Transcript Document

Total Internal Reflection Fluorescence Microscopy of Single Rhodamine B Molecules
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
By using the techniques of high resolution fluorescence microscopy, it is possible to track the location of
single molecules in amorphous hosts. In crystalline environments, the fluorescence images of single
molecules reveal the three dimensional dipole orientations. With the help of annular illumination, these
experiments allow imaging single molecules having dipole moments in a direction perpendicular to the
substrate as well as parallel. Moving 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.
Here we analyzed single Rhodamine B molecules embedded in polymer thin films. We observed different
patterns of emission which provides information about the three dimensional dipole moment orientation of
molecules. They are doughnut-like structures, rings, asymmetric rings, or spots. We also imaged the
diffusion of single Rhodamine B molecules.
Rhodamine B, PMMA
 The molecule undergoes an intersystem crossing to its lowest triplet state T1. The transition
accompanies 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 triplet state is much higher then the fluorescence lifetime. The average
fluorescence lifetime of Rhodamine B is ~2 ns [2].
Photobleaching
 An irreversible chemical reaction that occurs while the electron is in its excited state. It results with
final disappearance of 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 [3].
Blinking and Photobleaching
S1
Rhodamine B
(C28H31 N2O 3 Cl)
CH3
Blinking
Blinking
During the past 15 years, single molecule studies grew rapidly, particularly in its application to biological
systems and chemical processes. Single molecules have been used as markers and local probes for local
nano physical and chemical properties of molecular processes in their environments.
Photophysical Properties
CH3
CH3
Cl-
T1
Fast relaxation
Intersystem crossing
S0-S1 mixing
COOH
Radiative transition
S0
570
Oxygen (O)
Carbon (C)
Nitrogen (N)
575
580
585
590
595
600
605
2
1
3
Photobleaching
Absorption
Intensity (a.u.)
N+
Fluorescence
CH3
1m
6
5
4
There is a 3 seconds time interval between consecutive images.
610
Wavelength (nm)
Fluorescence Emission Spectrum of Rhodamine B
PMMA Polymer Thin Films
Image of a Single Dipole Depending on the Orientation
PMMA (C5O2H2)n is a clear, colorless polymer of
methyl methacrylate.
For an oscillating electric dipole with amplitude vector , in a medium with refractive index n0 and distance
z0 above a planar interface, the electric field amplitude of the dipole at position z > 0 is given by the planewave representation [1];
PMMA (Poly (methyl methacrylate)) thin films are
amorphous hosts in which Rhodamine B molecules
don not have a preferred orientation.

Sample Preparation

 3mg/ml PMMA in chloroform (CHCl3) solution is prepared
 A small amount of 0.2 nM Rhodamine B in methanol was added to the solutions.

 Glass substrates were put into the UV- Ozone Cleaner for 30 minutes before spin
coating.
ES
EP
E P = c1 () cos  sin   c2 () sin  cos  cos(  )
ES = c3 () sin  sin(   )

c1 () =  1 ()  r p ()()
E
 Solutions were spin-coated onto glass substrates at 2000 rpm, for 1 minute.
E =
c 2 () =  1 ()  r p ()()
E

c3 () =   1 ()  r s () ()
 The solvents were dried in a pressure oven.
() = e
1
EP
cos 
1
E =
ES
cos 

Fourier Transform
 ikn2 cos 
[1]
Experimental Setup
Microscope
Objective
60x,NA=1.4, oil
CCD image plane
Dichroic mirror

E

k
1.5X magnification
element

k
Total Internal Reflection (TIR)
CCD
camera
Filter
=10°
=0°
532 nm
Nd:YAG Laser
A continuous wave laser (l=532nm) was used for excitation in inverted geometry. A high numerical
aperture microscope objective (N.A.=1.4, 60x oil) was used for excitation. The collimated laser beam was
focused to the back aperture of the microscope objective for wide-field illumination. The angle of incidence
of the laser beam to the polymer-air interface was further adjusted to observe total internal reflection. The
achieved “annular illumination” enabled the excitation of molecules with dipole orientations perpendicular
to the substrate as well as parallel [4]. The emitted fluorescence was collected by the same microscope
objective (epi-fluorescence set up) and transmitted through a dichroic mirror, a 1.5x magnification element
and a bandpass filter. TIRF microscopy images were detected by a thermoelectrically cooled charge coupled
device (CCD) camera.
=20°
=30°
Images obtained with electromagnetic calculations and characterization of optical system.
Diffusion Properties of Single Molecules
Consecutive
specific path
path followed
followed by
by aa single
singleRhodamine
RhodamineBBmolecule
molecule
Consecutive TIRF
TIRF microscopy
microscopy images
images reveal
reveal the
the specific
diffusing
diffusing in
in PMMA
PMMAfilm.
film.
There
There is
is aa 0.2
0.2 second
second time
time interval
interval between
between consecutive
consecutive images.
images.
Single Molecules in Polymer Host PMMA
We observed diffusing as well
as stationary single molecules.
Stationary single molecules
revealed different emission
patterns
(doughnut-like
structures, rings, asymmetric
rings, or spots) due to the
different dipole orientations
(Fig.1). While some shapes
possessed circular symmetry,
some shapes were circularly
asymmetric. The asymmetry
was due to the tilt of the
emission dipole with respect to
the optical axis. The observed
variety of images was explained
by calculating the emission
pattern of a dipole located
below a dielectric-air interface
[1].
1
2
3
Trajectories fallowed by a diffusing single molecule
Conclusions
Using total internal reflection fluorescence microscopy, we determined the 3-D dipole orientation and
diffusion properties of single Rhodamine B molecules embeded in a PMMA thin film. We are planning to
use this method to explore the morphology of different polymeric thin films.
References:
[1] M.A. Lieb, Single Molecule Orientations Determined by Direct Emission Pattern Imaging, J. Opt. Soc. Am. B./Vol. 21, No:6/June 2004
[2] M. Böhmer, J. Enderlein, Orientation Imaging of Single Molecules by Wide-field Epifluorescence Microscopy, J. Opt. Soc. Am. B./Vol. 20, No:3 /March 2003
[3] Ch. Zander, J. Enderlein, R. A. Keller, Single Molecule Detection in Solution, WILEY-VCH, 2002.
[4] R. J. P. Zimmermann, C. Hettich, I. Gerhardt, A. Renn, V. Sandoghdar, “Aligned Terrylene Molecules in a Spin Coated Crystalline Film of p-Terphenyl”,
Chemical Physics Letters, January 2004.
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).
Single molecule images obtained by TIRF microscopy
Koç University Nano-Optics Research Laboratory, Rumeli Feneri Yolu, Sariyer, Istanbul 34450 Turkey • [email protected]