A safe, simple, and facile staining method using polysiloxanes for high-contrast visualization of gelator aggregates by transmission electron microscopy

Article abstract of DOI:10.1246/bcsj.20180072

The staining of TEM samples using Si atoms was investigated using aggregates of loose gels formed by twelve structurally different gelators in several solvents. Thirteen commercially available siloxanes were used as stains. TEM images of non-stained and OsO4-stained samples of molecular aggregates formed by the dodecamethylenediamide of N-methacryloyloxyethylaminocarbonyl- L-isoleucine in 1-propanol were poorly defined and low-contrast. However, the image of a methacryloyloxypropyl- terminated polydimethylsiloxane (S1)- stained sample was characterized by very clear bundles of fine fibers. The staining effect was explained by the wrapping of fibers, the stabilizing of the individual fibers, and reinforcing by S1. An S1 concentration of more than 5mgmL^-1 was found to be necessary for satisfactory contrast. S1 was successfully applied to the observation of aggregates of eleven other gelators. S1 worked universally as an aggregate stain regardless of the gelator or solvent polarity. The staining effect was observed for other siloxanes. This effect was found to depend on the molecular weight of the siloxane (>1, 000) rather than the kind of siloxane employed. Energy-dispersive X-ray spectroscopy indicated that the molecules of S1 gather on the surface of the fibers during drying, wrapping them. The results indicate that the present staining method guarantees reproducibility and universality.

Full text of DOI:10.1246/bcsj.20180072

Selected Paper
A Safe, Simple, and Facile Staining Method Using Polysiloxanes
for High-Contrast Visualization of Gelator Aggregates
by Transmission Electron Microscopy
Kenji Hanabusa,*1,2 Masashi Nakashima, Eriko Funatsu, Sachiyo Kishi, and Masahiro Suzuki
3
3
3
1
1Interdisciplinary Graduate School of Science and Technology, Shinshu University, Ueda, Nagano 386-8567, Japan
2Division of Frontier Fibers, Institute for Fiber Engineering, ICCER, Shinshu University,
Ueda, Nagano 386-8567, Japan
3Faculty of Textile Science & Technology, Shinshu University, Ueda, Nagano 386-8567, Japan
E-mail: hanaken@shinshu-u.ac.jp
Received: March 4, 2018; Accepted: April 9, 2018; Web Released: July 4, 2018
Kenji Hanabusa
In 1979, Kenji Hanabusa started his academic carrier at Shinshu University as an Assistant Professor. He
received his Ph.D. from Osaka University in 1981 in Japan. In 1987, he was promoted to an Associate
Professor, and since 1999, he has been a Full Professor in the Interdisciplinary Graduate School of Science
and Technology at Shinshu University. His research interest is the development of low-molecular weight
gelators and thickeners on the basis of supramolecular chemistry.
Abstract
The staining of TEM samples using Si atoms was inves-
1
. Introduction
tigated using aggregates of loose gels formed by twelve struc-
turally different gelators in several solvents. Thirteen commer-
cially available siloxanes were used as stains. TEM images of
Transmission electron microscopy (TEM) is a technique in
which a beam of electrons is transmitted through a sample to
form an image. The sample investigated is typically an ultrathin
section or a suspension mounted on a grid. An image results
from the interaction between the electrons and the sample as
the beam is transmitted through the sample. The image is
magnified and focused onto an imaging device, such as a
fluorescent screen or a sensor such as a charge-coupled device
(CCD). Historically, TEM was first demonstrated by Max
Knoll and Ernst Ruska in 1931, and the first TEM with higher
resolution than optical microscopes was built by Rusk in 1933,
with the first commercial version being completed in 1939. In
1986, Ruska was awarded the Nobel Prize in physics for the
development of TEM.
non-stained and OsO -stained samples of molecular aggregates
4
formed by the dodecamethylenediamide of N-methacryloyl-
oxyethylaminocarbonyl-L-isoleucine in 1-propanol were poorly
defined and low-contrast. However, the image of a meth-
acryloyloxypropyl-terminated polydimethylsiloxane (S1)-
stained sample was characterized by very clear bundles of fine
fibers. The staining effect was explained by the wrapping of
fibers, the stabilizing of the individual fibers, and reinforcing
¹
1
by S1. An S1 concentration of more than 5 mg mL was found
to be necessary for satisfactory contrast. S1 was successfully
applied to the observation of aggregates of eleven other
gelators. S1 worked universally as an aggregate stain regardless
of the gelator or solvent polarity. The staining effect was ob-
served for other siloxanes. This effect was found to depend on
the molecular weight of the siloxane (>1,000) rather than the
kind of siloxane employed. Energy-dispersive X-ray spectros-
copy indicated that the molecules of S1 gather on the surface of
the fibers during drying, wrapping them. The results indicate
that the present staining method guarantees reproducibility and
universality.
TEM can provide images at significantly higher resolutions
than those available using optical microscopy because the
wavelength of electrons is smaller than those of light. As a
result, TEM is a major analytical method in biology, physics,
and chemistry. As demonstrated by the breadth of literature on
TEM, it has made significant contributions to science, par-
1­6
ticularly the biological sciences. However, the contribution
of TEM to chemistry has also rapidly increased in recent years.
The earliest examples of TEM usage in chemistry are a series
of pioneering studies by Kunitake’s group on synthetic bilayer
membranes in which TEM was used to obtain images of the
structures of artificial amphiphiles negatively stained with
Keywords: Gelator aggregate
j
Polysiloxane
j
TEM
7
­10
uranyl acetate.
1176 | Bull. Chem. Soc. Jpn. 2018, 91, 1176–1185 | doi:10.1246/bcsj.20180072
© 2018 The Chemical Society of Japan

TEM is now widely and routinely used in polymer science.
