Scanning Electron Microscopy

Zhan-Ting Li and Xin Zhao

Chinese Academy of Sciences, Shanghai, China

1 Introduction
2 Supramolecular Gels
3 Supramolecular Liquid Crystals
4 Supramolecular Polymers
5 Vesicles
6 Nanotubes, -Pores, -Ribbons, -Rods, and -Spheres
7 Supramolecular Chirality
8 Conclusion
References

1
2
5
6
7
8
11
12
13

INTRODUCTION

The scanning electron microscope (SEM) is an electron

microscope that uses electron beams to scan a sample and
form an image of the sample surface.13 The electrons
interact with the atoms of the sample to produce various
signals, which include secondary electrons, primary backscattered electrons, characteristic X-rays, Auger electrons,
cathodoluminescence, and specimen current and transmitted
electrons, providing information about the samples surface topography, composition, and electrical conductivity.
Although all these signals are present in SEM, only the
first three signals, especially the secondary electrons, are
commonly used in commercial instruments, which can produce very high-resolution images of a sample surface. The
first SEM image was obtained by Knoll in 1935,4 while
the first commercial instrument was developed in 1965 by
the Cambridge Instrument Company, named Stereoscan.

Since then, it has found wide applications in the biological,

materials, and chemical researches.5
There are many advantages of using SEM in the detection
of a sample: (i) It is relatively cheap and widely available.
(ii) Owing to the very narrow electron beam, it has a
large depth of field, which allows a large amount of
the sample to be in focus at one time and yields a
characteristic three-dimensional appearance. (iii) SEM can
produce images of very high resolution. Greater than
500 000 times magnification can be achieved, which means
that several nanometers in size can be revealed. (iv) The
magnification rate of SEM can be easily set successively.
Therefore, for a sample, a low magnification can be used
to obtain the whole picture, while the high magnification
may be set to observe the detailed structures. (v) SEM has
a large depth of focus, which yields a characteristic threedimensional image that is useful in obtaining information
on the surface structure of a sample.
Since the secondary electrons are of low energy, their
trajectories can be easily affected by electromagnetic fields.
As a result, a charge buildup on the surface of the sample
can change the path of the secondary electrons. To avoid
this, the surface of the sample must be conductive. Thus,
the electroconductive sample can be measured directly. For
non-electroconductive samples, shadowing methods have
been developed to coat the samples with a thin layer
of metal. Thus, the preparation of the sample for SEM
measurement is, in general, simple.
The conventional SEM measurement carried out in a
vacuum has disadvantages and may also result in artifacts.
Moreover, the coating can obscure the fine surface structure
details of some non-electroconductive samples. In the
1980s, the environmental scanning electron microscope
(ESEM) was developed,6 which permits the imaging of
wet systems with no prior specimen preparation. Since the
sample environment can be dynamically altered, hydration

Supramolecular Chemistry: From Molecules to Nanomaterials, Online 2012 John Wiley & Sons, Ltd.
This article is 2012 John Wiley & Sons, Ltd.
This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470661345.smc044

Techniques

and dehydration processes can be followed as they happen

in the sample chamber. Also in the 1980s, cryo (cold)
SEM became commercially available,7 allowing samples
to be preserved and recorded at below ambient temperature
(typically between 100 and 175 C). Another improved
technique, the field emission (FE) SEM can produce clearer,
less electrostatically distorted images with spatial resolution
down to 1.5 nm.3
Supramolecular chemistry focuses on the chemistry
beyond the molecules and the bottom-up building of
ordered assembled systems from molecular subunits or
components. The binding process of a synthetic receptor
for a single molecular or ionic species is difficult to observe
directly by any electron microscopic techniques. However,
an ocean of assembled systems exhibit ordered morphologies on the nano- to microscales. With more and more interest focusing on the complicated systems based on molecular
recognition and self-assembly, the structural information
within this range of scales is becoming increasingly important. SEM has become a powerful and routine analytical
technique for collecting such information. In most studies,
SEM is used together with other microscopic techniques
such as AFM, TEM, and STM, which guarantees a complete understanding of the morphology of the sample.

SUPRAMOLECULAR GELS

Gels are a colloidal phase state in which a small amount of

a solid-like network immobilizes the bulk flow of a larger
amount of liquid phase, which may be considered as a special supramolecular system. Both polymeric and molecular
building blocks can form the three-dimensional networks.
Typically, the networks consist of fibrillar structures. For
molecular gels, these fibers are formed by the self-assembly
of molecular blocks by complementary noncovalent interactions. The formation of the gels is usually judged simply by
visual observations (vial inversion test). However, their fine
structures can be investigated only by using various microscopes. The size of the fibers of gels generally ranges from
several nanometers to a few tens of micrometers, which is
well covered by SEM. Therefore, SEM is widely used for
observing the morphology of the fibers.
An early example concerns organogels of compound
1 with aliphatic solvents, reported by Terech et al.8
The small-angle X-ray and neutron scattering techniques
demonstrated that the gel networks resulted from the entanglement of long, solid-like rigid fibers, while SEM was used
to characterize the morphologies of the xerogels (Figure 1).
Owing to collapses of the brittle structures in the 3D network during the shrinking step caused by evaporation of
the liquid, SEM focused on the general shapes and morphologies rather than on the absolute quantities such as the

