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Scanning Electron Microscopy

Zhan-Ting Li and Xin ZhaoChinese Academy of Sciences, Shanghai, China

1 Introduction 1

2 Supramolecular Gels 23 Supramolecular Liquid Crystals 5

4 Supramolecular Polymers 6

5 Vesicles 7

6 Nanotubes, -Pores, -Ribbons, -Rods, and -Spheres 8

7 Supramolecular Chirality 11

8 Conclusion 12

References 13

1 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.1–3 The electrons

interact with the atoms of the sample to produce various

signals, which include secondary electrons, primary back-

scattered electrons, characteristic X-rays, Auger electrons,

cathodoluminescence, and specimen current and transmitted

electrons, providing information about the sample’s sur-

face 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 pro-

duce 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 detectionof 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 three-

dimensional 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.

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2 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 resolutiondown to 1.5nm.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 morpholo-

gies on the nano- to microscales. With more and more inter-

est focusing on the complicated systems based on molecular

recognition and self-assembly, the structural information

within this range of scales is becoming increasingly impor-

tant. SEM has become a powerful and routine analyticaltechnique for collecting such information. In most studies,

SEM is used together with other microscopic techniques

such as AFM, TEM, and STM, which guarantees a com-

plete understanding of the morphology of the sample.

2 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 spe-

cial 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 interac-

tions. 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 micro-

scopes. 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 entan-glement 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 net-

work during the shrinking step caused by evaporation of

the liquid, SEM focused on the general shapes and mor-

phologies rather than on the absolute quantities such as the

OH

H 1

10 µm200 µm

20 µm

10 µm

2 µm

20 µm

(a)

(b)

(c)

(d)

(e)

(f)

Figure 1 SEM images of fractured xerogels of 1 in cyclo-hexane: (a) random repartition of very long and rigid fibers;(b) detail showing the presence of thinner and more flexiblefibrils; (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 (0.1– 0.2µm) which are entangled in a porous

matrix. Figure 1(b) reveals that thinner, more flexible fib-

rils (0.05µm) 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 solid–liquid-phase separation, which is

also confirmed by SEM (Figure 1d–f), because the micro-

graphs 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 throughthe 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,




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Scanning electron microscopy 3

N

N

O O

OO

OCH3O17

N

EtEt

Et Et

N

N

OO

O O

O

17

N

Et Et

EtEt

2

OCH3

0.5 µm

5 µm

200 nm

2 µm

(a) (b)

(d)(c)

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

mixture (80: 20)) at different magnifications. (a) Nanoporousstructure; inset: vial inversion test. (b) Image at higher magni-fication showing the three-dimensional network of nanofibers.The smallest fibers are about 6.1 nm in diameter (yellow arrow).(c) Whirls with diameters of 10–15 µm. (d) Directional arrange-ment of fibers within a “microstream” in the gel. (Reproducedfrom 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 (subtract-ing the thickness of the metal layer used for gel imag-

ing −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 supramolecu-

lar fibers within the gel. On the basis of these observations

and also molecular modeling, the authors proposed a hier-

archically assembling mechanism for the gelation process.

At the first level, the hydrophobic and stacking interac-

tions caused face-to-face stacking of compound 2 into smallaggregates of 8–10 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 hydropho-

bic 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 hydropho-

bic 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 stud-

ied by using SEM. For example, Zhang et al. found

that compound 3 gelated cyclohexane through the for-mation of an entangled network of thin solid fibers with

diameters of about 40–80 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 tube-

like structure with diameters of about 20 –60 nm, which was

clearly shown by using SEM (Figure 3b).

For rigid and partially rigid gelators, their fibrous struc-

tures can be readily observed by using SEM. However,

SEM focuses on the three-dimensional surface information.

For proposing a rational assembling pattern, in many stud-

ies, 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

25– 100 nm width, which further cross-linked to form three-

dimensional 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. Two-or 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




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4 Techniques

S

S

S

SS

S

SNH

NH

O

3

(a) (b)

NONE SEI 300 V WD 6.7 nm1 µm× 10.000NONE SEI 300 V WD 6.7 nm1 µm× 4.10

1 µm

100nm

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 clearlyshows the tube-like superfine structures of the complex gel. (Reproduced from Ref. 10. © American Chemical Society, 2005.)

(a)

(b)

2 4 6 8

Cu-KR (2q / °)

3.01 nm

1.49 nm

1 µm

(c)

(d)

(e)

3.01 nm

I n t e n s i t y ( a . u .

