School of Biomedical Engineering, Science & Health Systems V 1.0 SD [020327] Kambiz Pourrezaei,...

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School of Biomedical Engineering, Science & Health Systems

V 1.0 SD [020327]

Kambiz Pourrezaei, Ph.D.Drexel UniversitySchool of Biomedical Engineering,Science & Health Systems

NANO-OPTICS FOR CELLULAR INVESTIGATION

Focus on Microscopy 2004

University City Sheraton

Philadelphia, Pennsylvania



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OVERVIEWOptical Nanoprobes in NSOM/Raman: Membrane and Intracellular Detection Mark Contarino, Ed Keough, Irwin Chaiken, Som Tyagi, Kambiz Pourrezaei

Surfaced Enhanced Raman Spectroscopy: Label-Free Detection using SERS Optimized Nanoprobes Vishal Kamat, Ed Keough, Mark Contarino, Elisabeth Papazoglou, Kambiz Pourrezaei, Som Tyagi

Quantum Dots: Biological Applications of Fluorescent NanoparticlesBahar Edrissi, Amir Rezvan, Mark Contarino, Johan Verjans, Chris Reutelingsperger, Jagat Narula, Som Tyagi, Elisabeth Papazoglou, Peter Lelkes



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Fiber Optic Nano-probe Fabrication

• By exposing the silica core, the fiber is pulled into nano probes using the P2000 Micropipette Puller.

• Fiber tips are typically 30-60nm in diameter.• Fibers are cleaned using a wet chemical dip-process.• By varying the layering method and deposition parameters, we can have

smooth & uniform gold film or uniform gold blobs on the surface of these nano probes.

P2000 Micropipette Puller

30nm Au Sputtered Using Standard Smooth Layering

E-Beam Evaporation of 100nm Au



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Nanonics/Renishaw combination SPM/Raman system

• Simultaneous AFM/NSOM Topographic Information

• Nanoprobe Dimensions Allow for Sub-Wavelength (< 100 nm) Investigations

• Evanescent Illumination Reduces Background Fluorescence

• System Provides Controlled Probe Insertion



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Φ = 500 nm

λExcitation = 488nm

λEmission = 518nm

NSOM Fluorescence Imaging

Schematic of monochromatic light delivered through tapered nanoprobe aperture that excites FITC (green) labelled IgG (red) physi-adsorbed on glass slide. [concentration = 50ug/ml]



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Topographic Data

Simultaneous collection



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Fluorescence Recovery

Wavelength (nm)

Co

un

ts

Fluorescence collected of FITC-labeled anti-5-His IgG using Nanonics/ Renishaw platform under 488nm excitation through metallic coated nanoprobe (500nm aperture)



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Intracellular Targets

Si

O

O

Si

Si

OH

OH

OH

Si

CH3O

CH3O

CH3O

O O

SiSi

O

O

Si

Si

O

O

O

O O

NH2 Ab

SiSi

O

O

Si

Si

O

O

O

OOH

NH

Ab

+

Silica surface

3-Glycidoxypropyl-trimethoxysilane

1. o/n

2. 50C

Borate bufferpH 9.5

o/nRT

Y

Y Y

YY YYY

Y

Y

YYY

YYY YY

Y

YY

YY

Labeling procedure conjugates only exposed silica aperture, capturing intracellular fluorescent targets.

membrane



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Antibody Functionalization of Silica Surface

Phase contrast and fluorescent contrast images of control fiber (left) and FITC labeled antibody functionalized fiber (right). The fluorescence in the control is due to a defect in the fiber, as seen in the phase image.



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Cell under inverted microscope

Collect/Count Photons

Quantify Concentration

UV light

needle-like fiber

Y Y

<150 nm

Cullum and Vo-Dihn, 2000



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Preliminary Cell Viability

S2 Viability w ith Trypan Blue Exclusion

0

2

4

6

8

10

12

1 2 3 4 5 6 7 8

S2 Cell Num ber

Tim

e un

til b

lue

(max

10

min

)

38 nm tip

34 nm tip

31 nm tip

48 nm tip

Trypan blue experiments with four probe diameters. After several cells were probed with the same tip, there was an observed residue buildup on the tip. After this event, subsequent probes resulted in membrane adherence to the probe, rupturing cellular integrity



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SERS• Surface Enhanced

Raman Spectroscopy

Metallic clusters of < 200nm have shown to enhance the normally weak Raman scattering by as much as 1010!