OH
1

10 m

(a)

200 m

(d)

2 m

(b)

20 m

(e)

20 m

(c)

10 m

(f)

Figure 1 SEM images of fractured xerogels of 1 in cyclohexane: (a) random repartition of very long and rigid fibers;
(b) detail showing the presence of thinner and more flexible
fibrils; (c) collapsed network of the phase-separated solid of
1/heptane gel, showing fibers emanating from a central point;
(d) thick bundles of fibers; (e and f) high orientation degree of
fibers. (Reproduced from Ref. 8. American Chemical Society,
1998.)

diameters, lengths, or topologies of the fibers. Figure 1(a)

shows the formation of very long and rigid fibers of variable
thickness (0.10.2 m) which are entangled in a porous
matrix. Figure 1(b) reveals that thinner, more flexible fibrils (0.05 m) are also present, while Figure 1(c) exhibits
a special morphology where fibers are emanating from a
central point. When stored for a long time, the gel can
evolve toward a solidliquid-phase separation, which is
also confirmed by SEM (Figure 1df), because the micrographs show that the phase-separated solid is made of fibers
associated in locally aligned bunches. This observation also
confirms that the true equilibrium state of such very long
and rigid fibers is the separated biphasic system, which
has a higher degree of orientation than the xerogel through
the formation of oriented bundles by collapses of separated
fibers of the xerogel.
The magnification rate of SEM can be set successively.
This is not necessary for observing the morphology of
supramolecular gels. However, it does provide access to
recording images at different magnifications. For example,

Supramolecular Chemistry: From Molecules to Nanomaterials, Online 2012 John Wiley & Sons, Ltd.
This article is 2012 John Wiley & Sons, Ltd.
This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470661345.smc044

Scanning electron microscopy

Et
O
Et
O

Et
O

Et
N

N
N

O
17

Et
O
Et

0.5 m

(a)

5 m

(c)

OCH3

17

CH3O

O
Et

O
Et

200 nm

(b)

2 m

(d)

Figure 2 Cryo-SEM images of the gel of 2 (8 mM, water/THF

mixture (80 : 20)) at different magnifications. (a) Nanoporous
structure; inset: vial inversion test. (b) Image at higher magnification showing the three-dimensional network of nanofibers.
The smallest fibers are about 6.1 nm in diameter (yellow arrow).
(c) Whirls with diameters of 1015 m. (d) Directional arrangement of fibers within a microstream in the gel. (Reproduced
from Ref. 9. American Chemical Society, 2009.)

Rybtchinski reported that compound 2 gelated water/THF

(tetrahydrofuran) mixtures due to the hydrophobically
driven aromatic stacking.9 Cryo-SEM images revealed an
interconnected porous structure of the gel (Figure 2a),
in which nanofibers created a three-dimensional network
(Figure 2b). It also showed that the smallest fibers had
widths of about 6.1 nm, and their actual width (subtracting the thickness of the metal layer used for gel imaging 0.6 nm) was about 5.5 nm, which is similar to the
size observed in the case of the solution-phase network.
Furthermore, thicker fibers of various diameters were also
observed. The images at lower magnifications revealed the
presence of whirls and streams (Figure 2c and d). These

anisotropic regions are several micrometers large, and

demonstrate a certain long-range order of the supramolecular fibers within the gel. On the basis of these observations
and also molecular modeling, the authors proposed a hierarchically assembling mechanism for the gelation process.
At the first level, the hydrophobic and stacking interactions caused face-to-face stacking of compound 2 into small
aggregates of 810 molecules. The stacked molecules were
shifted with respect to each other, due to the steric bulk of
the aliphatic chains. At the second level, the hydrophobic effect was the driving force for further aggregation,
which was driven by the aliphatic side chains that, as a
result of aromatic stacking, formed a substantial hydrophobic domain. Their interaction resulted in fibers with distinct
segmentation. Then, the fibers assembled into entangled
bundles, while branching out of these bundle accounted for
the creation of junctions.
The superfine structures of organogels can also be studied by using SEM. For example, Zhang et al. found
that compound 3 gelated cyclohexane through the formation of an entangled network of thin solid fibers with
diameters of about 4080 nm and lengths up to tens
of microns (Figure 3a).10 The structure of the gel was
changed when 7,7,8,8-tetracyanoquinodimethane (TCNQ)
was added because it formed a charge-transfer complex
with the tetrathiafulvalene unit of 3. Subsequently, the
entangled thin fibers of the gel was transformed into a tubelike structure with diameters of about 2060 nm, which was
clearly shown by using SEM (Figure 3b).
For rigid and partially rigid gelators, their fibrous structures can be readily observed by using SEM. However,
SEM focuses on the three-dimensional surface information.
For proposing a rational assembling pattern, in many studies, other techniques have to be used.11, 12 Lu et al. have
reported that compound 4 gelated hydrocarbons (Figure 4).
SEM demonstrated that the molecules in the gel phase
were self-assembled into one-dimensional nanofibers with a
25100 nm width, which further cross-linked to form threedimensional networks (Figure 4a).11 The small-angle X-ray
diffraction of the xerogel illustrated that the molecules
were packed into the lamellar structure (Figure 4b), which,
together with semiempirical quantum calculation, supported
that the molecules adopted a one-dimensional molecular
packing pattern to self-assemble into thin fibers (Figure 4d),
which were further wound or laced to give the wide
nanofibers (Figure 4a).
Most of the reported gelators are single molecules. Twoor multicomponent systems can also gelate liquids if they
are able to assemble three-dimensional networks through
the formation of similar fibrous structures, which can also
be characterized by using SEM.13, 14 For example, Smith
et al. found that both compound 5 and its mixture with
diamine 6 gelated toluene.13 The SEM images of their

