1) Nie, S. and S.R. Emory, Probing single molecules and

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

2. Outline • Metal Enhancement of Raman, SHG • 2) Dendrimer Encapsulated nanodots

3. Extinction coefficient ε: Strong absorbers have ε between 20,000-100,000 Absorption cross section δ also used: 1x 10-16 cm2 = 23,000 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

4. Implication of oscillator strength and absorption spectra Oscillator strength must be conserved Spectra with large maximum must be narrow Broad spectra will have smaller extinction coefficient

5. 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 Effect Metal acts like antenna, concentrates electromagnetic energy Charge Transfer Process : Between metal electrons and dye

6. Colloidal Gold Absorption Spectra Surface Plasmon Resonance Small particles blue shift, broaden 1240/eV =nm Whetten, J. Phys. Chem, 1997

7. NLO Imaging of NIE-115 Neuroblastoma Cells TPEF SHG SHG much weaker than TPEF: Very hard imaging Improve by SPR with gold?

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

9. 100 nm Gold Nanoparticle-Dye Conjugates Polymer Coated (styrene, methacrylic acid mixture) Gold Colloids linked to Styryl ANEPPS via Succinimydyl Ester •Well-defined distance between dye and metal •Hope to be less toxic Dye-Nanoparticle Conjugates are unique: Both Components can SHG under the Right conditions

10. TEM images of 100 nm Particles Polymer Coated: 3 nm thick uniform Uncoated Thickness controlled by relative polymer concentrations

11. Depend on dye and gold?

12. For dye concentration

13. Fluorescence QY Lifetime shorter If quenching

14. Enhancement Factor was 20

16. Will use for CARS (two weeks) Surface enhancement of spontaneous Raman also Provides large enhancements.

17. Laser overlaps With absorption Band: Enhances but Will now bleach May be Necessary for Adequate S/N Just like resonance enhanced SHG of dyes

18. 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

19. SPR Enhancement: overlap fluorescence/SHG/Raman excitation with SPR of metal surface Silver is bluer, more narrow than gold: Silver usually better enhancement than gold

20. 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 Nie, Science 1997

21. Size and Shape of “hot particles”? Examine by AFM Hot cylinder Brightness Is Raman intensity Hot faceted sphere Hot aggregate Panel A: 1,2 hot; 3, 4 were not: 100 nm vs 35 nm C,D also hot different shapes: No obvious correlation Probably edges: lightning rod enhancement Nie, Science 1997

22. Strong SERS Polarization Excitation dependence SERS, ordinary Raman similar spectra (with cm-1) Consistent with electric dipole, Surface plasmon interaction Nie, Science 1997

23. Strong SERS Polarization signal dependence Excitation was scrambled polarization Signal polarization selected (dichroic) Signal polarized along long axis of dye By contrast, Bulk SERS largely depolarized Unique aspect of nanoparticle SERS Nie, Science, 1997

24. Time dependent SERS Spectrum of one particle Different bands for same particle come and go and change intensity Probably Changes in orientation Dye finally bleaches (resonance Raman 514 nm) Nie, Science, 1997

25. Relative Single Molecule SERS and fluorescence Intensities B= dye bound to nanoparticle A= dye bound to surface (non-metallic) Integrated single molecule SERS 4 fold larger than single molecule fluorescence Nie, Science, 1997

27. 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, although • Broadened in solution) • Small clusters (few atom aggregates) have discrete • energy levels • Quantum confined like Semiconductor Quantum Dots

28. 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 than • electron-hole Bohr radius • Spectrum Red Shifts for larger particles: like dyes • Blue shifts for small particles • Select desired wavelength by size of particles • Spin forbidden emission~longer lifetimes 40 ns (NOT fluorescence)

29. Bioimaging

30. 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?

31. Common Problems with Quantum Dots • Normal synthesis have hydrophobic ligands for • Stability 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-specifically • when intracellular (even if ok in solution)

33. 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

34. But: free metal nanodots do not emit in solution Water quenches emission completely Only when usrface bound: protected and fewer nonradiative decay pathways Particles on Films limited in use as probes or biosensors 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

35. General Scheme for Dendrimer Formation Generation (e.g. G2 or G4) is number of branched layers Ions reduced to neutrals by white light activation Also being investigated as drug delivery devices Balogh et al

36. Absorption of Dendrimer Encapsulated Silver Clusters NaBH4 reduction makes Larger clusters: SPR nonemitting (1) No NaBH4 reduction for Emitting species Fluorescent dendrimers are photoactivated: photoreduced From ions to neutrals (3) Emitting species have a few silver atoms, <8 Dickson, JACS 2002

37. 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

38. Emission Spectra of Silver nanodot Dendrimers in solution: 400 nm excitation Distinct spectral types: average to bulk AgO surface bound nanodots Only 5 sizes substantially contribute Dickson, JACS 2002

39. 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

40. Absorption Emission of Gold Nanodots/G4 Dendrimers n=8 No surface plasmon peak particles <2 nm 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 Dickson, JACS 2003

41. Fluorescent Lifetimes of Gold Nanodot/Dendrimers Au8 Short (nanosecond): singlet-singlet (dsp) 93% Long (microsecond):triplet-singlet emission Analogous to fluorescent dyes Dickson, JACS 2003

42. Size tunable Au: dendrimers –small particles blue shift Analogous to semiconductor quantum dots Dashed=Absorption Solid=Emission Larger Sizes prepared by increasing Au concentration Dickson, Phys. Rev. Lett 2004

43. Size dependence of photophysical properties of Au/ dendrimers 330 nm 765 nm Smaller particles shift towards the blue (like QDs and larger Gold colloids): Have larger quantum yields Larger sizes have more non-radiative decay pathways (librations) Lower emission quantum yields (like red fluorescent dyes) Consistent with “energy gap” law: nonradiative rate increases At lower energy separation (probability) Dickson, Phys. Rev. lett 2004

44. Classify emission: fluorescence or luminescence? Like dyes or quantum dots? Natural lifetime: τ/QY 4.9 22 Longer lifetime at longer wavelengths consistent with Spontaneous emission: just like fluorescent dyes, τ~λ3 Unlike quantum dots consistent with dye type fluorescence emission

45. Size scaling of emission for nanodots and Quantum Dots Small Au nanodot spectra fit well to “Jellium” model: continuous sea of d electrons scale as n-1/3 (number of atoms) Quantum confinement in metals and semiconductors Have different mechanisms: QD are pseudo-one electron atoms: n-2/3 scaling for electron-hole formation Dickson, Phys. Rev. lett 2004

46. Advantages of Au, Ag dendrimers over • semiconductor quantum dots • Water soluble without coatings • Simple synthesis, no high temperatures, pressures, • Molecular beam epitaxy, multiple layers • 3) Maintain polarization (QD’s do not): better sensors of • Environment? • 4) Comparable brightness to quantum dots • 5) Can do FRET with nanodots: QD absorption too broad • But will not bleach likes dyes

47. TEM imaging of Cells labeled with Silver Nanodot Dendrimers 3T3s In cytoplasm On surface U937 In vesicles On surface Balogh, Nanoletters

48. Live Cell Imaging with Silver Nanodot Dendrimers Aqueous With silver Aqueous Without silver labeled cells DIC fluorescence Control cells fluorescence DIC