11­19
2. Experimental
For example, the morphology of block copolymers,
blend
2
0­22
23­25
26
polymers,
dendrimers,
spun core/shell fibers, co-
2.1 Instrumentation. Elemental analysis was performed
with a Perkin-Elmer 240B analyzer. Infrared spectra were
recorded on a Jasco FTIR-7300 spectrometer using KBr plates.
27
28
polymer latex particles, graft-copolymers on natural rubber,
and composite particles of epoxy-polymers29 have been studied
using TEM.
1
H NMR spectra were obtained on a Bruker AVANCE 400
In general, TEM samples need high-atomic-number stains
for enhanced contrast. This staining material absorbs or scatters
part of the electron beam. Heavy-metal-containing compounds
are used prior to TEM observation to selectively deposit
spectrometer. TEM was performed with a JOEL IEM-2010
electron microscope. Energy dispersive X-ray spectrometry
(EDS) was performed using a NORAN Voyager 1000 attached
to a JOEL TEM-2010.
7
­10,23,24
electron-dense atoms on the sample. Uranyl acetate,
2.2 Reagents. 9-Decenol, triphosgene, and 2-chloro-4,6-
dimethoxy-1,3,5-triazine (CDMT) were purchased from Tokyo
Chemical Industry Co., Ltd. Poly(ethylene glycol) (average
Mn = 10,000), Karstedt’s catalyst solution, 1,1,1,3,5,5,5-hepta-
methylsiloxane, and 2-chloro-1,3,2-dioxaphospholane 2-oxide
were supplied by Aldrich. The siloxanes methacryloyloxy-
propyl-terminated polydimethylsiloxane (S1), 1,3-bis(3-meth-
acryloyloxypropyl) tetramethylsiloxane (S2), aminopropyl-
terminated polydimethylsiloxane (S3 and S4), carbinol-
terminated polydimethylsiloxane (S5), octamethylcyclotetra-
siloxane (S12), and decamethylcyclopentasiloxane (S13) were
obtained from Gelest Inc. KF-96-10cs (S6), KF-96-50cs (S7),
KF-96-100cs (S8), KF-96-300cs (S9), KF-50-100cs (S10), and
KF-54 (S11) were purchased from Nacalai Tesque Inc. Osmium
1
1,16,19,20,22,26,28
osmium tetroxide (OsO ),
ruthenium tetroxide
phosphotungstic acid
are widely employed as stains. Even though
4
1
3­15,17,18,21,27
(
(
RuO4),
H PW O )
and
2
9­34
3
12
4
uranyl acetate is a radioactive compound and both OsO4 and
RuO4 are extremely toxic oxidizing agents that must be
handled with utmost care, surprisingly little research effort
has been dedicated to developing non-hazardous stains for
TEM. As a rare example, Stara et al. reported oleum, a H SO
2
4
3
5
solution of SO , as a new and simple staining method. How-
3
ever, oleum is a strongly toxic oxidizing solution. Furthermore,
Mann et al. reported two potentially useful stains, i.e., the
commercially available Gd(fod)3, where fod is 1,1,1,2,2,3,3-
heptafluoro-7,7-dimethyl-4,6-octadione, and a molybdenum
complex prepared from t-butylammonium molybdate and
docosylphosphonic acid.36
tetroxide (OsO ) was purchased from Wako Pure Chemicals.
4
2.3 Gelation Test. Gelation tests were performed using the
inverted test-tube method. A typical procedure was as follows:
A weighed sample and 1 mL of solvent in a septum-capped test-
tube with an internal diameter of 14 mm was heated until the
solid dissolved. The resulting solution was allowed to cool and
held at 25 °C for 2 h and the gelation was then checked visu-
ally. When no fluid ran down the wall upon inversion of the
test-tube it was judged to be a gel. Gelation ability was evalu-
ated in terms of the minimum gel concentration, which is
In recent years, considerable research effort has been dedi-
cated to the development of low-molecular-weight gelators that
physically gel water or organic solvents. Accordingly, with
advances in supramolecular chemistry, an increasing number of
3
7­57
studies on gelators have been published in recent years.
Low-molecular-weight gelators exhibit a unique characteristic
in that they immobilize substantial volumes of solvent at low
concentration. Gelators are also characterized by thermally
reversible sol-gel transitions. These features can be attributed
to the formation of three-dimensional (3D) network structures
mediated by noncovalent interactions such as hydrogen bond-
ing, electrostatic interactions, van der Waals interactions, and
π-π interactions. The visual imaging of 3D network structures
is very important in the investigation of gelation mechanisms.
Accordingly, electron microscopic observation is routinely
used to obtain information on the network structures of aggre-
gates formed by gelators. To the best of our knowledge, the first
study of gels using scanning electron microscopy (SEM) was
reported by Tachibana et al. in 1969,58 who obtained SEM
images of the left- and right-handed twists of the D- and L-
forms of 12-hydroxystearic acid in an ethanol gel. The obser-
vation of aggregates in gels using TEM was first reported in
¹
1
defined as the concentration in g L (gelator/solvent) neces-
sary for gelation at 25 °C.
2.4 Synthesis.
acryloyloxyethylaminocarbonyl-L-isoleucine (G1):
Dodecamethylenediamide of N-Meth-
To a
solution of 42.98 g (0.162 mol) of N-benzyloxycarbonyl-L-
isoleucine in 150 mL of ethyl acetate was added 36.73 g (0.178
mol) of N,N¤-dicyclohexylcarbodiimide at 0 °C. After the mix-
ture was stirred for 1 h at 0 °C, a solution of 16.22 g (0.081 mol)
of 1,12-dodecamethylenediamine in 150 mL of chloroform was
added, and this solution was stirred for 2 h at 0 °C for a further
2 h at room temperature. The resulting mixture was stirred for
10 h at 40 °C and then at 55 °C overnight. After removing the
formed dicyclohexylurea by hot filtration, the filtrate was
cooled to room temperature and the gel formed was suction
filtered and dried. The crude product was dissolved in 1.2 L
of hot 1-propanol and cooled to room temperature. The gel
formed was broken well using a mechanical stirrer and suc-
tion filtered. After repeating this purification with 1-propanol,
45.48 g (81%) of the dodecamethylenediamide of N-benzyloxy-
carbonyl-L-isoleucine was obtained.