Supramolecular Chemistry: From Molecules to Nanomaterials, Online 2012 John Wiley & Sons, Ltd.
This article is 2012 John Wiley & Sons, Ltd.
This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470661345.smc044

Techniques
O
S

N
H

N
H

100nm
1 m

NONE

SEI

300 V 4.10 1 m WD 6.7 nm

(a)

SEI 300 V 10.000 1 m WD 6.7 nm

NONE

(b)

Figure 3 SEM image of (a) the cyclohexane gel of 3 and (b) that of the charge-transfer complex of 3 with TCNQ. The inset clearly
shows the tube-like superfine structures of the complex gel. (Reproduced from Ref. 10. American Chemical Society, 2005.)

O
O

OH HO

O
O

O
O

4
(c)

1 m

(a)

Intensity (a.u.)

3.01 nm
3.01 nm
(d)

1.49 nm

2
(b)

Cu-KR (2q /)

8
(e)

Figure 4 (a) SEM image and (b) SAXD pattern of xerogel 4 obtained from cyclohexane. (c) Molecular structure of 4. (d) Unimolecular
stacking with a length of 3.01 nm in the gel. (e) Proposed molecular packing model along the growing direction of the gel fiber.
(Reproduced from Ref. 11. American Chemical Society, 2009.)

xerogels revealed that both samples formed fibrous structures (Figure 5) and the addition of the second component dramatically changed the nanoscale morphology of
the assembled superstructure. On the basis of the observations, they proposed that the formation of the complex made
the network more interpenetrate and the nanoscale fibers
narrower because of the presence of more connecting

points, that is, the acid-amino electrostatic interactions of

their complex.
Gels usually remain in their phase state for some time
and eventually collapse to amorphous precipitates. Tang
et al. found that the hydrogel of the 1 : 2 mixture of 7
and 8 could spontaneously transfer into macroscopic crystals during storage.15 One related issue was to observe

Supramolecular Chemistry: From Molecules to Nanomaterials, Online 2012 John Wiley & Sons, Ltd.
This article is 2012 John Wiley & Sons, Ltd.
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DOI: 10.1002/9780470661345.smc044

Scanning electron microscopy

O

O
N
H

C11H23

N
H

O
C11H23

N
5 H

OH NH(CH2)12NH2

HO

(a)

C11H23
O

C11H23
N
H 5

(b)

Figure 5 SEM images of 5 in the absence ((a) scale bar =

20 m) or presence ((b) scale bar = 2 m) of 6. (Reproduced from
Ref. 13. Royal Society of Chemistry, 2005.)

the transferring process from the gels to the crystals. As

it is difficult to use conventional SEM to obtain highresolution images of naturally hydrated gels under high
vacuum, ESEM was applied to study the transition. The
samples were observed by using ESEM at constant vapor
pressures of 560 Pa (for hydrated samples) and 410 Pa (for
dehydrated samples) (Figure 6). On storing for 1 h, the sample displayed an entangled fibrillar network filled with water
(Figure 6a), which is typical of ordinary gels. After 12 h,
fibers and crystals were present simultaneously in the sample (Figure 6b). After 36 h, only prismatic crystals remained
(Figure 6c). The transition was clearer for dehydrated samples (Figure 6a c ) and could also be observed using SEM,
but only for the xerogel.

3
H O

N
8

100 m

(a)

a'

8
N

OH

100 m

Crystal

Fiber

50 m

(b)

(c)

50 m

b'

100 m

c'

SUPRAMOLECULAR LIQUID
CRYSTALS

OH

O
O

O
O
H

100 m

Figure 6 ESEM images of hydrated samples after storing the

gel (formed at 5 wt%, 7 : 8 = 1 : 2) for (a) 1 h, (b) 12 h, and
(c) 36 h. a , b , and c represent the dehydrated samples after 1 h,
12 h, and 36 h storage, respectively. (Reproduced from Ref. 15.
American Chemical Society, 2008.)

