Transcript High-Resolution Imaging of Single Fluorescent Molecules with the

High-Resolution Imaging of Single
Fluorescent Molecules with the Optical
Near-Field of a Metal Tip
H. G. Frey, S. Witt, K. Felderer, and R. Guckenberger, Phys. Rev. Lett. 93, 200801 (2004).
Marc McGuigan
Journal Club
Monday, April 10, 2006
Outline
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Introduction
 Near Field Microscopy
 Purpose
Experimental Setup
Sample Preparation
Results
Data Model
Conclusion
Beating the Diffraction Limit
d min 

2 NA
d min visible  0.2  0.5m
Alternatives
•Scanning Tunneling Microscope
•Atomic Force Microscope
•Scanning Electron Microscope
•Transmission Electron Microscope
Why use visible light?
•Contrast
•Easier Sample Preparation
History
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(1)
1928 – Synge Idea
 Strong light source behind thin metal film
 100 nm diameter hole to illuminate biological sample
 Sample less than 100 nm away from source
(2)
 Discusses ideas in letters to Albert Einstein
(3)
1972 – E. A. Ash and G. Nicholls
 Passed microwaves (3 cm) through 1.5 mm aperture
 Scanned over grating and were able to resolve 0.5 mm lines and 0.5 mm
gaps in grating
1984
 Pohl, Denk, Duerig (IBM) (SNOM)
 Lewis group (Cornell) (NSOM)
 Subwavelength aperture at apex of sharp transparent probe tip that is
coated with metal
Diagram Source: Molecular Expressions Optical Microscopy Primer, http://micro.magnet.fsu.edu/primer/index.html
Evanescent Waves
Wave vectors propagating in k space
Total Internal Reflection
n1 sin 1   n2 sin  2
 n2 

n
 1
 c  sin 1 
k  k  n1k 
2
1x
2
1z
2
k22x  k22z  n2k 
2
(4)

E1  ei k1x xk1z z t 

E2  ei k2 x xk2 z z t 
n
cos 2   1
n2
E2  e


in1k 


2
 n2 
   sin 2 1 
 n1 
 n2

 n1
2



 sin 2 1 x sin 1  z  it



k1z  k 2 z    n1k sin 1 
E2  exi z t 
k2 x  n2k cos2 
n 
  n1k sin 1    2 
 n1 
Diagram Source: K. Iizuka, Elements of Photonics: In Free Space and Special Media, Volume 1 (John Wiley & Sons, New York, 2002).
2
2
Evanescent Waves
Evanescent Waves on a Corrugated
Metal Surface
kx2  kz2  nk
Evanescent Waves on an Array of Metal
Pins
2
To satisfy boundary
conditions:
 zm
2
 2    
2
k xn      m 
   d 
2
md
k zm 
This can be re-written as:
2
The value of kxn is imaginary for
high values of m and the waves
are evanescent waves