1
987 by Terech et al,59 who published both TEM and SEM
images of a native steroid in a cyclohexane gel. Over the last
two decades, our group has developed several low-molecular-
weight gelators and succeeded in imaging their 3D network
structures by SEM and TEM.6
0­65
We herein report a novel, simple, and safe staining method
for visualizing molecular aggregates of gelators in TEM
using polysiloxanes as stains, which are commercially avail-
able, inexpensive, non-toxic, and provide high-contrast images.
This is the first attempt to use polysiloxanes as stains for
TEM.
The dodecamethylenediamide of N-benzyloxycarbonyl-L-
isoleucine (45.48 g, 0.065 mol) was hydrogenated in the
presence of Pd-C in 500 mL of 1-propanol for 5 h at room
temperature under a hydrogen atmosphere. After confirming
the complete removal of the protecting group by silica gel TLC
Bull. Chem. Soc. Jpn. 2018, 91, 1176–1185 | doi:10.1246/bcsj.20180072
© 2018 The Chemical Society of Japan | 1177

¹
1
(chloroform/methanol/acetic acid, 95:5:1, v/v/v) using nin-
by IR (910 cm ), the solvent was removed and the resulting
matter was dissolved in 150 mL of hot methanol followed by
charcoal treatment. The filtrate without charcoal was left to
stand at room temperature and the gel formed was broken well
and filtered. Charcoal treatment twice of the methanol solu-
hydrin visualization, the solution was filtered. The filtrate was
evaporated and recrystallization from a mixture of 100 mL THF
and 200 mL of petroleum ether provided 19.69 g (70%) of the
dodecamethylenediamide of L-isoleucine.
To a hot solution of 1.06 g (3.39 mmol) of the dodecame-
thylenediamide of L-isoleucine in 80 mL of toluene was added
¹1
tion gave 3.93 g (72%) of G3. FT-IR (KBr, cm ): 3294 (νN-H
amide), 2850 (νCH2), 1691 (νC=O urethane), 1645 (νC=O,
amide I), 1050 (νSi-O-Si), disappeared 910 (δC-H terminal
olefin). Found: C 63.47, H 12.05, N 3.71%. Calcd for
1
.44 g (6.78 mmol) of 2-methacryloyloxyethylisocyanate drop-
wise. After stirring for 30 min at 80 °C, the product was pre-
cipitated by adding 100 mL of hexane. Recrystallization from
1
C H N O Si : C 64.06, H 11.52, N 3.56%. H NMR (400
4
2
90
2
5
3
5
0 mL of 2-methoxyethanol gave 2.20 g (88%) of G1. FT-IR
MHz, DMSO-d , TMS, 25 °C): δ = 5.87 (d, 1H; -OCONH-),
5.19 (t, 1H, -CONH-), 4.04 (t, 2H, -OCH2-), 3.89 (t, 1H,
6
¹1
(KBr, cm ): 3282 (νN-H amide), 2849 (νCH ), 1722 (νC=O
2
ester), 1631 (νC=O, amide I), 1568 (δN-H amide II). Found: C
-COCHNH-), 3.25 (q, 2H, -NHCH -) 1.87 (m, 1H, -CH- iso-
2
6
9
1.51, H 8.97, N 11.51%. Calcd for C H N O : C 61.93, H
.30, N 11.40%. H NMR (400 MHz, DMSO-d6, TMS, 25 °C):
leucyl), 1.66 (m, 2H, -OCH CH ), 1.59 (m, 2H, -SiCH CH -),
1.48 (m, 2H, -NHCH2CH2-), 1.25 (s, 44H, -CH2- and CH3CH2-
38
68
6
8
2
2
2
2
1
δ = 0.78­0.82 (d, 12H; CH -isoleucyl), 1.22 (br, 24H; alkyl),
isoleucyl), 0.92 (q, 6H, CH in isoleucyl), 0.88 (t, 3H, CH in
3
3
3
1
.39 (m, 4H; CONHCH CH ), 1.56 (m, 2H; -CH-isoleucyl),
octadecyl), 0.45 (t, 2H, -SiCH -), 0.15 (s, 3H, -SiCH ), 0.083
2 3
2
2
1
.88 (s, 6H; COC(CH )=CH ), 3.02 (m, 4H; CH NHCO), 3.29
(s, 18H, -Si-CH3).
1-Undecylcarbonylamino-3,5-bis(rac-sec-butylaminocar-
bonyl)benzene (G6): A solution of 200 mL of pyridine
3
2
2
(m, 4H; NHCONHCH ), 3.98 (d, 2H; NHCHCO), 4.06 (t, 4H;
2
NHCH CH O), 5.68, 6.06 (s, 1H; COC(CH )=CH ), 6.10
2
2
3
2
(
d, 2H; NHCONH), 6.22 (t, 2H; NHCONHCH ), 7.92 (t, 2H;
containing 54.35 g (0.30 mol) of 5-aminoisophthalic acid was
cooled in an ice-water bath and then 69.37 g (0.30 mol) of
dodecanoyl chloride was added by portions over 1 h. After
stirring for 3 h at room temperature, evaporation of pyridine
followed by recrystallization from 300 mL of methanol pro-
vided 85.60 g (79%) of 5-undecylcarbonylaminoisophthalic
2
CH NHCO).