Supramolecular liquid crystals are a state of matter that

consists of two or more components and exhibit properties between those of a conventional liquid and those
of a solid crystal. The molecular components bind each
other to form a liquid crystalline mesophase, which is
typically characterized by polarized optical microscopy
(POM), X-ray diffraction (XRD), and differential scanning calorimetry (DSC). SEM is useful for studying their
assembling structures on the surface and helps establish
their assembling patterns. For example, Yagai et al. have
reported that POM, XRD, and DSC experiments on the
films revealed that complexes of 9 and 10a and b formed
identical fan-shape textures characteristic of columnar liquid crystalline phases.16 The complexes might form a 1 : 1
Hamilton-type complex or extended supramolecular polymers. Dynamic light scattering (DLS) provided evidence
that the Hamilton-type complexes were formed in millimolar concentrations. Optical microscopy (OM), POM,
and SEM images were further obtained for their solutions in cyclohexane (Figure 7). OM and POM images
(Figure 7ad) showed that the fibers that were in parallel
to either the polarizer or the analyzer (those indicated by
arrows) were not visible under crossed-polarizer condition,
indicating the uniaxial nature of the fibers. The FE-SEM
image illustrated that the fibers were composed of bundled
thinner fibers with diameters less than 100 nm (Figure 7e),
and the cross-sectional image showed that the narrower
fibers possessed a solid interior (Figure 7f). Because the
fibers gave XRD peaks almost identical to those of their
films, the SEM results indicated that the fibers consisted of
hexagonally packed columns of disk-like 1 : 1 complexes,
and thus supported the formation of Hamilton-type binding
pattern.

Supramolecular Chemistry: From Molecules to Nanomaterials, Online 2012 John Wiley & Sons, Ltd.
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DOI: 10.1002/9780470661345.smc044

Techniques

O
HN

NR2
NH

RO

H
N
(CH2)n

H
N

N N N
N N
H
H
9
10a: n = 5 10b: n = 7 NR2
R2N
R = n -C12H25
n N
R2N N N
N NR2
H
H
O
N N
N N
H
H
N N
N
N
H
H
O
O
RO
OR
9
10
(i) Hamilton-type complexes
NR2

OR

Ar
N NR2
N
H
H
9
O
N
N
N
N
O
O
H
H
N
N
H
H
N
N
N
N
H
N
H
10
HN
H
O
10
O
N N
N N
O
H
H
R2N N N
N N NR2
9
R2N
Ar
Ar
8
R2N

R2N

Ar
N

(ii) Extended supramolecular polymers

100 m

100 mm
(a)

(b)

A
P
(c)

100 m

(d)

5 m
(e)

4
P

100 m

The difference in the thin film morphology exhibited

on the SEM images can also be used to reveal the effect
of the molecular structures on the self-assembling properties. Asha et al. have reported that compound 11ad
produced a liquid crystalline phase.17 SEM images were
used to reveal the effect of the functional group ester versus amide and the flexible alkyl terminal chains on the
self-assembling properties (Figure 8). The image of 11a
(Figure 8a) showed one-dimensional rod-like supramolecular structures with diameters of 0.51.0 m and lengths of
several micrometers. The rods tended to aggregate into huge
rods, as shown in the inset. The image of 11b (Figure 8b),
which showed the maximum aggregation tendency in the
solution, gave supramolecular organization of rods stacked
several micrometers long. In regions where isolated rods
were identified, the thinnest rods had widths of about
150 nm and lengths of a few tens of micrometers, leading to aspect ratios (length over width) of magnitude 100.
This long aspect ratio compared to that of the unsubstituted 11a was attributed to the longer molecular length
of 11b with respect to 11a. The SEM of 11c (Figure 8c)
showed one-dimensional ball-like supramolecular structures
of 34 m diameter that were interconnected by nanometerscale rods (indicated by arrows), which was ascribed to
the bulky tridodecyl substitution at the terminal benzene
rings. The image of ester derivative 11d (Figure 8d) displayed a leaf-like pattern, 1.83 m in diameter and several micrometers in length, which is distinctly different
from the rod- or ball-like aggregates formed by the amide
series. It was proposed that the absence of an additional
building force such as the hydrogen bonding in the amide
series resulted in such a different morphology, because
11d had only interaction to aid in its self-assembly
process.

1 m
(f)

Figure 7 OM (a, b) and POM images (c, d) of nanofibers of

910a (a, c) and 910b (b, d) grown from cyclohexane (5.0 mM),
and SEM images of nanofibers of 910b (e, f). (Reproduced from
Ref. 16. American Chemical Society, 2007.)

