1) Nie, S. and S.R. Emory, Probing single molecules and single nanoparticles by surface-enhanced Raman scattering. Science, 1997. 275: p. 1102-1106.

2) Zheng, J. and R.M. Dickson, Individual water-soluble dendrimer-encapsulated silver nanodot fluorescence. J Am Chem Soc, 2002. 124(47) p. 13982-3.

3) Peyser, L.A., et al., Photoactivated fluorescence from individual silver nanoclusters. Science, 2001. 291: p. 103-106.

4) Alvarez, M.M., et al., Optical Absorption Spectra of Nanocrystal Gold Molecules. J. Phys. Chem. B, 1997. 101: p. 3706-3712.

Assigned Reading

Critique: Biophys. J, vol 89, 572-580 (2005) Makareva et al

Outline

1) Metal Enhancement of Raman, SHG

2) Dendrimer Encapsulated nanodots

 

Oscillator strength is integral of the absorption band

Sum rule: oscillator strength, f, for one electron is:1x 10-16 cm2 eV

Or n* 1x 10-16 cm2 eV for n electrons

Limits absorption strength of dye molecules

How to overcome for better contrast?Add or “borrow” more electrons

Extinction coefficient ε:

Absorption cross section δ also used: 1x 10-16 cm2 = 23,000

Strong absorbers have ε between 20,000-100,000

Implication of oscillator strength and absorption spectra

Oscillator strength must be conserved

Spectra with large maximum must be narrowBroad spectra will have smaller extinction coefficient

SHG and Raman Enhancement by Metals

·     Surface Enhancement of Second Harmonic Generation, Raman Scattering of dyes on Bulk Surfaces (factor of 105 ) 1972 · More Recently Extended to Nanoparticles (factors of 1-1014 )  (Nie, Feld groups, ~1999)Possible Mechanisms·     Surface Plasmon Resonance·     Metallic atoms have delocalized d orbitals·     Metal Colloids or Surfaces have sea of electrons·     Optical excitation is collective- huge absorptions

Induced dipole coupling to dye molecule

·     Corona or Lightning Rod EffectMetal acts like antenna, concentrates electromagnetic energy Charge Transfer Process : Between metal electrons and dye

Whetten, J. Phys. Chem, 1997

Small particles blue shift, broaden

Colloidal Gold Absorption SpectraSurface Plasmon Resonance

1240/eV =nm

SHG much weaker than TPEF:Very hard imagingImprove by SPR with gold?

SHG TPEF

NLO Imaging of NIE-115 Neuroblastoma Cells

Not well-defined experiment:Processes highly distant dependent:r-6

100 nm Gold Nanoparticle-Dye Conjugates

Polymer Coated (styrene, methacrylic acid mixture)Gold Colloids linked to Styryl ANEPPS via Succinimydyl Ester

Dye-Nanoparticle Conjugates are unique:Both Components can SHG under theRight conditions

•Well-defined distancebetween dye and metal

•Hope to be less toxic

TEM images of 100 nm Particles

Uncoated Polymer Coated:3 nm thick uniform

Thickness controlled by relative polymer concentrations

Depend on dye and gold?

For dye concentration

Fluorescence QY

Lifetime shorterIf quenching

EnhancementFactor was 20

Surface enhancement of spontaneous Raman alsoProvides large enhancements.

Will use for CARS (two weeks)

Laser overlapsWith absorptionBand:Enhances but Will now bleachMay be Necessary forAdequate S/N

Just like resonance enhanced SHG of dyes

Surface Enhanced Raman Scattering

•6 orders of magnitude larger than spontaneous Raman cross sections (10-30 cm2)

•More chemical/structural information than fluorescence(vibrational spectra, like CARS)

•May be more bleach resistant (off resonance)

•Arises from surface plasmon resonance, lightning rod effect

Nie, Feld groups showed some particles have enhancements of 1010-14: comparable to absorption cross sections (10-16 cm2) of fluorescent dyes

