nanoplasmonic biomolecular imaging ( 奈米電漿子生物分子影像 )

Nanoplasmonic Nanoplasmonic Biomolecular ImagingBiomolecular Imaging(( 奈米電漿子生物分子影像奈米電漿子生物分子影像 ))

Shean-Jen Chen (Shean-Jen Chen ( 陳顯禎陳顯禎 ))3-30-20063-30-2006

Adaptive Photonics Lab, NCKUAdaptive Photonics Lab, NCKU- Nanoplasmonic Biosensing & Molecular Imaging - Nanoplasmonic Biosensing & Molecular Imaging

- Adaptive Optics for Vision Science - Adaptive Optics for Vision Science

- Surface Plasmons & Particle PlasmonsSurface Plasmons & Particle Plasmons

- Label-free Nano-imagingLabel-free Nano-imaging : Surface Plasmon Resonance (SPR) Microscopy

- Amplified Optical Near-fieldAmplified Optical Near-field : Metal-tip Plasmon-enhanced Near Field Scanning Optical Microscope (NSOM) for Fluorescent or Raman Molecular Image

- Breaking Diffraction LimitBreaking Diffraction Limit : Nanoplasmonic Structured Metalayer

- Conclusions

OutlinesOutlines

Adaptive Photonics Lab, NCKUAdaptive Photonics Lab, NCKU

Advantages:Advantages: - Intuitive image interpretation- Applicable to samples in natural

environment- In general non-destructive- Easy to use

Optical MicroscopyOptical Microscopy

Disadvantages:Disadvantages:- Abbe diffraction limit- Large sample area

exposed to illumination light

1.2 if 2

0.61 min

NA

NA

Total Internal Reflection FluorescenceMicroscopy (TIRFM)

- Intensity Measurement: Controlling the depth resolution via changing incident angle or wavelength

Filter out scattering light

“ What bother the TIRF ?” It can provide a better depth resolution than confocal microscopyconfocal microscopy, especially when it comes to selecting fluorescent molecules close to the biosensing surface.

sin2

/1 d212

2122

1p

nn


Evanescent Wave

- Surface Plasmons & Particle PlasmonsSurface Plasmons & Particle Plasmons

- Label-free Nano-imaging : Surface Plasmon Resonance (SPR) Microscopy

- Amplified Optical Near-field : Metal-tip Plasmon-enhanced Near Field Scanning Optical Microscope (NSOM) for Fluorescent or Raman Molecular Image

- Breaking the Diffraction Limit : Nanoplasmonic Structured Metamaterial

- Conclusions

Outlines


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TE-cooler

P-wave light Detector

θ

SlideAu filmO-ring

SAM in SAM outReceptor inLigand in Ligand out

Flow cell

Receptor out

PrismSlideAu filmSAMReceptorsLigands

Surface Plasmon Resonance (SPR) Surface Plasmon Resonance (SPR) BiosensorsBiosensors

(Kretschmann-Raether Configuration)(Kretschmann-Raether Configuration)

sp00x ksinθεkk

21

210sp εε

ε εkk

(Surface plasmon wave at semi-infinite structure)

SPR condition:


• Label-free sample

• Real-time biomolecular interaction analysis (BIA)

• Kinetic study

• Characterize and quantify biomolecular interaction

• High sensitivity

(~ 1 pg/mm2)

• Potential of high

throughput screening

Advantages of SPR BiosensingAdvantages of SPR BiosensingTheoretical sensitivity

(Resolution unit: RIU; λ= 632.8 nm) Spectrum Intensity

Detection approach

Angular Wavelength Intensity Phase

Prismcoupler

5 x 10-7 2 x 10-5 5 x 10-5 2 x 10-7

Grating coupler

2 x 10-6 6 x 10-5 2 x 10-4 8 x 10-7

1. ATR coupler: BK7 / Au (48 nm) / analyze (1.32).2. Grating coupler: Pitch of 800 nm & depth of 70 nm.3. Angular resolution is 1 x 10-4 deg.4. Wavelength is 0.02 nm.5. Intensity resolution is 0.2 %.6. Phase resolution is π/200.


