Optical Metrology of Nanomaterials and Nano-assemblies Optical - PowerPoint PPT Presentation

Optical Metrology of Nanomaterials and Nano-assemblies Optical Metrology of Nanomaterials and Nano-assemblies for Quantitative Biophotonics for Quantitative Biophotonics The Fifth US-Korea Forum on Nanotechnology: The Fifth US-Korea Forum on Nanotechnology: Nano-Biotechnology Nano-Biotechnology Jeeseong Hwang Jeeseong Hwang jch@nist.gov jch@nist.gov Biophysics Group Biophysics Group Optical Technology Division Optical Technology Division Physics Laboratory Physics Laboratory

NIST Founded in 1901 (National Bureau of Standards) U.S. Commerce Department’s Technology Administration. Mission: To develop and promote measurement, standards, and technology to enhance productivity, facilitate trade, and improve the quality of life. www.nist.gov

What is ‘Biophotonics?’ Biophotonics is the study of the interaction of light with biological material , where “light” includes all forms of radiant energy whose quantum unit is the photon. Dennis Matthews NSF Center for Biophotonics Absorption of photonic energy by a human body Photodynamic surgery physics.upenn.edu

Ultimate goal of NANOBiophotonics for DYNAMICAL quantitative nanoscale imaging Nanomaterials: “Manipulated” photons Contrast agents or Manipulators I CROSOCPE LI GHT M cohesiondev.rice.edu

Measurement Strategy Optical Metrology for Biophtotonics and Biophysics (OMB 2 ) Diffraction-limited Diffraction-limited optical techniques Validated information Integrated tools optical techniques Integrated tools with enhanced with enhanced temporal resolutiion temporal resolutiion Nanoprobes Nanoprobes Nanoscale Nanoscale Measurement details Measurement details confidence confidence

Quantum Dot (QD) Attractive fluorophores fluorophores for bio for bio- - Attractive Attractive fluorophores for bio- imaging due to its broad imaging due to its broad Optical imaging due to its broad Properties? absorption and narrow absorption and narrow absorption and narrow ? symmetric emission spectra symmetric emission spectra symmetric emission spectra Higher quantum yield and more Higher quantum yield and more Higher quantum yield and more photostable than than photostable photostable than conventional organic dye conventional organic dye conventional organic dye CdSe/ ZnS Functional Coating Size and composition dependent Size and composition dependent Size and composition dependent tunable absorption and tunable absorption and tunable absorption and emission pattern 2nm 8nm emission pattern emission pattern Bio- -functional Coating functional Coating Bio Bio-functional Coating

Why single QD characterization? “Sensor” for nanoscale environment. • Probe electron-hole separation/recombination kinetics Conduction responsible for fluorescence intermittency Band Local environment and fluorescence Band Gap • ZnS monolayers - Nirmal et al, Nature, 383:802, 1996 . - addition of ZnS monolayers result in greater fluorescence Valence - masks surface imperfections/defects Band - prevents air/solvent molecules from interacting with CdSe surface • Oxygen/Argon atmosphere - Koberling et al, Adv Mater, 13:672, 2001 . - fluorescence quenched in presence of oxygen - oxygen traps electrons at quantum dot surface • β -Mercaptoethanol - Hohng et al, JACS, 126:1324, 2004 . - near 100% blinking suppression - thiol moiety donates electrons What is the effect of the BIOCONJUGATION, functional coating on the fluorescent properties of single quantum dots?

Coming up… Measurable I: Surface hydrophilicity Measurable I: Surface hydrophilicity � Single particle tracking on QDs � Single particle tracking on QDs interacting with a lipid membrane interacting with a lipid membrane An application An application Measurable II: Distance and orientation Measurable II: Distance and orientation Nanosensor assembly � Fluorescence Energy Transfer using Nanosensor assembly � Fluorescence Energy Transfer using and characterization of QDs as donors and characterization of QDs as donors bacteriophage/QDs bacteriophage/QDs Measurable III: electrostatic environment nanocomplexes Measurable III: electrostatic environment nanocomplexes � Intermittency in fluorescence, � Intermittency in fluorescence, “blinking” of single QDs “blinking” of single QDs Measurable IV: Local concentration Measurable IV: Local concentration � Fluorescence from “clustered” QDs � Fluorescence from “clustered” QDs

Measurable I : Surface hydrophilicity Nanoparticles interacting with a lipid membrane Nanoparticle vs membrane interactions Hydrophobic vs Hydrophilic

single particle tracking of single nanocrystals Nanoshells trapped inside a lipid vesicle 785 nm DIC Combined 785 nm DIC and Fluorescence Fluorescence

