Transcript Document

Single Molecule Tracing to Analyze the Surface Morphology of Block-Copolymer Thin Films
Mustafa Yorulmaz(1), Alper Kiraz(1), A.Levent Demirel(2)
(1)Department of Physics, Koç University, Rumelifeneri Yolu, 34450 Sariyer, Istanbul, Turkey (2)Department of
Chemistry, Koç University, Rumelifeneri Yolu, 34450 Sariyer, Istanbul, Turkey
Motivation
Observation of Different Diffusion Dynamics
and Diffusion Along Channels
During the past 15 years, single molecule studies grew rapidly for their applications especially in biological systems and
chemical processes [1]. Single molecules are sensitive probes that can provide detailed information on their host matrices.
By using the techniques of high resolution fluorescence microscopy, it is possible to track the location of single molecules
in amorphous hosts. Diffusing as well as stationary molecules can be observed by Total Internal Reflection Fluorescence
(TIRF) microscopy. The diffusion properties of molecules in different polymeric hosts can be understood by observing the
dynamics of single molecules.
1
In our studies, we use Terrylene dye molecules [2] embedded in poly(butadiene(1,4 addition)-b-ethylene oxide) (PB-PEO)
diblock copolymer thin films [3]. We try to determine the physical surface properties of PB-PEO by analyzing thin films
that are prepared from polymer/dye solution via the TIRF microscopy technique. The information about the structure is
gained as the probe molecules diffuse inside the polymeric nanostructure. We observe different types of motions such as
normal diffusion, confined motion, partially confined motion and diffusion in channels in each movie.
PB-PEO
CH2 CH === CH
Terrylene
CH2
m
b
CH2 CH2
Sample Preparation
O
Samples are prepared by spin
coating a highly diluted (~20 nM ) dye
molecule/Toluene solution which is
mixed with PB-PEO/Toluene solution
(typical dispersion 40 mg/mL) at 2000
rpm for 1 minute.
n
(163000g/mol)
(32000g/mol)
 Thin films are then annealed at 65
C for several hours for better channel
formation. Resulting channels are
observed with diameters of ~30 nm.
Diffusion along parallel channels and partially confined motion
of a single dye molecule from different movies recorded using the
same sample
PB
AFM image of the poly(butadiene)-poly(ethylene oxide) (PB-PEO) polymer
surface which shows the PB cylindrical channels inside the PEO matrix
parallel to the substrate.
Photophysical Properties
kISC
kFL
Fluorescence (ns-ms)
or non-radiative decay
PB
PB
PEO
PB
1
2
4
3
PB
Blinking
Internal conversion (ps)
hu
Excitation
Diffusion along a channel and random walk of a single dye
molecule from the above movie
PB
Molecular structure of
Terrylene (C30H16)dye
molecule
S1
2
T1
 The molecule undergoes an intersystem crossing to its lowest triplet state T1. The
transition is accompanied by a spin flip of the excited electron and is thus
symmetrically disfavored.
 Intersystem crossing rates are low, one crossing for every 105-106 excitations.
 However the average lifetime of the triplet state is much higher than the
fluorescence lifetime. The average fluorescence lifetime of Terrylene is ~3.8 ns [4].
Photobleaching
 Photobleaching is an irreversible chemical reaction that occurs while the electron
is in its excited state. It results in the final disappearance of the molecule from
observation. Typical fluorescent dye molecules survive about 105 to 106 excitation
cycles until photodestruction, although this number can vary widely and strongly
depends on the nature of the embedding medium .
kT
Phosphorescence (ms-s)
or non-radiative decay
S0
Experimental Setup
O
DM
M
T
CCD
camera
F
532 nm
Nd:YAG Laser
Illustration of the experimental setup. O, microscope objective
(60X, NA=1,49, oil); DM, dichroic mirror; M, 1.5X
magnification element; T, 2X telescope; F, filters (LP 550 and
HQ 605/90).
A continuous wave laser (l=532nm) is used for
excitation in the inverted geometry. The collimated
laser beam is focused to the back aperture of a high
numerical aperture microscope objective (N.A.=1.49,
60x) for wide-field illumination. The angle of
incidence of the laser beam to the polymer-air
interface is further adjusted to observe total internal
reflection. The fluorescence is collected by the same
microscope objective and transmitted through a
dichroic mirror, a 1.5x magnification element, a 2x
telescope and a bandpass filter. TIRF microscopy
images are recorded with an Electron Multiplied
Charge Coupled Device camera (Hamamatsu –
ImagEM).
Calculation of the mean square displacement (MSD) is performed by averaging all steps corresponding to
a lag time t ;
 r  t  
2