SUPRAMOLECULAR POLYMERS

A supramolecular polymer is a polymer whose monomeric

repeating units are held together by noncovalent bonds. For
main-chain supramolecular polymers, the linear backbones
may form ordered structures. If the size of these assembled architectures falls into the range covered by SEM,
then SEM can be used to investigate their morphology on
the surface, which provides useful information for establishing the assembling patterns. Haino et al. reported that
compounds 12 and 13 formed supramolecular polymers
in solution through the calix[5]areneC60 stacking interactions.18 The size and morphology of the composite on
the surface were confirmed by the SEM image of their 1 : 1
solution (Figure 9a and b). The thicker entwined fibers had
lengths of more than 100 m and widths of 250500 nm
(Figure 9b), indicating that ditopic host 13 iteratively bound

Supramolecular Chemistry: From Molecules to Nanomaterials, Online 2012 John Wiley & Sons, Ltd.
This article is 2012 John Wiley & Sons, Ltd.
This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470661345.smc044

Scanning electron microscopy

O
11a: X =

O
11b: X =

N
H

N
H

O
11c: X =

N
H

OC12H25
O

OC12H25

11d: X =

O
OC12H25

OC12H25
OC12H25

7 kV

5,000 5 m 2690 RRLSEM

(a)

15 kV

6,000 2 m

10 kV

5,000

2670 RRLSEM

(b)

10 kV

5,000

5 m 2680 RRLSEM

(c)

5 m 2683 RRLSEM

(d)

Figure 8 Scanning electron micrograph (SEM) images of self-assembled (a) 11a, (b) 11b, (c) 11c, and (d) 11d drop-cast from toluene.
(Reproduced from Ref. 17. American Chemical Society, 2008.)

to dumbbell fullerene 12 to create a two-dimensional

nanonetwork.
Yagai et al. have reported that compounds 14 and 15
formed supramolecular polymers through intermolecular
triple hydrogen bonding interactions between the melamine
and cyanuric acid functionalities.19 In nonpolar solvents
such as methylcyclohexane, the perylene bisimide unit
in 14 stacked strongly to form filamentous precipitates.
FE-SEM revealed that the filaments were composed of
intertwined thinner fibrils (Figure 10a and b), while a
magnified image showed that the fibrils had ribbon-like
morphology with widths of about 100 nm (Figure 10c),
which has been attributed to the ordered stacking of the
perylene bisimide unit. In contrast, SEM images revealed
that a quadruple hydrogen-bonding-driven supramolecular
polymer with no aromatic unit in the backbone forms
cotton-like structures.20

5 VESICLES
The formation of vesicles in solution can be characterized
by DLS. To get more insight into their structures and assembling mechanisms, their surface morphology is usually also
studied by using a combination of microscopic methods.
Zhao et al. have reported that compound 16a and b formed
supramolecular polymers that were stabilized by hydrazidebased quadruple hydrogen bonding motifs.21 In decalin, the
supramolecular polymers further self-assembled into vesicular structures. SEM images clearly showed the formation
of spherical entities of average diameters of about 0.6 and
1.0 m, as shown in Figure 11(a) and (b), respectively. The
vesicles may exhibit defects, which may be regarded as an
evidence for their hollowness.22 For 16a and b, the fluorescence and TEM micrographs (Figure 11c and d) further
supported their hollow feature, because obvious luminance
differences and membranes were observed.

Supramolecular Chemistry: From Molecules to Nanomaterials, Online 2012 John Wiley & Sons, Ltd.
This article is 2012 John Wiley & Sons, Ltd.
This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470661345.smc044

Techniques

NR2

OC12H25
H
N

O
N
H
12

O
C12H25O

RHN

O
O

13

NH

X
CH3

X=
OH HO
OH

OH

H
N

O
C12H25

O
N

C12H25
O

(a)

000045

10 kV 500.000

0.5 m

N
H

H
N

N
N

H
NR
N

NR2

000063

(b)

Figure 9 SEM images of the cast-film of the mixture of 12 and

13 (1 : 1) on a glass plate. (Reproduced from Ref. 18. American
Chemical Society, 2005.)

Most observed vesicles on the surface are flattened

because the evaporation of the encapsulated solvents causes
the ball to collapse. When vesicles are very stable, they may
retain their three-dimensional shape on the surface, which
can be observed by SEM.22, 23 Kim et al. have reported
that the donoracceptor complex of 17 and 18 could be
encapsulated into the cavity of cucurbit[8]uril (CB[8]).23
The resulting ternary complex 19 formed large vesicles in
water, as evidenced by SEM and TEM images (Figure 12).
The SEM image showed large spheres with a diameter
ranging from 20 nm to 1.2 m and the typical vesicle size
lied between 400 and 950 nm. The vesicles maintained the
spherical shape, indicating their robust stability. In contrast,
the TEM image did not exhibit a comparably clear threedimensional contrast.