2
1
,1,1,3,5,5,5-Heptamethylsilyldecyloxycarbonyl-L-isoleu-
cylaminooctadecane (G3): Pyridine (2.64 g, 33.3 mmol) and
1
9
cooled in an in ice-water bath in 5 portions over a period of 1 h.
The solution was stirred at room temperature for 3 days and
then 100 mL of hexane was added. The precipitated pyridinium
chloride was filtered off and the resulting solution was evapo-
rated. Vacuum distillation using a glass tube oven gave 17.86 g
5.63 g (100 mmol) of 9-decenol were added to a solution of
.89 g (33.3 mmol) of triphosgene in 50 mL of dichloromethane
¹
1
acid. FT-IR (KBr, cm ): 3268 (νN-H amine), 2921 (νCH ),
3
2849 (νCH ), 1722 (νC=O carboxylic acid), 1662 (νC=O,
2
amide I), 1543 (δN-H amide II). Found: C 52.74, H 7.83, N
2.52%. Calcd for C H NO : C 66.09, H 8.04, N 3.85%.
2
0
29
5
1
H NMR (400 MHZ, DMSO-d6, TMS, 25 °C): δ = 13.19 (s, 2H,
¹
1
(82%) of 9-decenoxycarbonyl chloride (1779 cm for νC=O
COOH), 10.22 (s, 1H, NH), 8.43 (s, 2H, 2-benzene), 8.14
(s, 1H, 4-benzene), 2.33 (t, 2H, NHCOCH CH ), 1.60 (m,
in chloroformate).
2
2
L-Isoleucylaminooctadecane66 (13.87 g, 36.3 mmol) and
2H, NHCOCH CH ), 1.26 (m, 16H, CH CH ), 0.84 (t, 3H,
2 2 2 3
4
.41 g (43.6 mmol) of triethylamine were dissolved in 80 mL
CH3).
of THF and then 7.93 g (36.3 mmol) of 9-decenoxycarbonyl
chloride was added at 0 °C. After stirring for 1 h at room
temperature, the triethylammonium chloride was removed and
the solvent evaporated. The residue was dissolved in 200 mL
of hot methanol and cooled. The gel formed was broken well
using a mechanical stirrer and suction filtered. Purification
twice with methanol gave 14.75 g (72%) of 9-decenoxycar-
A solution of 3.63 g (0.010 mol) of 5-undecylcarbonylami-
noisophthalic acid and 3.51 g (0.020 mol) of CDMT in 100 mL
of dry THF was cooled in a salt-and-ice-water bath, 2.5 mL
(0.022 mol) of 4-methylmorpholine was added dropwise at ¹5
to 0 °C. After stirring for 3 h at 0 to 3 °C, 1.46 g (0.020 mol)
of rac-sec-butylamine was added dropwise at ¹5 to 0 °C. The
resulting mixture was stirred for 2 h at 0 to 3 °C and then over-
night at room temperature. The residue after evaporation was
dissolved in 100 mL ethyl acetate and washed successively
with water, 1 M NaOH, water, 1 M HCl, and water. After eva-
poration of ethyl acetate, recrystallization from a small amount
¹
1
bonyl-L-isoleucylaminooctadecane. FT-IR (KBr, cm ): 3292
(
(
νN-H amide), 2850 (νCH ), 1691 (νC=O urethane), 1645
νC=O, amide I), 910 (δC-H terminal olefin). Found: C 73.59,
2
H 12.95, N 5.03%. Calcd for C H N O : C 74.41, H 12.13,
3
5
68
2
3
1
¹1
N 4.96%. H NMR (400 MHz, DMSO-d6, TMS, 25 °C): δ =
.85 (d, 1H; -OCONH-), 5.80 (t, 1H, -CONH-), 5.22 (m, 1H,
CH =CH-), 4.96 (d, 2H, CH =CH-), 4.04 (t, 2H, -OCH2-),
of ethyl acetate gave 4.24 g (87%) of G6. FT-IR (KBr, cm ):
5
3295 (νN-H amine), 2924 (νCH ), 2853 (νCH ), 1636 (νC=O,
3
2
amide I), 1536 (δN-H amide II). Found: C 71.01, H 10.02, N
9.14%. Calcd for C H N O : C 71.00, H 10.00, N 8.87%.
2
2
3
.09 (t, 1H, -COCHNH-), 2.05 (m, 1H, CH CH CH-), 1.90
3 2
29 47
3
3
1
(
q, 2H, CH =CH-CH -), 1.60 (m, 2H, -OCH CH ), 1.49 (m,
H NMR (400 MH , CDCl , TMS, 25 °C): δ = 8.62 (s, 1H,
Z 3
2
2
2
2
2
H, -NHCH CH -), 1.26 (s, 42H, -CH -), 0.92 (q, 6H, CH in
NHCOCH CH ), 8.27 (s, 2H, 2-benzene), 7.93 (s, 1H, 4-
2 2
2
2
2
3
isoleucyl), 0.88 (t, 3H, CH in octadecyl).
benzene), 6.31 (d,2H, CONHCH(CH )CH CH ), 4.10 (m, 2H
3 2 3
3
Karstedt’s catalyst solution (60 ¯L) was added to a solution of
.00 g (7.08 mmol) of 9-decenoxycarbonyl-L-isoleucylamino-
octadecane in 150 mL of dry toluene and then 2.05 g (9.20
mmol) of 1,1,1,3,5,5,5-heptamethylsiloxane was added slowly
followed by stirring for 2 days at 55 °C under an argon atmos-
phere. After confirming the disappearance of the terminal olefin
(CONHCH(CH )CH CH ), 2.42 (t, 2H, NHCOCH CH ), 1.70
3 2 3 2 2
4
(m, 2H, NHCOCH2 CH2), 1.57 (m, 4H, CH(CH3)CH2CH3),
1.31 (d, 6H, CH(CH )CH CH ), 1.24 (m, 16H, CH CH ), 0.96
3
2
3
2
3
(t, 3H, CH CH CH ).