But most do nothing

SPR Enhancement: overlap fluorescence/SHG/Ramanexcitation with SPR of metal surface

Silver is bluer, more narrow than gold:Silver usually better enhancement than gold

Nie, Science 1997

SERS of Single Rhodamine Molecules on Ag Nanoparticles

Light scattering No dye

10-11 M

10-9 M

10-9 M less than 1 dye per nanoparticle

Size and Shape of “hot particles”?Examine by AFM

Nie, Science 1997

Panel A: 1,2 hot; 3, 4 were not: 100 nm vs 35 nmC,D also hot different shapes:No obvious correlationProbably edges: lightning rod enhancement

Hotaggregate

Hotcylinder

Hot facetedsphere

BrightnessIs Ramanintensity

Nie, Science 1997

Strong SERS Polarization Excitation dependence

SERS, ordinary Raman similar spectra (with cm-1)

Consistent with electric dipole,Surface plasmon interaction

Nie, Science, 1997

Strong SERS Polarization signal dependence

Excitation was scrambled polarizationSignal polarization selected (dichroic)

Signal polarized along long axis of dye

By contrast, Bulk SERS largely depolarizedUnique aspect of nanoparticle SERS

Nie, Science, 1997

Time dependent SERS Spectrum of one particle

Different bands for same particlecome and go and change intensityProbably Changes in orientation

Dye finally bleaches (resonance Raman 514 nm)

Nie, Science, 1997

Relative Single Molecule SERS and fluorescence Intensities

B= dye bound to nanoparticleA= dye bound to surface (non-metallic)

Integrated single molecule SERS4 fold larger than single molecule fluorescence

Metal Particle Size effects leading to SPR:

•Sizes>~2-10 nm required for true surface plasmon

•Resulting absorption spectrum is broad

•Continuous distribution of excited states: conductor(unlike dyes which have discrete states, althoughBroadened in solution)

Small clusters (few atom aggregates) have discreteenergy levelsQuantum confined like Semiconductor Quantum Dots

Quantum Dot Overview

•Semiconductor Nanocrystals: CdSe, ZnSe 1-5 nm(invented in mid 1980’s at Bell Labs, Brus, Alivisatos, Bawhendi)

•Broad Absorption spectrum (UV)-electron hole pair narrow emission (visible)

Quantum confinement: particle size smaller thanelectron-hole Bohr radius•Spectrum Red Shifts for larger particles: like dyes•Blue shifts for small particlesSelect desired wavelength by size of particles

• Spin forbidden emission~longer lifetimes 40 ns (NOT fluorescence)

Bioimaging

First Applied to bioimaging in 1998

•10-50 fold brighter than organic dyes

•High quantum efficiency ~ “70%”

Highly photostable: “bleach free”: no bonds to break

Labeling not specific without functionalization

Replace organic dyes?

Common Problems with Quantum Dots

•Normal synthesis have hydrophobic ligands forStability against aggregation; not water soluble

•Exchange with polar species for solubility:Lose stability against aggregation

Reduced luminescence for hydrophilic QDs

•Multi-layer coatings are somewhat more stable:Arduous fabrication

•Can coat with proteins, conjugates

Still can aggregate and bind non-specificallywhen intracellular (even if ok in solution)

•Small silver and gold nanoclusters or nanodots (few atoms) have strong absorption (SPR like):Much stronger than organic dyes

•Absorption coefficient Comparable to Semiconductor Quantum dots (CdSe)

•Strong emission when surface bound (none in solution)

•Not true SPR (too small) but energy of bands has same spectral size dependence:

As SPR and (and quantum dots): smaller particles blue shift

How to exploit optical properties of gold and silver nanoparticles for biology?