Supramolecular polymer

500 m

10 kV 500

NHR

NR2

O
N

O
N
H

NH
N
H

NR2

O
RN
H

OH

C12H25

15

N
C12H25O
CH3
CH3
CH3

H
N

OC12H25

N
C12H25O

Bu

R = C8H17
O

OC12H25

Et
14: R =

N
H
O

O
X

N
N

NANOTUBES, -PORES, -RIBBONS,

-RODS, AND -SPHERES

The supramolecular self-assembly of organic nanotubes and

related structures has received increasing interest since the
discovery of carbon nanotubes in 1991. SEM is a powerful technique for direct observation of their formation. For

10 m

(a)

1 m

(b)

1 m

(c)

Figure 10 FE-SEM of the filamentous precipitates of the

supramolecular polymer of 14 and 15 formed from methylcyclohexane (0.3 mM). The length of the bar across the ribbon in
(c) is 100 nm. (Reproduced from Ref. 19. American Chemical
Society, 2007.)

example, Bo et al. have reported that compound 20 selfassembled into nanotubes and layered sheets, which were
driven by stacking and hydrogen bonding between
the amide units.24 The materials were prepared by heating its suspension in THF to reflux until all the solids
were completely dissolved and then allowing it to cool
gradually to room temperature. SEM study of its air-dried
suspension showed the formation of fibril assemblies with
a high aspect ratio (Figure 13a). Their open-ended feature
clearly revealed the tubular structure (Figure 13b). TEM
images were also utilized to support the presence of the

Supramolecular Chemistry: From Molecules to Nanomaterials, Online 2012 John Wiley & Sons, Ltd.
This article is 2012 John Wiley & Sons, Ltd.
This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470661345.smc044

Scanning electron microscopy

OR
H
N
HN

RO
H
N
NH

O
HN

NH

C11H23

HN
N
H
OR

C11H23
20

O
O

NH
N
H
RO

O
16a: R = CH2CH(CH3)2
16b: R = n -C8H17

10 m

(a)

5 kV 7,000 2 m

02/ MAR/09

(a)

100 nm

(b)

10 kV 16,000 1 m

(b)

500 nm

(c)

200 nm

(d)

2.6 nm
10 m

50 nm

(c)

(d)

Figure 11 SEM images of the vesicles of (a) 16a (1.0 mM) and
(b) 16b (1.0 mM); (c) fluorescence micrograph of the vesicle of
16b (20 mM); and (d) TEM image of the vesicle of 16b (0.4 mM)
in decalin. (Reproduced from Ref. 21. Elsevier, 2010.)
+
C16H33 N

+
N C16H33

+
HO

18

O
CB[8] =

N CH2
H
N

N
H
N

C16H33

+
N

+
N

(f)

Figure 13 Morphology of 20. (a) SEM image of a sample

prepared by dropping its THF suspension (0.1 mg ml1 ) onto a
silicon substrate followed by air drying and coating with Pt;
(b) high-magnification SEM image with an open-ended cross
section; (c) TEM image; (d) high-magnification TEM image; (e
and f) high-magnification SEM images: internal and external
screw ends of the self-assembled tubes.24

OH

17

100 nm

100 nm

(e)

C16H33

O
HO
=

500 nm

(a)

OH
19
(Ternary complex)

1 m

2 m

(b)

Figure 12 (a) SEM and (b) TEM images of ternary complex

19. Samples (6.9 104 M) were negatively stained with uranyl
acetate (2 wt% in water) for observation by TEM.23

nanotubes (Figure 13c and d). Magnified SEM images further revealed the rolled-up style of the nanotubes with a

scrolling structure (Figure 13e), which was further confirmed by the terminal types of tubes (Figure 13f). SEM
images were also obtained for samples at different concentrations, which revealed that nanotubes were formed when
the concentration was low and transformed to layered sheets
at high concentrations. On the basis of the observations, the
authors proposed a hierarchical self-assembling mechanism;
that is, the one-layer nanosheets were first formed, which
might further roll up to generate the nanotubes or stack to
give layered sheets.
Jiang et al. have utilized SEM to study the morphology of the aggregates of porphyrin 21.25 The samples were
prepared by drop-casting a 6 mg ml1 chloroform solution
onto the surface of SiO2 substrate or quartz. It is worthy to note that the obtained films were annealed in a
chloroform-vapor-saturated desiccator. Only after annealing, the molecules self-assemble into long microtubes. The

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This article is 2012 John Wiley & Sons, Ltd.
This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470661345.smc044

10

Techniques
C10H21
N

NH

CH3COS(CH2)5O

C10H21

C10H21
N

N
O(CH2)5SCOCH3

M
N

22a: M = 2H
22b: M = Zn

HN
(A)

21

(B)

C10H21
a

50 nm

1 m

1 m
(C)

(D)

50 nm
50 nm
1 m
e

Figure 14 SEM images of microtube dendrites of 21 from

chloroform-vapor annealing. (Reproduced from Ref. 25. American Chemical Society, 2010.)