2
2
3
1-Undecylcarbonylamino-3,5-bis(1-ethylpropylaminocar-
bonyl)benzene (G7): This compound was prepared using 2-
1178 | Bull. Chem. Soc. Jpn. 2018, 91, 1176–1185 | doi:10.1246/bcsj.20180072
© 2018 The Chemical Society of Japan

ethylpropylamine by the same procedure as that used for G6.
¹
1
Drawing and
dropping
Yield; 70%. FT-IR (KBr, cm ): 3264 (νN-H amine), 1636
νC=O, amide I), 1560 (δN-H amide II). Found: C 71.54, H
Adding solvent
Heating
Solvent
(
9
8
1
7
.79, N 8.52%. Calcd for C H N O : C 71.81, H 10.25, N
30 51 3 3
1
.87%. H NMR (400 MH , CDCl , TMS, 25 °C): δ = 10.08 (s,
Z
3
Gelator
Cu grid
H; NHCO), 8.12 (s, 2H; 2-benzene H), 8.10 (d, 2H; CONH),
.85 (s, 1H; 4-benzene H), 3.78 (m, 2H; CONHCH), 2.31
Solution
Polysiloxane
(
8
t, 2H; NHCOCH ), 1.59 (m, 2H; NHCOCH CH ), 1.50 (m,
H; CH(CH CH ) ), 1.26 (m, 16H; C H CH ), 0.86 (t, 15H;
2 3 2 8 16 3
Figure 1. Illustration for staining of TEM sample using
polysiloxane.
2
2
2
CH CH ).
2
3
Table 1. Preparation conditions of TEM samples
PEG-Containing Gelator (G8): A mixture of 3.30 g (ca.
.33 mmol) of poly(ethylene glycol) (average Mn = 10,000),
.37 g (0.67 mmol) of N-(6-isocyanatohexylaminocarbonyl)-L-
0
0
Solvent1)
Specimen #
Gelator
Siloxane
1
2
3
4
5
6
7
8
9
G1 (5 mg)
G1 (5 mg)
G1 (5 mg)
G1 (5 mg)
G1 (5 mg)
G1 (5 mg)
G2 (10 mg)
G3 (5 mg)
G4 (1 mg)
G5 (5 mg)
G6 (0.3 mg)
G7 (0.3 mg)
G8 (1 mg)
G9 (3 mg)
G10 (1 mg)
G11 (1 mg)
G12 (4 mg)
G4 (5 mg)
G9 (3 mg)
G1 (5 mg)
G1 (5 mg)
G1 (5 mg)
G1 (5 mg)
G1 (5 mg)
G1 (5 mg)
G1 (5 mg)
G1 (5 mg)
G1 (5 mg)
G1 (5 mg)
G1 (5 mg)
G1 (5 mg)
G12 (4 mg)
G1 (5 mg)
G1 (5 mg)
none
none
1-Propanol
1-Propanol
1-Propanol
1-Propanol
1-Propanol
1-Propanol
Cyclohexane
Hexane
Toluene
Toluene
Toluene
Toluene
1,4-Dioxane
1,4-Dioxane
1,4-Dioxane
Dodecane
Water
67
isoleucylaminooctadecane, and 4 mg (6.7 ¯mol) of dibutyl
tin(IV) dilaurate in 30 mL of chloroform was refluxed for 24 h
under an argon atmosphere. The filtrate from the resulting hot
mixture was concentrated and poured into 100 mL of methanol.
The precipitate was suction filtered and dried. Yield: 3.3 g
2)
S1 (5 mg)
S1 (1 mg)
S1 (10 mg)
S1 (30 mg)
S1 (5 mg)
S1 (5 mg)
S1 (5 mg)
S1 (5 mg)
S1 (5 mg)
S1 (5 mg)
S1 (5 mg)
S1 (5 mg)
S1 (5 mg)
S1 (5 mg)
S1 (5 mg)
none
¹
1
(89%). FT-IR (KBr, cm ): 3375 (νN-H, urea), 3275 (νN-H,
amide), 1685 (νC=O, urethane), 1679 (νC=O, amide I), 1560
(
δC=O, amide II).
Polydimethylsiloxane-Containing Gelator (G9):
A
10
11
mixture of 3.30 g (ca. 0.66 mmol) of carbinol terminated poly-
dimethylsiloxane (Mw = 4500­5500), 0.74 g (1.34 mmol) of
N-(6-isocyanatohexylaminocarbonyl)-L-isoleucylaminooctade-
cane, and 8.5 mg (13.4 ¯mol) of dibutyltin(IV) dilaurate in 50
mL of chloroform was refluxed 24 h under an argon atmos-
phere. The resulting hot mixture was filtered and the filtrate was
concentrated and poured into 100 mL of methanol. The formed
gel was broken and suction filtered. Yield: 2.91 g (72%). FT-IR
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Toluene
none
1,4-Dioxane
1-Propanol
1-Propanol
1-Propanol
1-Propanol
1-Propanol
1-Propanol
1-Propanol
1-Propanol
1-Propanol
1-Propanol
1-Propanol
1-Propanol
Water
¹
1
(
(
KBr, cm ): 3335 (νN-H, urea), 3298 (νN-H, amide), 1682
νC=O, urethane), 1627 ν(C=O, amide I), 1571 (δC=O,
S2 (5 mg)
S3 (5 mg)
S4 (5 mg)
S5 (5 mg)
S6 (5 mg)
S7 (5 mg)
S8 (5 mg)
S9 (5 mg)
S10 (5 mg)
S11 (5 mg)
S12 (5 mg)
S13 (5 mg)
S5 (5 mg)
S1 (10 mg)
S9 (10 mg)
amide II).