)

O

OO

O OO

O

O

HOOH

N N

4

Figure 4 (a) SEM image and (b) SAXD pattern of xerogel 4 obtained from cyclohexane. (c) Molecular structure of 4. (d) Unimolecularstacking 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 struc-tures (Figure 5) and the addition of the second compo-

nent dramatically changed the nanoscale morphology of

the assembled superstructure. On the basis of the observa-

tions, 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 crys-

tals during storage.15 One related issue was to observe




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NH

O

NH

C11H23

C11H23

O

HO

O

NH(CH2)12NH2NH

C11H23

O

NH

C11H23

O

OH

O5 6 5

(a) (b)

Figure 5 SEM images of 5 in the absence ((a) scale bar =20µm) or presence ((b) scale bar = 2µm) of 6. (Reproduced fromRef. 13. © Royal Society of Chemistry, 2005.)

O

O

O

O

O

O

O

O

H

H

H

H

N

N

OH

OH7

8

8

100 µm100 µm

100 µm 100 µm

50 µm 50 µm

(a)

(b)

(c)

a'

b'

c'

Crystal

Fiber

Figure 6 ESEM images of hydrated samples after storing thegel (formed at 5 wt%, 7 : 8 = 1:2) for (a) 1h, (b) 12h, 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.)

the transferring process from the gels to the crystals. As

it is difficult to use conventional SEM to obtain high-

resolution 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 sam-

ple 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 sam-

ple (Figure 6b). After 36 h, only prismatic crystals remained

(Figure 6c). The transition was clearer for dehydrated sam-

ples (Figure 6a–c ) and could also be observed using SEM,

but only for the xerogel.

3 SUPRAMOLECULAR LIQUID

CRYSTALS

Supramolecular liquid crystals are a state of matter that

consists of two or more components and exhibit prop-

erties 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 scan-

ning 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 liq-

uid crystalline phases.16 The complexes might form a 1 : 1

Hamilton-type complex or extended supramolecular poly-

mers. Dynamic light scattering (DLS) provided evidence

that the Hamilton-type complexes were formed in mil-

limolar concentrations. Optical microscopy (OM), POM,

and SEM images were further obtained for their solu-

tions in cyclohexane (Figure 7). OM and POM images

(Figure 7a–d) 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 narrowerfibers 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.




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6 Techniques

NR2

N N

OO

N

N

N

N

RO

R2N N

H

H

HO

N

N

N

N

OR

NR2N

H

H

H

n

(i) Hamilton-type complexes

R2N

N N

OO

N

N

N

N

R2N N

H

Ar

H

H

O

N

N

N

N

NR2N

H

Ar

H

H

R2N

N N

OO

N

N

N

N

R2N N

H

Ar

H

HO

N

N

N

N

NR2N

H

Ar

H

H

(ii) Extended supramolecular polymers

910

10

9

9

8

10

R2N

HN NH

OO

O

9

NH

N

NNRO

NR2

NH

(CH2)n

HNN

N N OR

NR2

HN

10a: n = 5 10b: n = 7

R = n -C12H25

100 µm

100 µm

5 µm 1 µm

100 µm

A

P

A

P

(a)

(c)

(e)

(b)

(d)

(f)

100 mm

Figure 7 OM (a, b) and POM images (c, d) of nanofibers of 9·10a (a, c) and 9·10b (b, d) grown from cyclohexane (5.0mM),and SEM images of nanofibers of 9·10b (e, f). (Reproduced fromRef. 16. © American Chemical Society, 2007.)

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 prop-

erties. Asha et al. have reported that compound 11a– d

produced a liquid crystalline phase.17 SEM images were

used to reveal the effect of the functional group ester ver-

sus amide and the flexible alkyl terminal chains on theself-assembling properties (Figure 8). The image of 11a

(Figure 8a) showed one-dimensional rod-like supramolecu-

lar structures with diameters of 0.5–1.0µm 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

150nm and lengths of a few tens of micrometers, lead-

ing to aspect ratios (length over width) of magnitude 100.

This long aspect ratio compared to that of the unsubsti-

tuted 11a was attributed to the longer molecular lengthof 11b with respect to 11a. The SEM of 11c (Figure 8c)

showed one-dimensional ball-like supramolecular structures

of 3–4µm diameter that were interconnected by nanometer-

scale 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) dis-

played a leaf-like pattern, 1.8–3 µm in diameter and sev-

eral 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 π –π interaction to aid in its self-assembly

process.

