High-Resolution Imaging of Single Fluorescent Molecules with the Optical Near-Field of a Metal TipH. G. Frey, S. Witt, K. Felderer, and R. Guckenberger, Phys. Rev. Lett.

93, 200801 (2004). Marc McGuiganJournal ClubMonday, April 10, 2006

Outline

Introduction Near Field Microscopy Purpose

Experimental Setup Sample Preparation Results Data Model Conclusion

Beating the Diffraction Limit

NAd

2min

mvisibled 5.02.0min

Alternatives

•Scanning Tunneling Microscope

•Atomic Force Microscope

•Scanning Electron Microscope

•Transmission Electron Microscope

Why use visible light?

•Contrast

•Easier Sample Preparation

History

1928 – Synge Idea (1)

Strong light source behind thin metal film 100 nm diameter hole to illuminate biological sample Sample less than 100 nm away from source Discusses ideas in letters to Albert Einstein

(2)

1972 – E. A. Ash and G. Nicholls (3)

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

2211 sinsin nn

1

21sinn

nc

tzkxki zxeE 111

tzkxki zxeE 222

2121

21 knkk zx

2222

22 knkk zx

1121 sin knkk zz

222 cos knk x

12

2

1

2

2

12 sincos

n

n

n

n

tizxn

nkin

eE

11

22

1

21 sinsin

2

tzixeE 2

2

1

21

21 sin

n

nkn

Diagram Source: K. Iizuka, Elements of Photonics: In Free Space and Special Media, Volume 1 (John Wiley & Sons, New York, 2002).

(4)

Total Internal Reflection

Wave vectors propagating in k space

Evanescent Waves

222 nkkk zx

dmzm 2

md

kzm

22

2 2

m

dkxn

To satisfy boundary conditions:

This can be re-written as:

The value of kxn is imaginary for high values of m and the waves are evanescent waves

Diagram Source: K. Iizuka, Elements of Photonics: In Free Space and Special Media, Volume 1 (John Wiley & Sons, New York, 2002).

2

cd

Above dc kx is always imaginary and all the waves in x are evanescent waves.

Evanescent Waves on a Corrugated Metal Surface

Evanescent Waves on an Array of Metal Pins

Modes of Near Field Imaging

(a) Aperture NSOM

(b) Aperture-less NSOM

(c) Scanning tunneling optical microscope

Different types of scanning near field optical microscopes

NSOM Configurations

(a) collection

(b) illumination

(c) collection/illumination

(d) oblique collection

(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).

NSOM SetupStandard NSOM Setup(a)

Illumination

(b) Collection and Redistribution

(c) Detection

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)

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 NSOMResolution: 50-100 nm

Problems (6)

• 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

Aluminum-coated aperture probes

(a), (b) prepared by pulling

(c), (d) prepared by etching

300 nm 300 nm

(a), (c) macroscopic shape, SEM and optical image(b), (d) SEM close-up of the aperture region

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

Resolution: 10-20 nm Causes for Enhanced Electric

Field: (7)

•Electrostatic lightning rod effect (depends on geometry)

•Surface plasmon resonances (depend on excitation wavelength and geometry)

Induced surface charge density in metal probe

Left: Incident wave polarized perpendicular to tip axis

Right: Incident wave polarized along tip axis

Need large near field enhancement so the signal can be detected in the far field

The incident field should be polarized along the tip axis to maximize field enhancement

Schematic of experimental setup for tip-enhanced near field

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

1000f

Aperture NSOM resolution ~ 50 nmTip-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

10002

S

f

Noise

Signal

Simultaneous topographic image (a) and near-field two-photon excited

fluorescence image (b) of J-aggregates of

PIC dye in PVS film on a glass substrate.

Diagram (left) Source: E. J. Sanchez, L. Novotny, and X. S. Xie, Phys. Rev. Lett. 82, 4014 (1999).

Purpose

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: “highly resolved optical imaging of single dyes” “high-resolution topographs”

“tip-on-aperture” probe

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)

3.5 nm Cr and 33 nm Al deposited by evaporation at 45o

Drawing of “tip-on-aperture probe with the DNA sample.

SEM images of a “tip-on-aperture” probe

(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

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

DNA Cy-3 fluorophores covalently bound to the

termini of DNA Samples prepared in a polymerase chain

reaction (PCR) Mica Sheets

20 μl of 400 mM NiCl2 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

Results

200 nm

25 nm25 nm

Fitted tip radius: 12 nm

Fluorescence image of single Cy-3 dye molecules, which appear mostly

as double maxima.

FWHM = 10 nm

Zoomed image of a dye molecule together with a section along the line (three lines average).

Enlarged image of a bleaching event from one scan line (oriented vertically) to the next one.

25 nm25 nm

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 aperturetip EE 10

•Dye dipoles oriented vertically experience maximum excitation directly below the tip

•Inclined dyes display asymmetric peaks

•Vertical dye under the tip displays a circular structure

•Dye oriented in sample plane displays two symmetric maxima

Data Model

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

01

30

3

Im23

1 En

qcd

iAl 0.157.44

56.2mica

Lifetime without mirror

Quantum yield

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

Measurements

Patterns calculated with parameters fitted to the measurements

Tilt angle: 0o Tilt angle: 68o

Tilt angle: 49o

Tilt angle: 20o

Tilt angle: 14o

Image size: 117 nm

Fitted tip radius: 22nm

•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 tip-dye distance increases by 5 nm

Fluorescence patterns of differently tilted dye molecules.

Results

Fitted tip radius: 12 nm

Analyzed dye molecules

Note: A fitted parabola has been subtracted from each scan line to flatten the data

(a) Topography together with calculated positions of 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)

(b) Fluorescence image

200 nm

(d) First approximation fits

Green: Good fits

Yellow: Problematic fits

DNA with Cy-3 labeled termini on mica and corresponding

data modeling.

Accuracy in dye positions: 0.5 nm standard deviation

Azimuth angle accurate to 5o

Accuracy of tilt angle better than 10o

ResultsFluorescence pattern of two dyes located close to each other

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)

Single dye molecule

Conclusion

Results Near-field optical image of single

fluorescent dye molecules at high resolution High resolution topographic image of dye

molecules Improvements

Optimize tip-aperture geometry to allow plasmon resonance Vary tip length Change material

Sharpen the metal tip to improve resolution

References

1. B. Hecht et al., J. Chem. Phys. 112, 7761 (2000).2. M. A. Paesler and P. J. Moyer, Near Field Optics: Theory,

Instrumentation, and Applications (John Wiley & Sons, New York, 1996).

3. Molecular Expressions Optical Microscopy Primer, http://micro.magnet.fsu.edu/primer/index.html

4. K. Iizuka, Elements of Photonics: In Free Space and Special Media, Volume 1 (John Wiley & Sons, New York, 2002).

5. P. N. Prasad, Nanophotonics (John Wiley & Sons, Hoboken, 2004).6. E. J. Sanchez, L. Novotny, and X. S. Xie, Phys. Rev. Lett. 82, 4014

(1999).7. A. Hartschuh, M. R. Beversluis, A. Bouhelier, and L. Novotny, Phil.

Trans. R. Soc. Lond. A. 362, 807 (2004).8. L. Novotny, R. X. Bian, and X. S. Xie, Phys. Rev. Lett. 79, 645 (1997).9. N. Anderson, A. Bouhelier, L. Novotny, J. Opt. A. 8, S227 (2006). 10. H. G. Frey, F. Keilmann, A. Kriele, and R. Guckenberger, Appl. Phys.

Lett. 81, 5030 (2002).