d
m
dc 

2
Above dc kx is always imaginary
and all the waves in x are
evanescent waves.
Diagram Source: K. Iizuka, Elements of Photonics: In Free Space and Special Media, Volume 1 (John Wiley & Sons, New York, 2002).
Modes of Near Field Imaging
Different types of scanning near field
optical microscopes
NSOM Configurations
(c) collection/illumination
(a) collection
(a) Aperture NSOM
(b) illumination
(d) oblique collection
(b) Apertureless NSOM
(e) oblique illumination
(f) Dark field
Diagram (left) Source: M. A. Paesler and P. J. Moyer, Near Field Optics: Theory, Instrumentation, and Applications (John Wiley & Sons, New York, 1996).
Diagram (right) Source: B. Hecht et al., J. Chem. Phys. 112, 7761 (2000).
(c) Scanning
tunneling optical
microscope
NSOM Setup
Standard NSOM Setup
(a) Illumination
Tips (5)
•Heating and pulling method - Optical fiber is heated
with CO2 laser and pulled on both sides of heated area
•Chemical etching method - Hydrofluoric acid used to
etch glass fiber
•Fiber coated with metal
•Nanoparticle (Tip Enhanced)
(b) Collection and
Redistribution
(c) Detection
Diagram (left) Source: B. Hecht et al., J. Chem. Phys. 112, 7761 (2000).
Diagram (right) Source: Molecular Expressions Optical Microscopy Primer, http://micro.magnet.fsu.edu/primer/index.html
Aperture NSOM
Resolution: 50-100 nm
Aluminum-coated aperture probes
Problems (6)
300 nm
(a), (b) prepared by pulling
(c), (d) prepared by etching
300 nm
(a), (c) macroscopic shape, SEM
and optical image
(b), (d) SEM close-up of the
aperture region
•
Difficult to create smooth aluminum
coating on nanometer scale
•
Flat ends of the probes are not good for
high resolution topographic imaging
•
Absorption of light by metal coating
causes significant heating
Diagram (left) Source: B. Hecht et al., J. Chem. Phys. 112, 7761 (2000).
Diagram (right) Source: Molecular Expressions Optical Microscopy Primer, http://micro.magnet.fsu.edu/primer/index.html
Tip-Enhanced NSOM
Schematic of experimental setup
for tip-enhanced near field
Induced surface charge density in metal probe
Left: Incident wave polarized perpendicular to tip axis
Right: Incident wave polarized along tip axis
Resolution: 10-20 nm
The incident field should be polarized along the
tip axis to maximize field enhancement
Need large near field enhancement so the
signal can be detected in the far field
Causes for Enhanced Electric Field: (7)
•Electrostatic lightning rod effect (depends
on geometry)
•Surface plasmon resonances (depend on
excitation wavelength and geometry)
Diagram (left) Source: A. Hartschuh, M. R. Beversluis, A. Bouhelier, and L. Novotny, Phil. Trans. R. Soc. Lond. A. 362, 807 (2004). (7)
Diagram (right) Source: L. Novotny, R. X. Bian, and X. S. Xie, Phys. Rev. Lett. 79, 645 (1997). (8)
NSOM and Fluorescence
Aperture NSOM resolution ~ 50 nm
Simultaneous topographic image (a) and near-field twophoton excited fluorescence image (b) of J-aggregates
of PIC dye in PVS film on a glass substrate.
Tip-enhanced
•better resolution
•high background signal
•Bleaching of dyes
One solution: two-photon excitation
Two-photon excitation is a nonlinear
process
Detected signal is proportional to the
square of the intensity enhancement
factor (6)
Illuminated area of
sample: S = 105 nm2
Intensity enhanced area
under tip: σ = 100 nm2
2
Signal
f

f  1000

 1000
Noise
S
Diagram (left) Source: E. J. Sanchez, L. Novotny, and X. S. Xie, Phys. Rev. Lett. 82, 4014 (1999).
Purpose
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Interest: “investigation of self-fluorescing or
fluorescence labeled macromolecules at the single
molecule level.”
Challenge: combine optical and topographical
resolution of NSOM with fluorophore sensitivity
Results:
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“highly resolved optical imaging of single dyes”
“high-resolution topographs”
“tip-on-aperture” probe
Drawing of “tip-on-aperture
probe with the DNA sample.
Thin optical fiber in etching solution (10)
Tip covered with Cr (for adhesion) then
200 nm Au for contrast in SEM
Focus electron beam of SEM on the center
of the aperture
Electron-beam-deposited tip (EBD)
formed (7 s, 8 kV)
SEM images of a “tip-on-aperture” probe
3.5 nm Cr and 33 nm Al deposited by
evaporation at 45o
(a)
Before metallization
Diagram (left) Source: H. G. Frey, F. Keilmann, A. Kriele, and R. Guckenberger, Appl. Phys. Lett. 81, 5030 (2002).
(b) After metallization
Experimental Setup
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Light Source: argon laser (514 nm)
Light coupled to glass fiber onto the sample
Light transmitted through the sample and collected by objective (0.95 NA)
on inverse light microscope
Light filtered by 550 nm long pass filter
Signal detected by APD
Sample scanned ~ 1 micron per second
Scanned at constant distance with shear force feedback
Polarization of incident laser light adjusted to optimize S/N
1/3 of probes provide good fluorescence results
Sample Preparation
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DNA
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Cy-3 fluorophores covalently bound to the termini of
DNA
Samples prepared in a polymerase chain reaction (PCR)
Mica Sheets
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20 μl of 400 mM NiCl solution in water
2 min later – solution bottled off and 30 μl drop of
DNA (with Cy-3 label) solution applied to the sheet
10 min later – washed in ultrapure water and dried with
nitrogen
2
Results
Fluorescence image of single Cy-3 dye molecules,
which appear mostly as double maxima.
200 nm
Fitted tip radius: 12 nm
FWHM = 10 nm
25 nm
Zoomed image of a dye molecule
together with a section along the
25 nm
line (three lines average).
25 nm
Enlarged image of a
25 nm
bleaching event from one
scan line (oriented vertically)
to the next one.
Data Model
•Dye molecule excitation proportional to
squared field component parallel to dipole
moment
•They believe that the field from the aperture
light does not substantially influence the
experiment
Etip  10Eaperture
•Dye dipoles oriented vertically
experience maximum excitation
directly below the tip
•Dye oriented in sample plane
displays two symmetric maxima
•Inclined dyes display asymmetric
peaks
•Vertical dye under the tip displays a
circular structure
Data Model
Quantum yield
d 
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Software: MATHLAB 6.5 (Mathworks)
Classical Mirror Image Calculation
Neglected:
 Retroaction of dye dipole on tip dipole
 Retardation effects
Emission in direction of objective used to
calculate final signal
 Al  44.7  15.0i
 mica  2.56
Lifetime without mirror



