 20 nm (standard deviation, s ¼6;
higher SWNT recoveries are obtained at pH values attributed to
number of samples, N¼60). These results indicate a substantial
bulkier BSA conformations [16,34]. Parameters such as surface
increase in diameter from 1.4 to 20 nm. We argue that the thicker
coverage are assumed to be important in the stabilization, e.g.
‘adsorbed layer’ yields the above increased diameter.
higher surface coverage increases the energetic barrier to
Using different dispersants (vide infra), the diameter of the
coagulation [9,18,35].
imaged SWNT-dispersant complexes is measured, and found to be
In this study we use cryo-TEM imaging, enhanced by staining
directly related (see Table 1).
procedures to explore the surface coverage, focusing on the
Gum Arabic consists of two main components: a highly
morphology of the dispersing BSA layer over the SWNT.
branched polysaccharide, which comprises the majority of a
commercial GA, and an arabinogalactan–protein complex (GAGP),
3.1. Contrast enhancement by negative staining (NS) which comprises the minority ( $10 wt%) [37]. This complex
(GAGP) was earlier shown to be the element responsible for
BSA-dispersed SWNT at pH5 are shown in Fig. 1. The contrast dispersing SWNT in aqueous media [38,39]. GA-dispersed SWNT
reversal, characteristic of negative staining, is shown in Fig. 2. imaged by cryo-NS technique are shown in Fig. 3 with a
Namely, SWNT are seen in white, while the solution phase is dark. calculation example of the average diameter of the imaged
This is in contrast to Fig. 1. SWNT from cryo-NS micrographs.
The contrast reversal shown in Fig. 2 indicates that the The diameter of the imaged SWNT is evaluated from the
presence of staining agent increases the electron density of the contrast profiles along a given line, as shown in Fig. 3. In
background, while the object itself depletes the staining agent comparison to the results for BSA (Fig. 2), here the dispersant is
from its vicinity [27,28]. Still, individual BSA molecules cannot be GA, and the average diameter of SWNT is found to be 11 nm
observed in this image. In a previous review [30], it was (s ¼3 nm, N¼25). See Table 1 for comparison. The conformation
mentioned that while molecular weight could be used as one of GAGP complex surrounding and stabilizing SWNT in solution
indicator of object size and observability, the smallest protein was studied as well [38]. It was found by scattering techniques
molecule imaged by cryo-NS (up to date) has a molecular weight that the thickness of the polysaccharide layer is $17 nm, much
of $ 120 kDa, twice the molecular weight of BSA. In other words, larger than the thickness of the adsorbed layer we find in Fig. 3
and in the analysis ((1 1 À1)/2–5 nm, Table 1). However, the
effect of ionic strength on the layer thickness (which is an artifact
of the NS technique, vide infra) has not been studied yet.
The Rg of polyelectrolytes such as GA is expected to be
suppressed by increased ionic strength [40]. Indeed, similar effect
has recently been reported for GA-dispersed SWNT in water in the
presence of latex particles [41]. At low salt concentrations (that is,
large Rg), the latex particles were depleted from the SWNT vicinity
due to the extended GA chains. However, upon increasing the salt
content (and the ionic strength) the depletion range was
significantly reduced (more than 20 nm reduction), indicating a
‘collapse’ of the GA chain on the SWNT surface. We suggest that
the same effect takes place in the staining procedure of GA in our
results, and this explains the decrease in GA layer thickness from
Fig. 2. (A) NS-cryo-TEM micrographs of BSA-dispersed SWNT at pH5. The contrast 17 [38] to 5 nm in our study.
reversal effect is demonstrated. Here, SWNTs (indicated by arrows) are seen as Nonetheless, there is a difference in imaged diameter between
white objects because their surroundings have higher electron density. (B) The
SWNT crosses the carbon layer of the grid, which results in contrast reversal back
samples of the same nanotubes but with different dispersants
to ‘regular’ contrast, because the difference here in electron density is changed. (DBSA 4DGA, Table 1). This could support the argument that the
[AM] ¼16 wt%. The arrow head indicates Ni and Y catalyst particles. increase in the complex diameter (i.e. dispersant-SWNT) between