4 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 assem-

bled 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 estab-

lishing the assembling patterns. Haino et al. reported that

compounds 12 and 13 formed supramolecular polymersin solution through the calix[5]arene– C60 stacking inter-

actions.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 100µm and widths of 250–500 nm

(Figure 9b), indicating that ditopic host 13 iteratively bound




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N N

O

O

O

O

X X

N

H

O

11a: X = N

H

O

11b: X =

NH

O

11c: X =

OC12H25

OC12H25

OC12H25

OC12H25

OC12H25 O

O

11d: X =

(a) (b)

(c) (d)

7 kV × 5,000 5 µm 2690 RRLSEM 15 kV × 6,000 2 µm 2670 RRLSEM

10 kV 10 kV× 5,000 5 µm 2680 RRLSEM × 5,000 5 µm 2683 RRLSEM

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-likemorphology with widths of about 100nm (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 assem-

bling 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 hydrazide-

based quadruple hydrogen bonding motifs.21 In decalin, the

supramolecular polymers further self-assembled into vesic-

ular structures. SEM images clearly showed the formation

of spherical entities of average diameters of about 0.6 and1.0µm, 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 fluo-

rescence and TEM micrographs (Figure 11c and d) further

supported their hollow feature, because obvious luminance

differences and membranes were observed.




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8 Techniques

X

O

XO

N

OC12H25

C12H25O

XO

OH

CH3NH CH3

HOOHOH

CH3

O

N

C12H25O

OC12H25

O

HN

OC12H25

C12H25O

O

NH

12

13

X

X =

CH3

OH

10 kV × 500 500 µm 000045 10 kV × 500.000 0.5 µm 000063

(a) (b)

Figure 9 SEM images of the cast-film of the mixture of 12 and13 (1 : 1) on a glass plate. (Reproduced from Ref. 18. © AmericanChemical 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 reportedthat the donor– acceptor 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 1.2µm 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 three-

dimensional contrast.

6 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 power-

ful technique for direct observation of their formation. For

N

N

O

O

O

O

H

N

C12H25

C12H25

C12H25

N NH

NH

O

O

O

N N

N NHR′

NR′′2

NH

NN

NR′HN

NR′′2

14: R′ =

R′′ = C8H17

Bu

Et

15

N

N

O

O

O

O

N

NN

NR′N

NR′′2

HH

NN

N O

O

O

H

HN

N N

N NR′

NR′′2

H H

N N

NO

O

O

H

H

n

Supramolecular polymer

(a) (b)

(c)

10 µm 1 µm

1 µm

Figure 10 FE-SEM of the filamentous precipitates of thesupramolecular polymer of 14 and 15 formed from methylcy-clohexane (0.3 mM). The length of the bar across the ribbon in(c) is 100 nm. (Reproduced from Ref. 19. © American ChemicalSociety, 2007.)

example, Bo et al. have reported that compound 20 self-

assembled into nanotubes and layered sheets, which were

driven by π –π stacking and hydrogen bonding between

the amide units.24 The materials were prepared by heat-

ing its suspension in THF to reflux until all the solidswere 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




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O

O

NH

NH

N

N

O

O

RO

RO

OO

HN

O

HNO

N

N

H

H

O

O

OR

OR

16a: R = CH2CH(CH3)2

16b: R = n -C8H17

H

H

5 kV × 7,000 2 µm 02/ MAR/09 10 kV × 16,000 1 µm

10 µm 50 nm2.6 nm

(a)

(c) (d)

(b)

Figure 11 SEM images of the vesicles of (a) 16a (1.0mM) 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.)

N NC16H33 C16H33

C16H33 C16H33

+ +

HO

OH

+

NN

N N

O

O

H H

CH2

n

CB[8] =

=

N N+ +

HOOH

17 18

19

(Ternary complex)

(a) (b)

500 nm1 µm

2 µm

Figure 12 (a) SEM and (b) TEM images of ternary complex19. Samples (6.9 × 10−4 M) were negatively stained with uranylacetate (2 wt% in water) for observation by TEM.23

nanotubes (Figure 13c and d). Magnified SEM images fur-

ther revealed the rolled-up style of the nanotubes with a

NH

OC11H23

HN

O

C11H23 20

10 µm 100 nm

200 nm500 nm

100 nm 100 nm

(a)

(c)

(e)

(b)

(d)

(f)

