NANO-OPTICS FOR CELLULAR INVESTIGATION Focus on Microscopy 2004 University City Sheraton

1. NANO-OPTICS FOR CELLULAR INVESTIGATION Focus on Microscopy 2004 University City Sheraton Philadelphia, Pennsylvania Kambiz Pourrezaei, Ph.D.Drexel UniversitySchool of Biomedical Engineering,Science & Health Systems

2. OVERVIEW Optical Nanoprobes in NSOM/Raman: Membrane and Intracellular Detection Mark Contarino, Ed Keough, Irwin Chaiken, Som Tyagi, KambizPourrezaei 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 Nanoparticles Bahar Edrissi, Amir Rezvan, Mark Contarino, Johan Verjans, Chris Reutelingsperger,Jagat Narula, Som Tyagi, Elisabeth Papazoglou, Peter Lelkes

3. 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

4. Simultaneous AFM/NSOM Topographic Information Nanoprobe Dimensions Allow for Sub-Wavelength (< 100 nm) Investigations Evanescent Illumination Reduces Background Fluorescence System Provides Controlled Probe Insertion Nanonics/Renishaw combination SPM/Raman system

5. NSOM Fluorescence Imaging Φ = 500 nm λEmission = 518nm λExcitation = 488nm Schematic of monochromatic light delivered through tapered nanoprobe aperture that excites FITC (green) labelled IgG (red) physi-adsorbed on glass slide. [concentration = 50ug/ml]

6. Topographic Data AFM Simultaneous collection NSOM

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

8. Intracellular Targets membrane Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Labeling procedure conjugates only exposed silica aperture, capturing intracellular fluorescent targets.

9. 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.

10. UV light needle-like fiber Cell under inverted microscope <150 nm Y Y Collect/Count Photons Quantify Concentration Cullum and Vo-Dihn, 2000

11. Preliminary Cell Viability 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

12. SERS Apertureless probes could bring a Raman signal inside cells, fingerprinting biomolecules with a unique Raman spectra • Surface Enhanced Raman Spectroscopy Metallic clusters of < 200nm have shown to enhance the normally weak Raman scattering by as much as 1010!

13. 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.

14. SERS for ZnO Observed enhancement is around 100x’s RED: Enhanced Raman signal from ZnO (435 cm-1) on the gold coated probe. BLUE: Standard Raman signal for ZnO

15. SERS for Collagen - Type I Amine Peak 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.

16. Future Goals & Achievements • Fabrication of 10-20 nm Gold beads at the tip of these nano probes to have Tip Enhanced Raman Spectroscopy (TERS). • TERS for single cell analysis. • Analysis of Protein Structure & its folding process using TERS. • Determination of intracellular signaling pathways using TERS. • Integrating Near Field Scanning Optical Microscope (NSOM) & TERS for cell analysis with 50nm resolution.

17. Q – Dots Biological Applications of Fluorescent Nanoparticles Elisabeth S. Papazoglou, Bahar Edrissi, Amir Rezvan, Mark Contarino, Johan Verjans, Chris Reutelingsperger, Jagat Narula, Kambiz Pourrezaei, Peter Lelkes

18. Unique Optical Properties • Continuous Absorption Profile • Narrow Gaussian-like Emission Profile • Highly Luminescent with Single Excitation Source

19. 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-dots in lungs Aggregation in cardiac vasculature Q-dot uptake in liver

20. Instrumental Setup

22. Q-Dot – Cell Interactions Case I: Absence of specific Targeting Functionality Collagen 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 Gelatin Coated 8-well slide Fluorescence – FITC Filter – 40X – 1s Exposure time

23. Q-Dot – Cell Interactions Case I: Absence of specific Targeting Functionality Matrix-remodeled Uptake in Carcinoma Cells HDC (Human Ductile Carcinoma) + Lake Placid Blue-Carboxyl Terminated (CT) – Transferred Cells Gelatin Coated 8-well slide Phase Contrast – 40X HDC + CT (48 hrs) – Transferred Cells Gelatin Coated 8-well slide Fluorescence – FITC Filter – 40X – 1s Exposure Time

24. Q-Dot – Cell Interactions Case 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 Phase Contrast – 10X 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

25. Q-Dot – Cell Interactions Case II: Specific Targeting Functionality – Our Data Dynamic Observation after Addition of 6:1 Molar Ratio of QD:EGF A431 (Human Squamous Carcinoma) + previously mixed 6:1 molar ratio of Streptavidin-Qdots (655 tracker) and Biotin-EGF – 40 min Phase Contrast – 10X A431 (Human Squamous Carcinoma) + previously mixed 6:1 molar ratio of Streptavidin-Qdots (655 tracker) and Biotin-EGF– 40 min Fluorescence – 10X – TRITC – 1s Exposure Time

26. 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