Total Internal Reflection Fluorescence Microscopy of Single Rhodamine B Molecules

1
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
Photophysical Properties
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. 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.
Blinking

• 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
Rhodamine B, PMMA
S1
Blinking
2
3
1
Photobleaching
S0
5
6
4
There is a 3 seconds time interval between
consecutive images.
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.
• PMMA (Poly (methyl methacrylate)) thin films are
  amorphous hosts in which Rhodamine B molecules
  don not have a preferred orientation.

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 gt 0 is given by the plane-wave
representation 1
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.
• Solutions were spin-coated onto glass
  substrates at 2000 rpm, for 1 minute.
• The solvents were dried in a pressure oven.

Fourier Transform
Experimental Setup
Images obtained with electromagnetic calculations
and characterization of optical system.
A continuous wave laser (l532nm) 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.
Diffusion Properties of Single Molecules
Diffusion Properties of Single Molecules
Consecutive TIRF microscopy images reveal the
specific path followed by a single Rhodamine B
molecule diffusing in PMMA film.
Consecutive TIRF microscopy images reveal the
specific path followed by a single Rhodamine B
molecule diffusing in PMMA film.
There is a 0.2 second time interval between
consecutive images.
There is a 0.2 second time interval between
consecutive 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.
2
3
1
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,
No6/June 2004 2 M. Böhmer, J. Enderlein,
Orientation Imaging of Single Molecules by
Wide-field Epifluorescence Microscopy, J. Opt.
Soc. Am. B./Vol. 20, No3 /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. TÜBITAK-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-GEBIP/2006-19).
Single molecule images obtained by TIRF
microscopy
Koç University Nano-Optics Research Laboratory,
Rumeli Feneri Yolu, Sariyer, Istanbul 34450
Turkey myorulmaz_at_ku.edu.tr