PC-L-Isoleucylaminooctadecane (G12): Phosphorylcho-
line (PC)-containing gelator G12 was prepared via two reac-
tions. A mixture of 13.0 g (34 mmol) of L-isoleucylaminoocta-
decane and 2.59 g (36 mmol) of β-propiolactone in 20 mL of
dry THF was refluxed overnight under an argon atmosphere.
After evaporating, recrystallization from a mixture of THF
and hexane gave 5.66 g (62%) of N-2-hydroxyethylcarbonyl-
28
29
30
31
32
¹
1
L-isoleucyl-aminooctadecane. FT-IR (KBr, cm ): 3285 (νO-H
and νN-H amide), 1634 (νC=O, amide I), 1543 (δN-H
amide II). Found: C 71.35, H 12.12, N 6.42%. Calcd for
C H N O : C 71.31, H 11.97, N 6.16%.
3
3
3
4
1-Propanol
1-Propanol
27
50
2
3
2
-Chloro-1,3,2-dioxaphospholane-2-oxide (0.47 g, 33 mmol)
1)The amount of solvent was fixed to be 1 mL. 2)The sample
was negatively stained by OsO4.
and 0.37 g (36 mmol) of triethylamine were added to a solution
of 1.50 g (33 mmol) of N-2-hydroxyethylcarbonyl-L-isoleucyl-
aminooctadecane in 40 mL of dry THF and stirred for 2 h. After
removing the triethylammonium chloride, 35 g of trimethyl-
amine solution (35% in acetonitrile) was added. The resulting
solution was heated for 12 h at 85 °C in a sealed glass vessel.
After evaporating, recrystallization from a mixture of 5 mL
of methanol and 20 mL of acetonitrile gave 1.33 g (65%) of
siloxane were placed in a test-tube and then 1 mL of solvent
was added and heated until the gelator dissolved. A droplet of
the hot solution was placed on a MICA-coated Cu-grid (400
mesh). When the siloxane was only partially miscible in the
solvent, the resulting suspension was dropped on a MICA-
coated Cu-grid. The solvent was evaporated spontaneously at
room temperature for 2 h, and the sample was dried under
vacuum overnight. In the case of specimen 2 (see Table 1), the
¹
1
G12. FT-IR (KBr, cm ): 3287 (νN-H amide), 1635 (νC=O,
amide I), 1557 (δN-H amide II), 1049 (νP=O). Found: C
6
1
2.21, H 11.08, N 6.45%. Calcd for C H N O P: C 62.01, H
32 66 3 6
0.73, N 6.78%.
.5 Sample Preparation for TEM. Sample preparation
2
dried sample was negatively stained with OsO vapor (2 wt%
4
is illustrated in Figure 1. A weighed sample of the gelator and
acetone solution) for 10 h.
Bull. Chem. Soc. Jpn. 2018, 91, 1176–1185 | doi:10.1246/bcsj.20180072
© 2018 The Chemical Society of Japan | 1179

3
. Results and Discussion
Negative staining is one of the most commonly employed
electron staining techniques for TEM. The heavy metals perme-
ate the gaps in the sample on the supporting film to provide
contrast for the TEM image. For instance, a solution containing
the material under investigation is dropped onto a supporting
film and then the solvent is removed from the sample. The
resulting samples are stained mainly by one of two methods.
One involves a staining solution containing heavy metals, such
as uranium acetate or phosphotungstic acid, and the other
involves deposition of heavy metals by vapor sublimation of
compounds such as OsO and RuO . The heavy metal materials
4
4
permeate the gaps in the sample, and, as a result, these regions
appear dark due to the strong scattering of incident electrons.
Because the sample itself is not stained, this technique is
termed negative staining. In the present study, we stained the
3
D network structures of gels formed by gelators using poly-
siloxanes instead of heavy metals. The present staining method
is sufficiently safe and easy to perform that the sample can
be prepared by simply drying after a solution containing the
polysiloxane is dropped onto a supporting film.
3
.1 Gelators and Siloxanes. The structures of the thirteen
siloxanes (S1­S13) and twelve gelators (G1­G12) used in this
study are shown in Schemes 1 and 2. G2, G4, G5, G10, and
G11 were previously reported by our group, and the other
gelators were prepared in this study for the first time. All the
siloxanes used as stains are commercially available liquids.
The TEM sample preparation conditions are summarized in
Table 1, where the italic specimen number of each sample
corresponds to the number inserted in the TEM images given
Scheme 1. Structures of siloxanes S1­S13.
Scheme 2. Structures of gelators G1­G12.
1180 | Bull. Chem. Soc. Jpn. 2018, 91, 1176–1185 | doi:10.1246/bcsj.20180072
© 2018 The Chemical Society of Japan

1
2
3
4
5
00 nm
200 nm
500 nm
200 nm
3
500 nm
200 nm
500 nm
200 nm
5
500 nm
500 nm
200 nm
6
Figure 2. TEM images of non-stained (specimen 1), OsO4-
stained (specimen 2), and S1-stained (specimen 3) samples
of molecular aggregates formed by G1 in 1-propanol.
Right images are enlarged partial detail.
later. The gelation test results for G1, G3, G6­G9, and G12 are
summarized in Table S1.
500 nm
3
.2 Comparison of Non-Stained, OsO -Stained, and
Figure 3. Images of G1 aggregates stained using S1 at
various concentrations corresponding to specimens 3, 4, 5,
and 6 in Table 1.
4
Siloxane-Stained Samples.
OsO -stained, and S1-stained samples of molecular aggregates
TEM images of non-stained,
4
formed by G1 in 1-propanol, are shown in Figure 2. Since the
¹
1
minimum gel concentration of G1 in 1-propanol is 10 mg mL
(
dried overnight. The image of specimen 3 is characterized by
bundles of fibers with widths of 40­200 nm consisting of fine
fibers with widths of approximately 10 nm. The inner and outer
parts of the fibers appear white and dark, respectively, so the
molecular aggregates are very clear. It can be assumed that S1
assembles around aggregates of G1 during the drying process.