Figure 13 Morphology of 20. (a) SEM image of a sampleprepared by dropping its THF suspension (0.1 mg ml−1) onto asilicon substrate followed by air drying and coating with Pt;(b) high-magnification SEM image with an open-ended crosssection; (c) TEM image; (d) high-magnification TEM image; (eand f) high-magnification SEM images: internal and externalscrew ends of the self-assembled tubes.24

scrolling structure (Figure 13e), which was further con-

firmed by the terminal types of tubes (Figure 13f). SEM

images were also obtained for samples at different concen-

trations, 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 morphol-ogy of the aggregates of porphyrin 21.25 The samples were

prepared by drop-casting a 6 mg ml−1 chloroform solution

onto the surface of SiO2 substrate or quartz. It is wor-

thy to note that the obtained films were annealed in a

chloroform-vapor-saturated desiccator. Only after anneal-

ing, the molecules self-assemble into long microtubes. The




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10 Techniques

21

N

NH N

HN

C10H21

C10H21

C10H21

C10H21

a

b

c

a

cb

50 nm

50 nm50 nm

Figure 14 SEM images of microtube dendrites of 21 fromchloroform-vapor annealing. (Reproduced from Ref. 25. © Amer-ican Chemical Society, 2010.)

outer diameter of the microtubes ranges from 0.12 to

6.9µm, and the wall thickness is about 50 nm (Figure 14).

The microtubes also displayed uniform orientation, sug-

gesting 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 aggre-

gate 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 consid-

ered 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 differ-ent components. Depending on the self-assembling condi-

tions, one building block system may generate different

types of assembled entities. Jiang et al. reported that com-

pound 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

N

N N

N

CH3COS(CH2)5O O(CH2)5SCOCH3M

22a: M = 2H

22b: M = Zn

(A)

(C)

(E) (F)

(D)

(B)

1 µm

1 µm 100 nm

10 nm18 nm

100 nm 100 nm

1 µm

e

Figure 15 SEM images of nanostructures formed by 22a andb. (A) Air bubbles by 22a in CHCl3. (B) 3D networks by 22b inCHCl3. (C) Hollow spheres by 22a in CH3OH. (D) Rods by 22b

in CH3OH. (E) Ribbons by 22a in n-hexane. (e) Zoom-in imageof the rectangle part in E. (F) Hollow spheres by 22b in n-hexane.(f) Zoom-in image of the rectangle part in (F). (Reproduced fromRef. 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 chloro-

form solution of zinc porphyrin 22b led to the forma-

tion of three-dimensional network structures (Figure 15B),

which supported the significant intermolecular interaction

owing to the Zn–O(=C) coordination bond. When inject-

ing a small volume of their chloroform solutions intoCH3OH, metal-free porphyrin 22a self-assembled into hol-

low 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 Zn–O coordination. When n-hexane was used

as the medium, SEM revealed a reverse morphology. For




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N

N N

N

N

N

N

N Zn

23

N

CH3

R

R

24a: R = H

24b: R = t -Bu

300 nm

500 nm

300 nm

300 nm

500 nm

1 µm

2 µm

5 µm

2.5 µm

3 µm

60

40

20

0

N u m

b e r

7.82 ± 2.07 µm

(a) (b)

(c) (d)

(e) (f)

2 4 6 8 10 12 14

Length (µm)

Figure 16 SEM images of (a) 23 tube, (b) C60 –23 rod,(c) C70 –23 rod, (d) 23 –24a rod, and (e) 23 –24b rod. (f) Thelength distribution of 23–24b 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 C 60, C70, and 24a

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 16b–e), 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

of 23–24b. 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 of 23 (Figure 16a),

the cross-sectional shape of the 23–C60 and 23–C70 rods

(Figure 16b and c) were hexagonal. In contrast, the 23 –24a

and 23– 24b 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 23–24a and 23–24b rods might

hamper the formation of hexagonal structures, leading to

the distorted structures.

7 SUPRAMOLECULAR CHIRALITY

In solution and gel state, supramolecular chirality is mainly

investigated by using the circular dichroism spectroscopy. If

the samples form ordered microstructures, the helical chiral-

ity 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] = 5– 15 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 formhelical 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




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12 Techniques

NN

O

ORO

25a: R = (CH2)2CH3

25b: R = (CH2)4N(CH3)3+Br−

500 nm

250 nm

(a)

(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. © RoyalSociety 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 theself-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.

NH HN

HNO

HN O

(S ,S )-26

NH HN

HNO

HN O

(R ,R )-26

Figure 18 SEM picture of (S,S )-26, showing the right-handedhelices formed in benzene. (Reproduced from Ref. 29. © RoyalSociety of Chemistry, 2002.)

3 µm 2 µm

(a) (b)

Figure 19 SEM images showing the details of the helicoidalfibers found in the benzene gels of (S,S )-26 (a) and (R,R)-26(b).30

8 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-assembledsystems, 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 struc-

tural distortion of the studied samples. Thus, the observed




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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 trans-

mission observations of wet samples in an ESEM.31 How-

ever, its application for supramolecular science is not

reported yet.

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