Single-molecule imaging of rotaxanes immobilized on glass substrates: Observation of rotary movement

Article abstract of DOI:10.1002/anie.200801431

Hula hoop: The rotary movement of a chromophore-modified α-cyclodextrin (α-CD) was studied in a rotaxane structure attached to a glass substrate. The rotary movement of the α-CD was demonstrated by defocused wide-field imaging with total internal reflecti

Full text of DOI:10.1002/anie.200801431

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Angewandte
Chemie
DOI: 10.1002/anie.200801431
Single Molecular Motors
Single-Molecule Imaging of Rotaxanes Immobilized on Glass
Substrates: Observation of Rotary Movement**
Dai Nishimura, Yoshinori Takashima, Hiroyuki Aoki, Toshiaki Takahashi, Hiroyasu Yamaguchi,
Shinzaburo Ito, and Akira Harada*
Biological molecular motors such as muscle fibers, flagella,
and cilia[1–8] are precisely designed in nature, and are
reminiscent of microscale electronic and mechanical devi-
ces.[9–10] Inspired by the direct observation of rotary molecular
motor systems,[11] including FoF1-ATP synthase and F1-
ATPase, some research groups have attempted to directly
observe artificial molecular motors.[12–16] For example, the
three-dimensional orientation of myosin V was determined
using bifunctional rhodamine as a probe.[17] Even smaller
artificial molecular motors are extremely difficult to see, and
thus, additional visualization techniques are necessary to
observe rotational behavior. Rotaxanes have a suitable
structure for the construction of molecular motors. We have
previously investigated the rotational motion of a cyclo-
dextrin relative to an axis molecule using NMR techniques.[18]
We describe herein a single rotaxane molecule based on a-
cyclodextrin (a-CD) functionalized with a fluorescence probe
immobilized on a glass substrate, and estimate the rotary
movement of a-CD by total internal reflection fluorescence
microscopy (TIRFM).[19] When a single fluorescent molecule
was observed by TIRFM, the luminescence spot measured
about 150 nm. Even if an a-CD bearing a chromophore could
show rotary movement centering around the molecular axis
of an immobilized rotaxane, it would be difficult to directly
observe the three-dimensional motion of the a-CD by
conventional fluorescence microscopic techniques.[20] We
have therefore employed the defocused wide-field imaging
technique,[21–22] which functions by defocusing on a chromo-
phore of about 1 mm. The orientation of the emission
transition dipole of a single chromophoric molecule is
determined by its characteristic emission intensity distribu-
tion. In this study we observe the rotary movement of the a-
CD component of a rotaxane by measuring the emission
dipole orientation of the chromophores.
To immobilize the fluorescent rotaxanes a glass substrate
coated with 3-(triethoxysilyl)propyl isocyanate (ICTES) and
N-propyltriethoxysilane (PTES) was functionalized with 4,4’-
diaminodiphenylacetylene. This was accomplished by
immersing the glass substrate first in a dimethylformamide
(DMF; 10 mL) solution of the diamine and then in an
aqueous solution of a-CD modified with rhodamine B (6-
RhB-a-CD) to give the pseudorotaxane on a glass substrate
(G-pseudo-R-CD, Figure 1). G-pseudo-R-CD was stoppered
Figure 1. Schematic diagram of the preparation of rotaxane (R-CD)
with rhodamine B-modified a-CD on a glass substrate.
[*] D. Nishimura, Dr. Y. Takashima, Dr. H. Yamaguchi,
Prof. Dr. A. Harada
by immersion in a solution of 5-isothiocyanate isophthalic
acid[23] to yield the rotaxane with 6-RhB-a-CD on a glass
substrate (G-R-CD, Figure 1; the preparation is described in
detail in the Experimental Section).[24] Before the glass
substrate was examined by TIRFM, it was rinsed and
sonicated in aq. HCl (pH 3.5), water, methanol, toluene,
acetone, and methanol to remove free 6-RhB-a-CD.
When the nonfunctionalized glass substrate was immersed
in an aqueous solution of 6-RhB-a-CD and subsequently
washed, few luminescent spots were observed upon irradi-
ation at 532 nm (Figure 2a), indicating that any 6-RhB-a-CD
physically adsorbed on the glass substrate had been removed.
When we used nonmodified a-CD instead of 6-RhB-a-CD for
the formation of G-pseudo-R-CD, luminescent spots were not
observed, indicating that 4,4’-diaminodiphenylacetylene
Department of Macromolecular Science
Graduate School of Science, Osaka University
Toyonaka, Osaka 560-0043 (Japan)
Fax: (+81)6-6850-5445
E-mail: harada@chem.sci.osaka-u.ac.jp
Homepage: http://www.chem.sci.osaka-u.ac.jp/lab/harada/
Dr. H. Aoki, T. Takahashi, Prof. Dr. S. Ito
Department of Polymer Chemistry
Graduate School of Engineering, Kyoto University
Katsura, Nishikyo-ku, Kyoto 615-8510 (Japan)
[**] This work was partially supported by a Grant-in-Aid for Scientific
Research (No. S14103015) from the Ministry of Education, Culture,
Sports, Science, and Technology (Japan).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200801431.
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Figure 3. a) Snapshots of experimentally observed defocused images
of a single R-CD molecule in the dry state over 0.8 s. b) Snapshots of
experimentally observed defocused images of a single R-CD molecule
in the wet state over 1.2 s. The contrast of these images was
enhanced. The calculated emission patterns with variable parameters
(q, f) are shown in (c) and (d): (0, 90) for images 1 and 3; (90, 90)
for images 2 and 4. The averaged pattern for random orientation
(q=908 and f=all-around angle) is shown as image 5.
Figure 2. Fluorescence images of 6-RhB-a-CD (a), single pseudo-R-CD
molecules and single R-CD molecules (b), and on glass substrates (c).
The glass plates were illuminated with a Nd:YVO4 laser at 532 nm with