Consequently, the aggregates are wrapped in S1. Because Si
belongs to the second period in group 14 of the periodic table,
the electron density of the aggregates wrapped in S1 is higher
than that in the rest of the sample. Moreover, since the bundles
in specimen 3 are clearly formed by the gathering of fine fibers,
it is reasonable that the wrapping of fibers by S1 stabilizes the
individual fibers and reinforces them. Thus, the predominant
staining effect of S1 is explained by these factors. Furthermore,
¹
1
see Table S1), the concentration (5 mg mL ) for preparing the
TEM samples is half the minimum gel concentration. There-
fore, it is assumed that the images in Figure 2 show fibers of
loose gels before actual gelation. Specimen 1 is an image of
non-stained G1, where low-contrast molecular aggregates with
widths of 10­200 nm are observed. In general, the contrast in
TEM images is brought about by the difference in the elec-
tronic dose of an electron beam transmitted through a sample.
Considering that both the reflection and transmission of an
electron beam depends on the electron density of a sample, the
contrast in the image observed for non-stained G1 is most
likely provided by the relatively high electron density of the
two methacryloyloxy segments. It seems that the thick mo-
lecular aggregates in specimen 1 result from the gathering and
fusing the several thin fibrous aggregates, although it is also
possible that they were flattened by the heat of the electron
S1 is a more useful staining agent compared with OsO , which
4
is both expensive and strongly toxic.
Figure 3 shows images of G1 aggregates stained using S1 at
various concentrations corresponding to specimens 3, 4, 5, and
6 in Table 1. The staining effect for specimen 4, which was
beam. Specimen 2 is an image of OsO -stained G1 obtained
from a dried sample of the loose G1 gel stained by OsO vapor
for 10 h. Though the contrast in specimen 2 is somewhat better
than that in specimen 1, undefined molecular aggregates with
widths of 10­200 nm are still observed. Specimen 3 is an image
of G1 stained by S1. A hot solution containing 5 mg G1 and
4
4
¹
1
stained using 1 mg mL S1, is only partially observed. The
staining effects for specimens 5 and 6, which were stained at 10
¹
1
and 30 mg mL , respectively, are highly defined, even at low
magnification. Thus, it can be concluded that an S1 concen-
¹1
5
mg S1 in 1 mL 1-propanol was dropped onto a Cu-grid and
tration higher than 5 mg mL is necessary to provide satisfac-
Bull. Chem. Soc. Jpn. 2018, 91, 1176–1185 | doi:10.1246/bcsj.20180072
© 2018 The Chemical Society of Japan | 1181

7
9
8
1
18
19
2
00 nm
200 nm
2
00 nm
200 nm
0
Figure 5. Images of non-stained aggregates of G3 in tolu-
ene (specimen 18) and G9 in 1,4-dioxane (specimen 19).
and G1169 are reported elsewhere. As shown in Figure 4, the
images of specimens 7, 8, and 9 are of aggregates formed by
G2 in cyclohexane, G3 in hexane, and G4 in toluene, where
fine fibers having almost uniform diameters are observed.
Although it is difficult to observe clear aggregates of G5 by
2
00 nm
200 nm
11
12
staining with OsO , we have succeeded in observing aggregates
4
of G5 for the first time by S1-staining (i.e., specimen 10) with
entangled fibers having widths of approximately 10 nm being
clearly detected. G6 and G7 are unique gelators that can form
thixotropic gels in toluene (see Table S1). Thixotropy refers to
a phenomenon whereby a gel transforms to a liquid sol by stir-
ring or vibration, and undergoes subsequent re-gelation upon
standing. The aggregates in specimen 11 are characterized by
an arabesque pattern composed of fibers tangled in knots or
snarls. The aggregates in specimen 12 apparently comprise fine
fibers with widths of several nanometers and reach widths of
approximately 100 nm. The aggregates formed by the polymer-
based gelators G8 and G9 in 1,4-dioxane are also observed as
definite fibers by S1 staining (specimens 13 and 14). The ionic
gelators (G10­G12) form helically knitted aggregates with
widths of approximately 20 nm (specimens 15, 16, and 17). It
is interesting to note that the aggregates formed by G12 in
water can be observed by S1 staining (specimen 17). This is
somewhat surprising because S1 is only sparingly soluble in
water. Thus, the results in Figure 4 demonstrate that S1 can
work universally as a stain regardless of the kind of gelator
observed or the polarity of the solvent.
2
00 nm
200 nm
1
3
14
2
00 nm
200 nm
1
5
16
2
00 nm
200 nm
1
7
3
.4 Self-Staining by Siloxane-Segment-Containing Gela-
tors. If Si atoms can scatter an electron beam efficiently, it
should be possible to observe aggregates of G3 and G9 clearly
without any staining because they contain siloxane segments.
Figure 5 shows images of aggregates of G3 in toluene and G9
in 1,4-dioxane, which were not stained. Compared to the fine
fibers with uniform diameters of approximately 20 nm observed
in specimens 9 and 14 (Figure 4), the images of specimens 18
and 19 are thick, poorly contrasted, and indistinct. Given that
indistinct thick fibers are the result of flattening by the heat of
the electron beam, staining by S1 is very significant from the
standpoint of the wrapping of fibers and the stabilization and
reinforcement of individual fibers. We can conclude that self-
staining due to the siloxane segment in G3 and G9 is insuf-
ficient owing to the Si atoms being homogeneously dispersed
throughout the aggregates. Figure 6 shows an illustration of the
aggregates for non-staining and S1 staining. When the samples
are not stained by S1, the aggregates are flattened and fused
by the heat of the electron beam and are consequently observed
as indistinct thick fibers (top of Figure 6). Conversely, if the
2
00 nm
Figure 4. Images of aggregates of G2­G12 stained by S1,
which correspond to specimens 7­17 in Table 1.
tory contrast. Given that the atomic numbers of Si and Os are
1
4 and 76, respectively, a relatively large amount of S1 is
necessary for efficient electron scattering.