- Reflection relationship:

22 01 12 1

01201 12 1

exp 2

1 exp 2

p pzp

p pz

r r ik dR r

r r ik d

1012 012 012tan Im Rep p pr r r

1 'tan 2r r n

expr r i

' 'Im r r

Intensity and Phase Variation at Intensity and Phase Variation at SPRSPR

- Phase variation analysis:

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Re

flect

ivity

SPR curve /Magnitude -Prism/Au/Protein/Water

p-wave

s-wave

6nm 18nm

55 60 65 70 75 80 85-4

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-2

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Ph

ase

sh

ift (

rad

ian

)

SPR curve /Phase -Prism/Au/Protein/Water

6nm 18nm

s-wave

p-wave


Particle Plasmon ResonanceParticle Plasmon Resonance (D.A. Schultz, 2003)


Plasmonic BiosensorsPlasmonic Biosensors

Four Plasmonic Effects:Four Plasmonic Effects:- Surface plasmons - Particle plasmons- Inter-particle coupling - Gap mode

→ → To increase sensitivity (less than 1 pg/mmTo increase sensitivity (less than 1 pg/mm22) ) → → To increase local EM field (about 10To increase local EM field (about 105 5 times)times)


0 0.1 0.2 0.3 0.4 0.5X 0.6

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4122.36

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1000

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0.21

4122.36

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X cut = 0.3 0.6

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0.21

4122.36

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Y cut = 0

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00.1

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G.-Y. Lin et al., Proc. SPIE 6095 (2006).

50 nm

Nanoparticle-enhanced Plasmonic Nanoparticle-enhanced Plasmonic BiosensorsBiosensors

• Grain size 4.0 nm & interval 2.0 nm

• Not analyte-tagged nanoparticles

• Not synthesizing

• Excitation of surface plasmons and particle plasmons

• Locally enhanced EM fields

• Providing more sensitive biosensors

Glass

Au film

Adaptive Photonics Lab, NCKUAdaptive Photonics Lab, NCKUW. P. Hu et al., Biosensors & Bioelectronics 19 (2004) 1465.S.-J. Chen et al., U.S. Patent Pending No. 10/660833, 2003.

Advanced Plasmonic Advanced Plasmonic BiosensingBiosensing

Biomolecular Imaging with Plasmonic Effects Biomolecular Imaging with Plasmonic Effects

What is Next? What is Next?


- Surface Plasmons & Particle Plasmons

- Label-free Nano-imagingLabel-free Nano-imaging : Surface Plasmon Resonance (SPR) Microscopy



- Conclusions

Outlines


Common-path Phase-Shift Common-path Phase-Shift Interferometry SPR ImagingInterferometry SPR Imaging

Adaptive Photonics Lab, NCKUAdaptive Photonics Lab, NCKUS.-J. Chen et al., Journal of Biomedical Optics 10 (2005) 034005.

- Long-term stability to reject external disturbances- Long-term stability to reject external disturbances

- Easy aligned and compact system- Easy aligned and compact system

Phase-shifting

Phase variation

Mapping to CCD

0 100 200 300 400-0.2

0

0.2

0.4

0.6

0.8

phas

e (

)

pixel

153

421

2

2tan),(

III

IIyx

15mer DNA SPR Phase Image15mer DNA SPR Phase Image- Five-step Phase-shift Interferometry - Five-step Phase-shift Interferometry

0 1/2π π 3/2π 2π

DNA DNA

- Phase Reconstruction- Phase Reconstruction


m 500

Y.-T. Su et al., Optics Letters 30 (2005) 1488.

- - SPR Phase MicroscopeSPR Phase Microscope for Living Cell Membrane Images for Living Cell Membrane Images with No Fluorescent Labelswith No Fluorescent Labels

- - Plasmon-enhanced TIR Fluorescence MicroscopePlasmon-enhanced TIR Fluorescence Microscope for for

Dynamic Living Cell Membrane ImagesDynamic Living Cell Membrane Images


SPR MicroscopySPR Microscopy

‘Surface Plasmons’ &

‘Particle Plasmons’


Cell Membrane ImagingCell Membrane Imaging

0 20 40 60 80 100 120 140 160 180 2000

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pixel

Inte

nsity

(a.u

.)