3qc3
1 
ImE0 
3
 20 n1

Index of refraction of medium with dipole
•Fit Parameters
•X, Y position of dye
•3D orientation of dye
•Normalization factor for dye
brightness and local background
•Parameters assumed constant
•Tip radius
•Tip-sample distance
•Quantum efficiency = 0.3
Results with Data Model
Fluorescence patterns of differently tilted dye molecules.
Measurements
Image size: 117 nm
Fitted tip radius: 22nm
Patterns calculated with parameters
fitted to the measurements
Tilt angle: 0o Tilt angle: 14o Tilt angle: 20o Tilt angle: 49o Tilt angle: 68o
•Tip-dye distance (calculated): 1 nm
•Tip-dye distance (approach curve): 2-3 nm
•Why the discrepancy?
•Treatment of quenching effects
neglects contributions with a stronger
dependence on distance becoming
important within 5 nm
•Tip apex flatter than a sphere
•Moon-like and ring-like patterns due to
strong quenching effects when tip-dye
distance below 3 nm
•As tip-dye distance increases central
minimum decreases in size
•Total number of photon counts per
pattern decreased by factor of 2 when tipdye distance increases by 5 nm
Results
DNA with Cy-3 labeled termini on mica
and corresponding data modeling.
(a) Topography together
with calculated
positions of analyzed
dye molecules
(b) Fluorescence image
Note: A fitted parabola has been
subtracted from each scan line to
flatten the data
Accuracy in dye positions:
0.5 nm standard deviation
Analyzed dye molecules
(c) Positions of dye
molecules in (a) with
tilt angles (upper
number) and
azimuth angles
(lower numbers)
from first
approximation fits in
(d)
Fitted tip radius: 12 nm
(d) First approximation
fits
Green: Good fits
Yellow: Problematic fits
200 nm
Azimuth angle accurate to 5o
Accuracy of tilt angle better than 10o
Results
Fluorescence pattern of two dyes located close to each other
Single dye molecule
300 nm
300 nm
(a) Experimental data
(b) Best fit when assuming
two dyes for the
encircled pattern
(c) Difference between
data (a) and fit (b)
Conclusion
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Results
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Near-field optical image of single fluorescent dye
molecules at high resolution
High resolution topographic image of dye molecules
Improvements
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Optimize tip-aperture geometry to allow plasmon
resonance
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Vary tip length
Change material
Sharpen the metal tip to improve resolution
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
B. Hecht et al., J. Chem. Phys. 112, 7761 (2000).
M. A. Paesler and P. J. Moyer, Near Field Optics: Theory, Instrumentation, and
Applications (John Wiley & Sons, New York, 1996).
Molecular Expressions Optical Microscopy Primer,
http://micro.magnet.fsu.edu/primer/index.html
K. Iizuka, Elements of Photonics: In Free Space and Special Media, Volume 1
(John Wiley & Sons, New York, 2002).
P. N. Prasad, Nanophotonics (John Wiley & Sons, Hoboken, 2004).
E. J. Sanchez, L. Novotny, and X. S. Xie, Phys. Rev. Lett. 82, 4014 (1999).
A. Hartschuh, M. R. Beversluis, A. Bouhelier, and L. Novotny, Phil. Trans. R.
Soc. Lond. A. 362, 807 (2004).
L. Novotny, R. X. Bian, and X. S. Xie, Phys. Rev. Lett. 79, 645 (1997).
N. Anderson, A. Bouhelier, L. Novotny, J. Opt. A. 8, S227 (2006).
H. G. Frey, F. Keilmann, A. Kriele, and R. Guckenberger, Appl. Phys. Lett. 81,
5030 (2002).