outer diameter of the microtubes ranges from 0.12 to

6.9 m, and the wall thickness is about 50 nm (Figure 14).
The microtubes also displayed uniform orientation, suggesting greater intermolecular order. Most importantly,
the images showed that the submicrometer-sized tubes
were often linked together to form Y-junctions, which in
turn constructed more complicated junctions, that is, the
dendrites with submicrometer-sized tube branches. These
submicrometer-sized tube dendrites might further aggregate to form larger dendrites, the submicrometer-sized tubes
of which spread out from its root and acted as a branch.
The assembling mechanism was thus proposed based on
the SEM observations. Since the microtube dendrites had a
curved 2D wall with thickness of 50 nm, they were considered to be formed by the rolling up of the higher ordered
multilayered crystalline film through porphyrin stacking,
which was stabilized by the interdigitation of the alkyl
chains.
There are many different forms of nanomaterials, which
can be assembled from single or two or more different components. Depending on the self-assembling conditions, one building block system may generate different
types of assembled entities. Jiang et al. reported that compound 22a and b aggregated into nanoribbons, -spheres,
or -rods from different solvents,26 as evidenced by SEM
(Figure 15). The image of 22a showed that its chloroform

100 nm

(E)

18 nm

(F)

10 nm

100 nm

100 nm

Figure 15 SEM images of nanostructures formed by 22a and

b. (A) Air bubbles by 22a in CHCl3 . (B) 3D networks by 22b in
CHCl3 . (C) Hollow spheres by 22a in CH3 OH. (D) Rods by 22b
in CH3 OH. (E) Ribbons by 22a in n-hexane. (e) Zoom-in image
of the rectangle part in E. (F) Hollow spheres by 22b in n-hexane.
(f) Zoom-in image of the rectangle part in (F). (Reproduced from
Ref. 26. American Chemical Society, 2008.)

solution formed a two-dimensionally ordered array of

air bubbles with highly monodispersed pores of about
500 nm in diameter (Figure 15A), reflecting the weakness
of its intermolecular interaction. In contrast, the chloroform solution of zinc porphyrin 22b led to the formation of three-dimensional network structures (Figure 15B),
which supported the significant intermolecular interaction
owing to the ZnO(=C) coordination bond. When injecting a small volume of their chloroform solutions into
CH3 OH, metal-free porphyrin 22a self-assembled into hollow spheres (Figure 15C), which was confirmed by the
two broken spheres, while the self-assembly of 22b led
to the formation of rods (Figure 15D) also as a result of
the above ZnO coordination. When n-hexane was used
as the medium, SEM revealed a reverse morphology. For

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This article is 2012 John Wiley & Sons, Ltd.
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DOI: 10.1002/9780470661345.smc044

Scanning electron microscopy

N
R
CH3
N
N

Zn

23

24a: R = H
24b: R = t -Bu

300 nm
1 m

2.5 m

300 nm

(a)

(b)

3 m

300 nm

500 nm

2 m

Number

7.82 2.07 m

(e)

SUPRAMOLECULAR CHIRALITY

(d)
60

500 nm

and b in the mixtures of DMF and acetonitrile.27 It was

revealed that all the samples formed nanotubes or nanorods,
but their sizes and structural characteristics were very
different. The zinc porphyrin itself produced tubes with
a large hollow hole (Figure 16a). In the presence of the
fullerenes, the hollow holes were completely closed to give
rods (Figure 16be), indicating that the fullerenes were
effectively encapsulated within the porphyrin tube. SEM
also helped to produce a detailed distribution of length and
diameter for the rods, as shown in Figure 15(F) for the rods
of2324b. The average sizes of the rods of the complexes
were estimated to be 15, 20, 60, and 80 nm, respectively.
The larger sizes of the rods of 24a and b suggested that an
encapsulation process of the fullerenes by the tube of 23
occurred after injecting. Similar to that of23 (Figure 16a),
the cross-sectional shape of the 23C60 and 23C70 rods
(Figure 16b and c) were hexagonal. In contrast, the 2324a
and 2324b rods (Figure 16d and e) adopted a distorted
polygonal shape. The difference has been ascribed to
the sizes of the nanoparticles. With increasing sizes of
fullerenes, the relative size of the flake assembly of 23
decreased. As a result, the macroscopic organization of the
assemblies of 23 in the 2324a and 2324b rods might
hamper the formation of hexagonal structures, leading to
the distorted structures.

(c)

11

5 m

40

20

0
(f)

6 8 10 12 14
Length (m)

Figure 16 SEM images of (a) 23 tube, (b) C60 23 rod,

(c) C70 23 rod, (d) 2324a rod, and (e) 2324b rod. (f) The
length distribution of 2324b rod. (Reproduced from Ref. 27.
American Chemical Society, 2009.)