.3 Staining of Aggregates of Different Gelators by S1.
3
G1 may be a unique gelator in terms of the inclusion of
methacryloyloxy segments. We evaluated the universality of S1
as a staining agent by using it to observe aggregates of other
gelators, i.e., G2­G12. Figure 4 shows images of aggregates of
G2­G12 stained by S1, which correspond to specimens 7­17 in
Table 1. G2­G7 are low-molecular-weight gelators and both
G8 and G9 are polymer-based gelators. G10 and G11 are salt-
type hydrogelators and G12 is a zwitterionic hydrogelator. The
preparation and gelation behavior of G2,62 G4, G5, G10,
61
68
69
1182 | Bull. Chem. Soc. Jpn. 2018, 91, 1176–1185 | doi:10.1246/bcsj.20180072
© 2018 The Chemical Society of Japan

2
2
2
0
2
4
21
23
25
Flattening and fusion of
fibers by heat of electron
beam
2
00 nm
200 nm
200 nm
200 nm
Flattened and fused aggregate
with low contrast
Wrapping, stabilizing,
and reinforcing of fibers
by S1 during drying
process
2
00 nm
Wrapped fine fibers with
high contrast
Figure 6. Process of sample preparation in the absence
(
above) and presence of S1 (below).
samples are prepared in the presence of S1, the aggregates are
wrapped during the drying process and both stabilized and
reinforced by the S1 (bottom of Figure 6). There is a possibility
that S1 has high affinity to the bundles of fibers through van der
Waals interactions.
200 nm
26
28
30
27
29
31
3
.5 Staining Effect of Different Siloxanes. S1 has two
methacryloyloxy segments at each end. However, we reasoned
that this unique structure may not necessarily be indispensable
for efficient staining. To establish the importance of the Si
atoms, the staining effects when other siloxanes are used were
investigated. Images of the aggregates of G1 stained by S2­
S13, which correspond to specimens 20­31 in Table 1, are
compared in Figure 7. First, we assessed the necessity of func-
tional groups at both ends of the siloxanes by using S4, S5, and
S8, which are aminopropyl-terminated polydimethylsiloxane,
carbinol-terminated polydimethylsiloxane, and ordinary poly-
dimethylsiloxane, respectively. The fine images observed for
specimens 22, 23, and 26 indicate that the staining effect is
not related to the presence or absence of the functional groups.
Conversely, fine images are not observed when S2, S6, S12, or
S13 are used as stains (see specimens 20, 24, 30, and 31). This
result can be explained by the volatility of these siloxanes
owing to their low molecular weight, i.e., S2 (Mw = 386.64),
S6 (Mw = ca. 1,000), S12 (Mw = 296.61), and S13 (Mw =
2
00 nm
200 nm
200 nm
200 nm
3
70.77). Sample preparation and observation for TEM are
performed under high vacuum, meaning that S2, S6, S12, and
S13 molecules after staining will be eliminated. The staining
effect of S3 (Mw = 900­1,000) is somewhat inferior to that of
S4 (Mw = ca. 3000), indicating that siloxanes with molecular
weights above 1,000 are necessary for efficient staining. It
should be mentioned that staining by S2, S6, S12, and S13
is not observed, even though ten-times the amount was used.
Thus, it is concluded from Figure 7 that the staining effect is
not related to the kind of siloxane used provided it possess a
molecular weight higher than 1,000.
200 nm
200 nm
Figure 7. Images of aggregates of G1 stained by S2­S13,
which correspond to specimens 20­31 in Table 1.
aggregate formed by G12 in water by S5, i.e., the carbinol-
terminated polydimethylsiloxane, which is more hydrophilic
than S1. Specimen 32 in Figure 8, in which specimen 17 is
also shown, is an image of the aggregate formed by G12 in
water. The image of specimen 32 is very similar to that of
specimen 17 in terms of the shape and size of the helically
knitted fibers. The similarity of specimens 17 and 32 strongly
suggests that the images observed in this study are not artifacts
In spite of the fact that the S1 is sparingly soluble in water, it
is effective for staining the aggregate formed by G12 in water,
and helically knitted fibers with widths of approximately 20
nm are observed (see specimen 17). We tried staining the
Bull. Chem. Soc. Jpn. 2018, 91, 1176–1185 | doi:10.1246/bcsj.20180072
© 2018 The Chemical Society of Japan | 1183

stain for the aggregates, regardless of the kind of gelator and
the polarity of the solvent. The staining effect was observed for
other siloxanes (S3, S4, S5, S7, S8, S9, S10, and S11) as well
as S1. Thus, the staining efficiency depends on the molecular
weight of the siloxane rather than its type, with an Mw of
17
32
>
1,000 being necessary. EDS indicated that molecules of S1
gather on the surface of the fibers during the drying process
and wrap around them. Thus, the results obtained in this study
indicate that the observed images are not artifacts and that
the present staining method guarantees reproducibility and
universality.
2
00 nm
200 nm
Figure 8. Images of aggregates of G12 in water, stained by
S1 (specimen 17) and S5 (specimen 32).
The present research was supported by JSPS KAKENHI
Grant Number JP24655101.
33
Supporting Information
0
keV
17
This material is available on http://dx.doi.org/10.1246/bcsj.
2
0180072.
2
00 nm
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0
keV
17
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Figure 9. Image of G1 stained by S1 (specimen 33) and
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