Make dendrimers (branched polymers) to encapsulate (and shield) nanoclusters (silver and gold)

New class of probes

But: free metal nanodots do not emit in solutionWater quenches emission completely

Only when usrface bound: protected andfewer nonradiative decay pathwaysParticles on Films limited in use as probes or biosensors

General Scheme for Dendrimer Formation

Also being investigated as drug delivery devicesBalogh et al

Ions reduced to neutrals by white light activation

Generation (e.g. G2 or G4) is number of branched layers

Dickson, JACS 2002

Fluorescent dendrimersare photoactivated: photoreducedFrom ions to neutrals (3)

Absorption of Dendrimer Encapsulated Silver Clusters

NaBH4 reduction makesLarger clusters: SPR nonemitting (1)No NaBH4 reduction for Emitting species

Emitting species have a few silver atoms, <8

Emission of Encapsulated Silver Nanodots in Solution

•Brightness increases as photoactivation occurs•Blinking is observed, single particles (like single dye molecules)•Anisotropic Emission, like surface bound•Very photostable over 30 minutes with Hg cw radiation•Emission is like dye fluorescence

Dickson, JACS 2002

Dickson, JACS 2002

Emission Spectra of Silver nanodot Dendrimers in solution: 400 nm excitation

Distinct spectral types: average to bulk AgO surface bound nanodotsOnly 5 sizes substantially contribute

Dickson, JACS 2003

Gold nanodot/dendrimers n=8 is “magic number” geometric shell closing Energetically favorable

Max is 360 nm-Not SPR band at 500 nm

Dickson, JACS 2003

Absorption Emission of Gold Nanodots/G4 Dendrimers n=8

High Quantum Yield: 45-50% ( at least 100 fold over free particles)Dendrimer shields nanoparticle from water,Greatly reduces quenching Smaller dendrimers (G2) do not adequately protect the nanodot:no emission

No surface plasmon peakparticles <2 nm

Fluorescent Lifetimes of Gold Nanodot/Dendrimers

Short (nanosecond): singlet-singlet (dsp) 93%Long (microsecond):triplet-singlet emissionAnalogous to fluorescent dyes

Dickson, JACS 2003

Au8

Size tunable Au: dendrimers –small particles blue shiftAnalogous to semiconductor quantum dots

Dickson, Phys. Rev. Lett 2004

Dashed=AbsorptionSolid=EmissionLarger Sizes prepared by increasing Au concentration

Size dependence of photophysical properties of Au/ dendrimers

Larger sizes have more non-radiative decay pathways (librations) Lower emission quantum yields (like red fluorescent dyes)

Consistent with “energy gap” law: nonradiative rate increasesAt lower energy separation (probability)

Dickson, Phys. Rev. lett 2004

Smaller particles shift towards the blue (like QDs and larger Gold colloids): Have larger quantum yields

330 nm

765 nm

Classify emission: fluorescence or luminescence?Like dyes or quantum dots?

Natural lifetime:τ/QY

4.9

22

Longer lifetime at longer wavelengths consistent withSpontaneous emission: just like fluorescent dyes,τ~λ3

Unlike quantum dots

consistent with dye type fluorescence emission

Size scaling of emission for nanodots and Quantum Dots

Quantum confinement in metals and semiconductors Have different mechanisms: QD are pseudo-one electron atoms:n-2/3 scaling for electron-hole formation

Small Au nanodot spectra fit well to “Jellium” model: continuous sea of d electrons scale as n-1/3 (number of atoms)

Dickson, Phys. Rev. lett 2004

Advantages of Au, Ag dendrimers over semiconductor quantum dots

1) Water soluble without coatings

2) Simple synthesis, no high temperatures, pressures, Molecular beam epitaxy, multiple layers

3) Maintain polarization (QD’s do not): better sensors ofEnvironment?

4) Comparable brightness to quantum dots

5) Can do FRET with nanodots: QD absorption too broadBut will not bleach likes dyes

TEM imaging of Cells labeled with Silver Nanodot Dendrimers

Balogh, Nanoletters

3T3s

U937

In vesicles

In cytoplasmOn surface

On surface

Live Cell Imaging with Silver Nanodot Dendrimers

Control cells

fluorescence DIC

fluorescence DIC

labeled cells

AqueousWith silver

AqueousWithout silver