Apertureless probes could bring a Raman signal inside cells, fingerprinting biomolecules with a unique Raman spectra



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SERS Optimized Nano-Probes

• The tapered probe is coated with gold film with the fabrication of well organized gold features on the surface of the tip (shown above).

• The tip of the probe will give optimum condition for maximum enhancement of the Raman signal.

• Functionalization of the tip with target specific antibody will enable us to detect any antigen in the cell.



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SERS for ZnO

RED: Enhanced Raman signal from ZnO (435 cm-1) on the gold coated probe.

BLUE: Standard Raman signal for ZnO

Observed enhancement is around 100x’s



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SERS for Collagen - Type I

In the inset : Red Enhanced Raman Spectrum of Collagen Type I.

Violet Normal Raman Spectrum of Collagen Type I

The enhancement factor is around 50x’s.

Amine Peak



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Future Goals & Achievementso Fabrication of 10-20 nm Gold beads at

the tip of these nano probes to have Tip Enhanced Raman Spectroscopy (TERS).

o TERS for single cell analysis.

o Analysis of Protein Structure & its folding process using TERS.

o Determination of intracellular signaling pathways using TERS.

o Integrating Near Field Scanning Optical Microscope (NSOM) & TERS for cell analysis with 50nm resolution.



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Elisabeth S. Papazoglou, Bahar Edrissi, Amir Rezvan, Mark Contarino, Johan Verjans, Chris Reutelingsperger, Jagat Narula, Kambiz

Pourrezaei, Peter Lelkes

Q – DotsBiological Applications ofFluorescent Nanoparticles



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• Continuous Absorption Profile• Narrow Gaussian-like Emission Profile• Highly Luminescent with Single Excitation

Source

Unique Optical Properties



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In vivo imaging of cardiac tissue apoptosis using Annexin-V targeted quantum dots.

-Ischemia/reperfusion studies in live animal murine model in collaboration from University Maastricht, Neth.

Q-dot uptake in liver

Aggregation in cardiac vasculature

Q-dots in lungs



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Instrumental Setup



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Q-Dot – Cell InteractionsCase I: Absence of specific Targeting FunctionalityCollagen Matrix Remodeling promotes Q-dot Uptake

BAEC (Bovine Aortic Endothelial Cells) + CT (48 hrs) – Transferred Cells

Gelatin Coated 8-well slide

Phase Contrast – 40X

BAEC + CT (48 hrs) – Transferred Cells


Fluorescence – FITC Filter – 40X – 1s Exposure time



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Q-Dot – Cell Interactions

Case I: Absence of specific Targeting FunctionalityMatrix-remodeled Uptake in Carcinoma Cells

HDC (Human Ductile Carcinoma) + Lake Placid Blue-Carboxyl Terminated (CT) – Transferred Cells



HDC + CT (48 hrs) – Transferred Cells


Fluorescence – FITC Filter – 40X – 1s Exposure Time



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Q-Dot – Cell InteractionsCase II: Specific Targeting Functionality

QD:EGF, through STV-Biotin

A431 (Human Squamous Carcinoma) + previously mixed 6:1 molar ratio of Streptavidin-Qdots (655 tracker) and Biotin-EGF – 5 min



Fluorescence – 10X – TRITC – 1s Exposure Time



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Q-Dot – Cell InteractionsCase II: Specific Targeting Functionality – Our Data

Dynamic Observation after Addition of 6:1 Molar Ratio of QD:EGF




Fluorescence – 10X – TRITC – 1s Exposure Time



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Q-Dots – Our Goals

I) Study internalization of EGF receptor in Squamous Cell Carcinoma

• Ligand mediated•Anti-body mediated (TJU- Dr. U. Rodeck)

II) Study IL-15 mechanism (Interleukin expressed in asthma)Protein Institute (Dr. I. Chaiken)

III) Combine with our Nanoprobes

School of Biomedical Engineering, Science & Health Systems V 1.0 SD [020327] Kambiz Pourrezaei,...

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