2
2
2
1
  r1  t     r2  t     r3  t   
n

1 n 2
  ri  t 
n i
Different types of random walk can be analyzed by looking at their corresponding MSD vs. t graph.
For instance, in trace 4, we observe a partially confined random walk and we confirm this by looking at
the MSD plot.
Calibration
Results of our calibration experiments
performed by translating a sample with
stationary Rhodamine B molecules using a
piezoelectric translation stage. In these
experiments the 20 nm step size of the
translation stage is measured as 21.83 nm
with 1.48 nm standard deviation. Standard
deviation shows the resolution of the
experimental setup in positioning single
dye molecules, i.e. positioning error.
We fitted the X-Y profiles of the image to a Gaussian function and determined the mean value of the
distribution, m = (xo,yo) and the positioning error  m . Standard positioning error is given as;
 si2 a 2 12 8p si4b 2 
 mi   
 2 2 
N
a N 
N
where N , a , b and si correspond to the number of collected photons, the pixel size of imaging
detector, the standard deviation of the background, the width of the distribution (standard deviation in
the direction, i ) respectively [5]. For our case 1.48 nm positioning error corresponds to an N/b ratio of
~1200.
The histogram of the angles between consecutive steps during the diffusion of a molecule provides
another proof for one dimensional diffusion. For a molecule diffusing along a 1D channel, the histogram
is expected to have peaks around 0 and p while for the case of random walk a flat histogram is expected.
Conclusions
We achieved a positioning resolution of ~1.5 nm with our experimental setup.
Using total internal reflection fluorescence microscopy, we observe different type of diffusion dynamics such as
confined random walk, partially confined random walk, diffusion inside a channel and diffusion along parallel
channels. Our results can be used in exploring the morphology of different polymeric thin films. In contrast to atomic
force microscopy, this technique is not fundamentally limited to the study of the morphology on the sample surface.
References:
[1] A. Zürner, J. Kirstein, M. Döblinger, C. Brauchle, “Visualizing Single-Molecule Diffusion in Mesoporous Materials”, Nature/Vol.450, No: 705/ November 2007
[2] R. J. Pfab, J. Zimmermann, C. Hettich, I. Gerhardt, A. Renn, V. Sandoghdar, “Aligned Terrylene Molecules in a Spin Coated Ultrathin Crystalline Film of p-Terphenyl”,
Chemical Physics Letters/ January 2004.
[3] A. L. Demirel, H. Schlaad, “Controlling the Morphology of Polybutadiene-Poly(ethylene oxide) Diblock Copolymers in Bulk and the Orientation in Thin Films by
Attachment of Alkyl Side Chains”, Polymer (2008), DOI: 10.1016/j.polymer.2008.05.041.
[4] T. Plakhotnik, W. E. Moerner, T. Irngartinger, and U. P. Wild, “Single molecule spectroscopy in Shpol'skii matrixes” Chimia/Vol.48 No: 31/1994
[5] A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, P. R. Selvin, “Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization”,
Science/ Vol. 300, No: 2061/June 2003
Acknowledgements:
This work was supported by the Scientific and Technological Research Council of Turkey (Grant No. TÜBİTAK-107T211). A. Kiraz acknowledges the financial support of the
Turkish Academy of Sciences in the framework of the Young Scientist Award program (Grant No. A.K/TÜBA-GEBİP/2006-19).
Koç University Nano-Optics Research Laboratory, Rumeli Feneri Yolu, Sariyer, Istanbul 34450 Turkey • [email protected]