SPRTIR

- GFP-tagged TM on the melanoma cell membrane near the chip surface is excited by the evanescent wave for TIR or surface plasmon wave for SPR;

- The enhancement of fluorescence is observed apparently btw the two images. The experimental results show that the fluorescence intensity can be enhanced about 3.0 fold.

- Because of the variant distance btw the cell membrane protein TM and the collagen-coated surface, different surface plasmon effects can be applied to interpret the phenomenon of fluorescence emission or quenching.

TIRFM Plasmon-enhanced TIRFM

Melanoma-GFP-tagged TM cell

L.-Y. He et al., Proc. SPIE 6088 (2006).

K.-F. Giebel et al., Biophysical Journal 76 (1999) 509.

A Goldfish Glial CellInterference Reflection Microscope

SPR Intensity Microscope

Ag Al

Lateral Resolution Limited by Lateral Resolution Limited by Propagation Length of Surface Plasmon WavePropagation Length of Surface Plasmon Wave

Al

Ag

Propagation Length @ λ=630nmAg = 19μm; Al = 1 μm




- Amplified Optical Near-fieldAmplified Optical Near-field : Metal-tip Plasmon-enhanced Near Field Scanning Optical Microscope (NSOM) for Fluorescent or Raman Molecular Image


- Conclusions

Outlines


Near-field Scanning Optical Microscope Near-field Scanning Optical Microscope (NSOM)(NSOM)

Operation:

Applications:• Single molecule to cell detection• Nanolithography• Super-resolution data storage• Near-field optical interaction on nanoparticles, nanoclusters, and localized surface plasmon.• Nanophotonics• Surface photochemistry

Evanescent Wave and Nano-scanning Tip Techniques to Break “Diffraction Limit”• Lateral spatial resolution: ~20nm• Longitudinal spatial resolution: ~50nm

• Confined by a metal aperture• Within short distance beyond the screen.


Single-molecule DetectionSingle-molecule Detection

FEBS Letters 573 (2004) 6.Journal of Cell Science 114 (2001) 4153.

Liquid operation of NSOM opens the way to directly visualise and quantify the size and composition of membrane domains, like lipid rafts, in solution. Fluorescence image of a dendritic cell in buffer solution collected in confocal mode (A) and NSOM mode (B).

Single molecule detection on cells by NSOM. This figure shows a 40 nm optical resolution near-field ‘zoom-in’ on the indicated area (3.2 mm2) in the bright-field image of a fibroblast expressing LFA-1-GFP. GFP excitation is accomplished using 488 nm light (Ar-Kr laser line) linearly polarized along 90°.

Apertureless NSOMApertureless NSOM

Adaptive Photonics Lab, NCKUAdaptive Photonics Lab, NCKUR. Fikri et al., Optics Letters 28 (2003) 2147.

In apertureless near-field scanning optical microscopy (ANSOM), the probe vibration is often used to increase the detected signal that can be detected at by a lock-in amplifier. The realistic model of ANSOM should take into account the scan of the probe as well as the probe vibration and the material properties.

• Amplified local EM fields

• To study protein conformational change

• To investigate biomolecular structures

Surface-Enhanced Raman Scattering (SERS)Surface-Enhanced Raman Scattering (SERS)


C. L. Lei et al., Mater. Sci. Eng. B 32 (1995) 39.

Metal tip-enhanced NSOM for Fluorescent Metal tip-enhanced NSOM for Fluorescent Molecular ImageMolecular Image

- Enhance local EM field & improve detecting signal- High resolution image for fluorescent DNA samples


DNA Fluorescent Image

H. Frey, PRL 93 (2004) 200801.

Metal tip-enhanced Raman SpectroscopyMetal tip-enhanced Raman Spectroscopy

N. Hayazawa et al., JAP 92 (2002) 6983.




- Breaking Diffraction LimitBreaking Diffraction Limit : Nanoplasmonic Structured Metamaterial

- Conclusions

Outlines


Stimulated Emission Depletion Stimulated Emission Depletion (STED) Microscopy(STED) Microscopy

Excitation pulses are followed by stimulated emission depletion pulses for fluorescence inhibition. After passing dichroic mirrors and emission filters, fluorescence is detected through a confocal pinhole by a counting photodiode.PNAS 97 (2000) 8207.

The role of the STED beam is to induce the transition L2 L3 by stimulated emission and to deplete the excited fluorescence.