an excitation power of 3 KWcmÀ2. Fluorescence emission was isolated
from laser light by a dichroic mirror and a bandpass emission filter
(550–610 nm), and imaged onto a cooled CCD camera. TIRF images
were acquired with 100 ms integration times.
q = 08, the calculated emission pattern shows a symmetric
circular ring. The defocused fluorescence image in the dry
state fits well to the calculated pattern with variable
parameters of q and f. For example, the molecule shown in
Figure 3a was evaluated to have the orientation of q = 758 and
f= 98 at time t = 0. Thus, the three-dimensional orientation of
individual molecules was determined by the defocused
fluorescence imaging method. The rotational motion of a
single molecule can be directly observed by the sequential
acquisition of the defocused images. As shown in Figure 3a,
the orientation of the 6-RhB-a-CD remains constant at
q ꢀ 758 and fꢀ 108, indicating that the molecule is immovable
in the dry state. Conversely, the circular defocused pattern in
the wet state cannot be fitted to the calculation with the set
values for q and f. Although the calculated image for q = 08
also shows concentric rings, the predicted diameter of the
inner circle is different from that observed. This result
indicates that the emission dipole rotates within the time
required for the image acquisition (300 ms). For comparison,
image 5 in Figure 3d was calculated by integration over the
azimuthal angle from 08 to 3608 (which assumes that 6-RhB-
a-CD spins around an axis faster than the frame rate of the
CCD), shows a pattern of concentric circles. This pattern
corresponds to the experimental data in the wet state. We
speculated that 6-RhB-a-CD swings around an axis and that
the rate of the rotary movement of 6-RhB-a-CD is faster than
3608/300 ms.
(lmax = 430 nm) acts soley as the rotor axis and is not
responsible for luminescent spots when irradiated at 532 nm.
G-pseudo-R-CD showed fewer luminescence spots than
G-R-CD (Figure 2b, c). We hypothesized that washing
removed most of 6-RhB-a-CD from the axis molecules of
G-pseudo-R-CD on the glass substrate due to the absence of
the stopper. In contrast, the rotaxane G-R-CD was stable on
the glass substrate even after it had been rinsed and sonicated
in certain solvents. The intensity and distribution of the
fluorescent spots depended on the concentration of 6-RhB-a-
CD, and, furthermore, each spot showed blinking and discrete
bleaching, indicating single-molecule detection (Figure S1 in
the Supporting Information).[25,26] These results indicate that
stable rotaxanes with a fluorescent molecular probe are
immobilized on the glass substrate; the observed luminescent
spots are attributed to the fluorescence from the rhodamine B
group of 6-RhB-a-CD.
To probe the orientation of the emission dipole of the
chromophore, G-R-CD was measured using defocused imag-
ing techniques under both sets of conditions. Figure 3 shows
typical emission patterns of a single rotaxane molecule under
dry and wet conditions. The emission of dry G-R-CD shows
two bilaterally symmetric two-lobe patterns that do not
change with time (Figure 3a). The emission patterns in the
wet state are different, revealing sets of concentric circles
(Figure 3b). The defocused pattern of a single dipole emitter
can be theoretically calculated.[19] Figure 3c,d shows the
emission patterns calculated for different polar and azimuthal
angles (q and f) of the transition dipole moment of
rhodamine B. At q = 908 and f= 908, the calculated emission
pattern showed a bilaterally symmetric two-lobe pattern. At
In conclusion, the present study has addressed the rotary
movement of 6-RhB-a-CD in a rotaxane structure adsorbed
on a glass substrate. The rotary movement of 6-RhB-a-CD
has been demonstrated by defocused wide-field imaging with
TIRFM. Although TIRFM does not have a sufficiently high
resolution to observe a substituent swinging on a rotor around
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[2]rotaxane formed consists of a-CD and diphenylacetylene with
stoppers of 5-isothiocyanate isophthalic acid.
Experimental Section
Preparation of derivitized surfaces.
Step A: Prior to monolayer formation, the glass substrates were
ultrasonicated in toluene, acetone, and methanol for 15 min, respec-
tively, and then dried with a stream of N2. The substrates were
subsequently cleaned with a UV-O3 cleaner (UV253, Filgen) for 1 h to
create a hydrophilic surface.
Step B: The cleaned substrates were immersed in a solution of
PTES (2.18 ” 10À3 mol), ICTES (1.48 ” 10À7 mol) in toluene (10 mL)
for 2 h at room temperature. The treated substrates were washed four
times with toluene and quickly taken to next step.
Step C: The substrates were immersed in a solution of 4,4’-
diaminodiphenylacetylene (5 mg) in DMF (10 mL) for 2 h at room
temperature. These substrates were washed four times each with
acetone and water.
Step D: The substrates were immersed in aqueous solutions
(10 mL) of a-CD (1.02 ” 10À4 mol) and 6-RhB-a-CD (5 ” 10À9 mol)
for 14 h at room temperature.
Step E: A solution of 5-isothiocyanateisophthalic acid (4 mg) and
NaHCO3 (3 mg) in water (1 mL) was added to the reaction solution
and the solution was left for 5 h.
Step F: The substrates were rinsed ten times with water. Addi-
tionally, the substrates were rinsed four times and shaken for 15 min
in the following solvents: aq. HCl (pH 3.5), water, methanol, toluene,
acetone, and methanol. The substrates were then dried with a stream
of N2 and immediately imaged using TIRF microscopy (see the
Supporting Information).
Received: March 26, 2008
Keywords: defocused imaging · fluorescent probes · rotaxanes ·
.
single-molecule studies · supramolecular chemistry
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