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Fig. 3. NS-cryo-TEM micrographs of gum arabic-dispersed SWNT solution. Contrast profiles along lines (i) and (ii) are plotted. An averaged diameter is calculated. From a
collection of such images the average diameter of a GA-dispersed SWNT is 11 nm (s ¼ 3 nm, N ¼25). [AM] ¼ 16 wt%.
SWNT in unstained (Fig. 1) and stained samples (Figs. 2 and 3) is
due to the adsorbed dispersants. However, more work is required
to establish this argument (see ‘artifacts and interpretations’ in
Section 3.3).
We now return to the BSA-dispersed SWNT case with the
intention of employing cryo-NS to study this system. In Fig. 4
cryo-NS image of such a system at pH10 is presented.
In Fig. 4 we also find a contrast reversal (as observed in Figs. 2
and 3), indicating negative staining. However, in contrast to our
results for pH5 (Fig. 2), here the protein layer is inhomogeneous:
in addition to a thin protein layer, small protein aggregates (or
patches) can be observed on the SWNT surface (indicated by
single white arrows). It results in smaller imaged SWNT diameter
Fig. 4. NS-cryo-TEM micrographs of BSA-dispersed SWNT at pH10. The contrast
(D¼6 nm, s ¼2 nm, N¼80) compared to pH5 (D¼20 nm, s ¼6 enhancement induced by the staining agent is evident ([AM] ¼ 16 wt%). In this case
nm). The SWNT imaged diameter distributions dispersed by the the SWNTs are covered by an inhomogeneous layer. The SWNT diameter is 6 nm
two BSA structures (at pH5 and pH10) are plotted in Fig. 5. (s ¼2 nm, N ¼ 80). White arrows in (A) indicate SWNT covered by thin protein
We conclude that cryo-NS is a viable route to enhance the layer and protein patches. The white double-arrow in (B) indicates a small SWNT
bundle; black arrows indicate Ni and Y catalyst particles. The big black particles in
contrast of SWNT dispersions and allows detection of some
the bottom left of A are surface contamination. Bar ¼200 nm.
features of the adsorbed layer. It was found that for BSA at pH5,
the imaged nanotube diameter is 20 nm thick, and a homogenous
layer of proteins covers the NT surface. When GA is used to situation, in which the electron density of the background area is
disperse SWNT, a homogenous layer is found as well, but the NT enhanced by heavy metal salt so that the specimen appears
thickness is found to be 11 nm. The reduced thickness is lighter in contrast to the darkly stained background [42]. Usually,
attributed to the different properties (e.g. Rg, Mw) of GA in NS and PS are attained by different staining agents, but here, we
comparison to BSA. We note that a collapse of the GA chains on found that the NS agent can be used for PS, if a much lower
the SWNT surface is due to the ionic strength, induced by the concentration is used. Interestingly, at low stain concentration,
staining agent. The homogeneity of the layer is disturbed when the staining agent appears to attach specifically to the object of
BSA at pH10 is used as the dispersant. Here, a diameter of 6 nm interest instead of being homogenously distributed in the
was evaluated and protein aggregates are randomly distributed solution. The origin of this phenomenon is not yet clear.
along the SWNT; the inhomogeneity could be a result of, e.g., In Fig. 6 BSA-dispersed SWNT at pH5 is stained as in Fig. 2 but
inter-protein electrostatic repulsion or (more likely) the different at a much reduced staining agent concentration (0.5 wt% instead
conformation of BSA. The results are summarized in Table 1. of 16 wt%, corresponding to 0.025 and 0.8 M, respectively, see
Experiment section).
Unlike unstained (Fig. 1) or heavily (NS) stained samples
3.2. Contrast enhancement by positive staining (PS) (Fig. 2), in Fig. 6a ‘labeling’ effect of BSA molecules is observed, i.e.,
positive staining. This labeling facilitates the localization of the
We now turn to discuss the second strategy adopted, that is, BSA molecules on the SWNT surface. In general, a thick and dense
direct contrast enhancement by positive staining. In positive layer of stained protein molecules is found on the NT surface
staining, the heavy metal salts attach to the macromolecules (i.e. (white arrows in Fig. 6 and illustrated in Fig. 6B). However,
the objects) in the specimen to increase their electron density and occasionally bare SWNT can be found, although this is an
the mass thickness contrast. This differs from the negative staining exception rather than the common observation (Fig. 6B).

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E. Edri, O. Regev / Ultramicroscopy 110 (2010) 751–757 755
Fig. 5. Imaged diameter histograms of SWNT dispersed by BSA at pH5 (A) and pH10 (B). The continuous line is a Gaussian fit to the results. The average image diameters
are 20 76 nm (pH5) and 6 72 nm (pH10).
Fig. 6. (A, B) Positively stained-cryo-TEM images of BSA-dispersed SWNT at pH5 ([AM] ¼ 0.5 wt%). The reduced staining agent concentration results in a ‘labeling’ of BSA
molecules. This allows their localization on the SWNT surface. A dense and thick layer of BSA molecules seems to cover the SWNT surface (white arrows). Black arrows
indicate an artifact: surface contamination. (C) An illustrated positively stained BSA-dispersed SWNT at pH5 (not to scale).
Fig. 7. (A, B) Cryo-PS images of BSA-dispersed SWNT at pH10. The adsorbed BSA molecules form a thin and dilute layer on the SWNT surface as is illustrated in (C) and
indicated by white arrows.
We applied the same procedure for BSA-dispersed SWNT at was found that at pH5 a dense core layer covers the SWNT surface
pH10 (Fig. 7). (Fig. 6). At pH10, on the other hand, BSA covers the SWNT in a thin
In Fig. 7 we find SWNT covered with a layer of BSA molecules and dilute layer.
as in Fig. 6 (pH5). However, the BSA layer at pH10 is thinner and These differences can be attributed to both the electric charge
more dilute than at pH5. This finding is in line with our negative and the structure of BSA. At pH5 the BSA is close to the isoelectric
staining results (Figs. 2 and 4; also Table 1). At pH10, the BSA point (IEP $ 5.1) and has a bulky structure [17], while at pH10 the
molecules are directly attached to the SWNT in a more dilute BSA has a more ‘loose’ structure and high electric charge
layer than at pH5, as is illustrated in Fig. 7C. ( $ 40 mV) [17,43]. The thick diameter of SWNT observed using
In summary, we have shown that the staining agent concen- NS at pH5 (Fig. 2, d ¼20 nm) goes hand in hand with the thick
tration can be used to ‘switch’ between an NS effect at high layer observed in PS imaging (Fig. 6). Similarly, at pH10, a thin
concentration to a ‘labeling’ effect or PS at low concentration. It layer is observed in both imaging techniques (PS and NS). The

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756 E. Edri, O. Regev / Ultramicroscopy 110 (2010) 751–757
agreement between the PS and NS results could suggest that the layer of BSA forms on the SWNT surface. This results in a smaller
ionic strength induced by the staining (an artifact) has a small imaged diameter of SWNT (d ¼6 nm, NS), and a thin BSA layer was
effect on the morphology of adsorbed BSA. observed (PS). These differences are attributed to protein electro-
static charge and conformation.
3.3. Artifacts and interpretations
References
We note that the staining procedure dramatically increases the
ionic strength of the solution (for NS [AM] $ 0.8 M) [30], which
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