Comparison btw Confocal & STEDComparison btw Confocal & STED

Reducing the fluorescence focal spot size to far below the diffraction limit: (a) spot of a confocal microscope (left) compared with that in a STED microscope (right) utilizing a y-oriented intensity valley for STED (upper right insert, not to scale) squeezing the spot in the x direction to 16 nm width. (b) The average focal spot size (squares) decreases with the STED intensity following a square-root law. Insert (right) discloses the histogram of the measured spot sizes rendering the 26 nm average FWHM.

PRL 94 (2005) 143903. Adaptive Photonics Lab, NCKUAdaptive Photonics Lab, NCKU

In 1968, Veselago first considered the case of a medium that had both negative dielectric permittivity and negative magnetic permeability.

Negative Refractive IndexNegative Refractive Index

Nature 420 (2002) 119. Adaptive Photonics Lab, NCKUAdaptive Photonics Lab, NCKU

Negative Refraction Makes a Perfect Negative Refraction Makes a Perfect LensLens

A negative refractive index medium bends light to a negative angle with the surface normal. Light formerly diverging from a point source is set in reverse and converges back to a point.

J. B. Pendry, PRL 85 (2000) 3699.

Flat Slab

Object Real Image


Microwave Imaging by Flat Lans using NegMicrowave Imaging by Flat Lans using Negative Refractionative Refraction

Metal Nanostructure with square copper split ring

resonators and copper wire strips

Photonic Crystal at Band-gap Edge for 9.3 GHz

The images can be observed only in a narrow frequency range, between 9.0 and 9.4 GHz.

R. A. Shelby et al., Science 292 (2001) 77.P. V. Parimi et al., Nature 426 (2003) 404.


N. Fang et al., Science 534 (2005) 308.

Sub-Diffraction-Limited Optical Imaging with a Sub-Diffraction-Limited Optical Imaging with a Silver SuperlensSilver Superlens

: SiO2

L B

Enhanced the Readout Enhanced the Readout Signal of Super-RENS Signal of Super-RENS

DiscDisc

J.-N. Yih et al., Applied Optics 44 (2005) 3001. Adaptive Photonics Lab, NCKUAdaptive Photonics Lab, NCKU

- Break diffraction limit & improve single particle detection without near-field tip- Lateral spatial resolution: ~100nm

Enhanced Lateral Resolution by Control of the Size Enhanced Lateral Resolution by Control of the Size & Distribution of Metal Nanoparticles& Distribution of Metal Nanoparticles

Coupled Waveguide-surface Plasmon Coupled Waveguide-surface Plasmon Resonance Biosensors Constructed Resonance Biosensors Constructed

with Sub-wavelength Gratingwith Sub-wavelength Grating

guided-layer( d = 250 nm)

Λ=380nm n1 / H2O

0.5Λ

n3 / BK7

substrate(1000μm)

White beam & normal incident (TM)

grating layer (δ = 25nm)

n2 / Ta2O5

biolayer: 10nm / 1.46Metal layer (δ = 60nm)

590 600 610 620 630 640 650 6600

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Red line: without a bio-layerBlue line: with a bio-layer

The reflectivity spectrum of CWSPR biosensors with sub-wavelength gratingThe reflectivity spectrum of CWSPR biosensors with sub-wavelength grating Localized surface plasmon mode and waveguide modeLocalized surface plasmon mode and waveguide mode Sharper dip-widthSharper dip-width Enhance sensitivityEnhance sensitivity


EM Wave transport below the Diffraction EM Wave transport below the Diffraction Limit in Metal Particle Plasmon Limit in Metal Particle Plasmon

WaveguidesWaveguides(S. A. Maier (S. A. Maier et alet al., 2003)., 2003)


• Establish the immobilization of biomolecules

• Build the real-time portable SPR and SERS metrology systems

• Using plasmonic biosensors to achieve the direct detection of drug

• Conformational & Structural information from CWSPR & SERS

• A total solution of the operation of advanced plasmonic biosensing

Anticipated Research OutcomesAnticipated Research Outcomes


Single Bio-molecule Detection for BIA

Molecular Scale Imaging of Living Cell

nanoplasmonic biomolecular imaging ( 奈米電漿子生物分子影像 )

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