Single Molecule Tracing to Analyze the Surface Morphology of Block-Copolymer Thin FilmsMustafa 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

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.

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.

TerryleneSamples 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.

Thin films are then annealed at 65 C for several hours for better channel formation. Resulting channels are observed with diameters of ~30 nm.

Sample Preparation

Experimental Setup

A continuous wave laser (=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).

Calibration

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 .

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].

Koç University Nano-Nano-OOpticsptics Research Laboratory Research Laboratory, Rumeli Feneri Yolu, Sariyer, Istanbul , Rumeli Feneri Yolu, Sariyer, Istanbul 3445034450 Turkey Turkey • • myorulmazmyorulmaz@ku.edu.tr@ku.edu.tr

Blinking

Photobleaching

ConclusionsWe 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 acknowledge s 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).

PB-PEObCH2 CH CH CH2=== CH2 CH2 O

m n(32000g/mol) (163000g/mol)

Photophysical Properties

Molecular structure of Terrylene (C30H16)dye

molecule 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.

S0

S1

Internal conversion (ps)

kISC

Fluorescence (ns-s) or non-radiative decay

kT

Phosphorescence (s-s) or non-radiative decay

T1h Excitation

kFL

532 nm Nd:YAG Laser

CC

D c

amer

a

O

DM

M

T

F

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).

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.

Observation of Different Diffusion Dynamics and Diffusion Along Channels

1

2

41

Diffusion along parallel channels and partially confined motion of a single dye molecule from different movies recorded using the

same sample

32

Diffusion along a channel and random walk of a single dye molecule from the above movie

2 2 2 2 21 2 3

1 1 n

ii

r t r t r t r t r tn n

Calculation of the mean square displacement (MSD) is performed by averaging all steps corresponding to a lag time ;t

Different types of random walk can be analyzed by looking at their corresponding MSD vs. graph. For instance, in trace 4, we observe a partially confined random walk and we confirm this by looking at the MSD plot.

t

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 while for the case of random walk a flat histogram is expected.

PB

PB

PB

PB

PB

PB

PEO

2 4 22

2 2

812i

i is s ba

N N a N

where , , and 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, ) respectively [5]. For our case 1.48 nm positioning error corresponds to an N/b ratio of ~1200.

N a b is

i

We fitted the X-Y profiles of the image to a Gaussian function and determined the mean value of the distribution, = (xo,yo) and the positioning error .Standard positioning error is given as;