Measurable II : Distance and orientation Fluorescence Energy Transfer using QD as donors Fluorescence resonance energy transfer is measured to study biomolecular K( θ ) 2 interactions such as DNA hybridization and antibody- antigen reaction. Cy5 QD ------Biotin-CG ACG GTA TAG ATG Si NP Cy5-GC TGC CAT ATC TAC---- Human Papilloma Virus gene detection

Measurable III : electrostatic environment Intermittency in fluorescence, “blinking” Fluorescent, “ON” state Trapped, “OFF” state “a particle in a box” Photoluminescence in Semiconductor conduction band surface “trap” states valence band An electron and a hole An electron or a hole recombine to emit a trapped on a surface photon photon “trap” state hole electron

Confocal Fluorescence Microscopy of Single QDs 1 µm

Surface functionalization results in shorter “on” periods of QD fluorescence due to increased surface traps Bare Carboxyl Amine 2 μ m bin time: 1ms

Surface conjugation chemistry of QDs Non-covalent bonding TOPO PEG Hydrophobic Interactions PE CdSe/ZnS >10 nm Thiol chemistry OR CdSe/ZnS + H 2 N-DNA CdSe/ZnS mercaptoundecanoicacid (MDA) - QD

Dynamical fluorescence analysis of a single bio-conjugated QD 8 0 c a f b d e 7 0 6 0 Counts/10ms 5 0 4 0 3 0 2 0 1 0 0 0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 2 0 0 0 0 2 5 0 0 0 3 0 0 0 0 3 5 0 0 0 T i m e [ m s ] 8 0 7 0 Intensity [arb. Units] 6 0 Counts/10ms 5 0 Spectral Shift 4 0 3 0 2 0 1 0 0 0 5 0 0 0 T i m e [ m s ] 8 0 7 0 6 0 Counts/10ms 5 0 4 0 3 0 550 560 570 580 590 600 610 620 630 2 0 1 0 Wavelength [nm] 0 1 4 0 0 0 1 4 5 0 0 1 5 0 0 0 1 5 5 0 0 1 6 0 0 0 1 6 5 0 0 1 7 0 0 0 T i m e [ m s ] Spectral diffusion 16.5 8 0 7 0 16.0 6 0 Counts/10ms 5 0 15.5 4 0 FWHM [nm] 3 0 15.0 2 0 14.5 1 0 0 3 0 0 0 3 0 0 5 0 0 3 1 0 0 0 3 1 5 0 0 3 2 0 0 0 3 2 5 0 3 0 3 0 0 0 3 3 5 0 0 3 4 0 0 0 3 4 5 0 0 3 5 0 0 0 14.0 T i m e [ m s ] 13.5 Increasing trap states 13.0 0 50 100 150 200 250 300 350 Decreasing quantum confinement size Time [sec]

“blinking” of a single QD ON OFF • Quantized levels in blinking � “counter” • No enhancement in the emission intensity � “measure cluster behavior”

Quantum Dot - Blinking Analysis Poisson Distribution <0.01% Background Counts: Mean = 2.33 counts/ms, σ = 1.52 mean+ σ = 3.86 +2 σ = 5.40 +3 σ = 6.92 +4 σ = 8.44 (Left) A 10 second background transient extracted from a 10 minute transient and collected from a dark region of the quantum dot sample. (Right) A histogram analysis of the background counts plotted relative to the mean and the standard deviation ( σ ) of the measurement. The mean+4 σ value was found to be above 99.99% of the background counts in the measurement and was employed as the threshold value. A threshold analysis procedure was performed following Kuno et al. J. Chem. Phys. 115(2):1028, 2001 .

Histogram Analysis (Log-Linear) of On and Off lengths 1 second counts/ms 41 16 5 260 106 4 308 ms On: 6 1 34 7 110 15 ms Off: ON OFF

Probability Density (Log-Linear) of On and Off lengths ON � OFF OFF � ON The on-time probability distribution (left) reflects the On � Off kinetics while the off-time probability distribution (rigt) reflects the Off � On kinetics. Curvature in the log-linear plot implies the blinking process is not exponential, therefore, a single recovery channel or single trap state is unlikely responsible for the blinking phenomenon.

Probability Density (Log-Log) of On and Off lengths OFF ON m off = -1.54 m on = -1.69 SD = 0.27 SD = 0.29 R = -0.94068 R = -0.94481 A linear log-log plot of the on-time (left) and off time (right) probability distribution implies that the blinking dynamics follow an inverse power law according to P( τ ) α τ - m . m can be extracted from the graph using a least Chi-square fit to the data (red line) and allows the blinking dynamics of the bare, carboxyl, and amine quantum dots to be quantitatively compared.