22a, ribbons of uniform size and orientation (Figure 15E),

with an average width of about 19 nm estimated from the
zoom-in image (Figure 15e), were assembled. In contrast,
the self-assembly of 22b gave hollow spheres (Figure 15F),
which was also evidenced by the broken spheres on the
zoom-in image (Figure 15f).
Sandanayaka et al. have used SEM to study the surfactant
(cetyltrimethylammonium bromide)-assisted self-assembly
of 23 in the presence and absence of C60 , C70 , and 24a

In solution and gel state, supramolecular chirality is mainly

investigated by using the circular dichroism spectroscopy. If
the samples form ordered microstructures, the helical chirality may be expressed and observed on the surface by SEM
or other microscopic methods. Shinkai et al. have reported
that the mixture of compound 25a and b gelated acetic
acid.28 When tetraethoxylsilane polymerization was carried
out in this gel (R = [25b/25a + 25b] = 515 mol%) and
the resulting polymer was calcinated, SEM showed that the
obtained silica not only retained the fibrous structure of
the gel but also possessed a right-handed helical structure
(Figure 17), characteristic of the supramolecular assemblies
of a chiral molecule. Since the inner diameter (about 10 nm)
of these helical fibers, estimated by TEM, was comparable
with that of the gel fibers, it was proposed that the chirality
in the organogel fibers was transcribed into these inorganic
silica fibers.
The above gels of compound 25a and b do not form
helical fibers that can be observed by SEM. However,
several chiral gelators do selectively self-assemble into
helicoidal fibers. For example, Escuder et al. have reported
that (S,S)-26 gelated several organic solvents.29 SEM of
the gel formed in benzene showed the presence of isolated
right-handed twisted ribbons of several micrometers of

Supramolecular Chemistry: From Molecules to Nanomaterials, Online 2012 John Wiley & Sons, Ltd.
This article is 2012 John Wiley & Sons, Ltd.
This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470661345.smc044

12

Techniques

O
RO

N
N

O
O

H
N
NH

25a: R = (CH2)2CH3

H
N

HN

H
N
NH

H
N
HN

(R,R )-26

(S,S )-26

25b: R = (CH2)4N(CH3)3+ Br

500 nm

(a)

Figure 18 SEM picture of (S,S)-26, showing the right-handed

helices formed in benzene. (Reproduced from Ref. 29. Royal
Society of Chemistry, 2002.)

250 nm

3 m

2 m

(b)

Figure 17 SEM images of silica structures prepared using gel

of 25a and 25b as a template: (a) SEM for R = 25 mol% and
(b) SEM for R = 10 mol%. (Reproduced from Ref. 28. Royal
Society of Chemistry, 2001.)

length and pitch, together with cylindrical objects and

longer fibers (Figure 18). SEM also revealed that, when the
gel was formed by fast cooling of the hot solution, more
helicoidal fibers were observed, but their sizes decreased.
In another study, Escuder et al. have prepared (R,R)26.30 SEM images showed that it produced helicoidal fibers
of opposite handedness in its benzene gel (Figure 19).
The SEM images of the precipitates of their racemic
and nonracemic mixtures were also recorded, which did
not show the presence of helicoidal aggregates. Since the
self-assembly of the fibers is affected by many factors,
it is nearly impossible to produce and observe fibers of
opposite handedness that have perfectly identical shape and
size. Thus, the above SEM results just illustrate that the
molecular chirality can be transferred and amplified to the
whole supramolecular system.

(a)

(b)

Figure 19 SEM images showing the details of the helicoidal

fibers found in the benzene gels of (S,S)-26 (a) and (R,R)-26
(b).30

CONCLUSION

SEM is a versatile technique for supramolecular science

to elucidate the microscopic structures of self-assembled
systems owing to its high lateral resolution and great depth
of focus. SEM is not in competition with other microscopic
techniques as it allows different imaging modes. In many
cases, it is used together with other microscopic techniques.
Since it covers the nano- to microscales, it is particularly
useful for observing the morphologies of the self-assembled
systems, but cannot be used to characterize the single
pure supermolecules such as rotaxane, catenane, knot,
and dendrimer.
Since the imaging is performed under high vacuum
for dried samples, conventional SEM cannot avoid structural distortion of the studied samples. Thus, the observed

Supramolecular Chemistry: From Molecules to Nanomaterials, Online 2012 John Wiley & Sons, Ltd.
This article is 2012 John Wiley & Sons, Ltd.
This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470661345.smc044

Scanning electron microscopy

morphologies are actually those of the collapsed samples
without solvents in them. To overcome this limitation, a
new scanning transmission electron microscopy (STEM)
imaging technique has been developed, which allows transmission observations of wet samples in an ESEM.31 However, its application for supramolecular science is not
reported yet.

13

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Supramolecular Chemistry: From Molecules to Nanomaterials, Online 2012 John Wiley & Sons, Ltd.
This article is 2012 John Wiley & Sons, Ltd.
This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470661345.smc044