High-Speed Imaging and Laser Optoinjection of Genes/Macromolecules into Living Cells

1. OSC Engineering in Cancer: Imaging and Diagnostics Workshop November 29, 2005 High-Speed Imaging and Laser Optoinjection of Genes/Macromolecules into Living Cells James F. Leary, Ph.D. SVM Professor of Nanomedicine Professor of Basic Medical Sciences and Biomedical Engineering Member: Purdue Cancer Center; Oncological Sciences Center; Bindley Biosciences Center; Birck Nanotechnology Center Purdue University, W. Lafayette, IN 47907 Email: jfleary@purdue.edu

2. Passive versus Interactive Imaging • Conventional imaging is passive. It processes the image of the cells but does not actively interact with the actual cells in the image. • Interactive imaging allows the user to act upon the cells in the image. For example we can laser ablate cells to remove them from the mixture, or we can laser opto-inject macromolecules or genes into selected cells. We can also interact more than once with the cells in the image. For example, we can laser opto-inject genes into cells, then eliminate (by laser ablation) the cells not laser optoinjected, a process which leaves only the laser opto-injected cells.

3. Interactive Imaging for Cancer Diagnostics and Therapeutics • Purge tumor cells, ex-vivo, for autologous bone marrow transplantation in cancer patients. • Select cancer cell clones for further growth and characterizations. • Select cancer cells on the basis of molecular fluorescence imaging for subsequent genomics or proteomics analyses. • Insert genes, transcriptional factors, RNAi probes, macromolecules into selected cancer cells for subsequent growth and/or characterizations.

4. High-throughput Cell Separation for Delivery of Highly Enriched Cell Subpopulations for Gene Expression Microarray Analysis of Nanoparticle-Treated Cells LEAP™ (Laser-Enabled Analysis and Processing) has throughputs greater than 100,000 events/sec, high cell purity, yield and viability. It can process several cells or a billion cells with an expanded cell range including fragile cells. Another advantage is that it can analyze and purify biohazardous cells without generating aerosols . Fluorescence collection optics of LEAP instrument Shooting at cells inside 384-well plates to eliminate undesired cells and capture desired cells for subsequent gene expression microarray analysis

5. Large areas imaged by rapid mirror deflections • F-theta lens is flat field corrected (in focus) over large area • Laser steering to hit specific cells via rapid mirror deflections • Major speed advantages • Laser-based manipulation enabled • Small areas imaged by slow sample movements • Refocus after each move • No laser or beam steering 1.5 mm Cyntellect core technology (LEAP™)F-theta lens approach permits high throughput F-theta 12 mm Microscope

6. Ultra High-Throughput Imaging 1.5 mm 12 mm 8.25 mm Four wells per image at 2.5X 3 min for 1536 wells in 2 colors

7. LEAP: Interactive Laser for Laser Ablation or Opto-injection The interactive laser is a pulsed Nd:YAG laser at 1064nm that can be frequency doubled to 532nm or frequency tripled to 355nm.

8. LEAP: Optical Path Legend ___ Laser Path ___Bright Field Path ___Excitation Path X- Galvo Y-Galvo Mirror Filter Wheel Camera 2 Mirror Filter Wheel Housing for Focusing Lens Prism Filter Wheel Camera 1 Beam Expander Objective Housing Excitation Lamp Mirror Stage Bright Field Lamp Objective Filter Wheel Filter Wheel Laser Filter Wheel ND Filter Beam Expander Mirror

9. LEAP: Robotic Sample Loading The robotic sample handler can process almost any format from slides to tissue culture dishes in manual mode and 24-, 96-, and 384 well plates for high-throughput processing.

10. LEAP Imaging System Laser Optoinjection for high-speed microinjection of genes and other molecules into selected cells Robotic delivery of multi-well dishes or other culture vessels for LEAP analysis Laser ablation or optoinjection of cells, in this case on a slide, under a cover slip

11. High-Speed laser Opto-Injection of Nanomaterials into Selected Single Cells LEAP (Laser Enabled Analysis and Processing) (Cyntellect, Inc.) laser opto-injection of nanoparticles into human cells for subsequent characterization of the global gene response to nanomaterials using gene expression microarrays human cell 532 nm laser beam (Ref: Clark et al., 2004) nanoparticle

12. High-Speed Laser Ablation of Non Opto-Injected Cells LEAP (Laser Enabled Analysis and Processing) (Cyntellect, Inc.) laser opto-injection of nanoparticles into human cells for subsequent characterization of the global gene response to nanomaterials using gene expression microarrays (after laser ablating non-optoinjected cells) Laser-ablated cell intact cell nanoparticle 532 nm laser beam

13. Microgenomics of Primary Human Adult Stem Cells versus Established Cell Lines Using gene expression microarray (“gene chip”) analyses of purified human stem cells, we can try to learn how to de-differentiate adult stem cells to make them more embryonic-like for improved regenerative medicine applications.

14. After Non-irradiated control Laser-Mediated Purification (Very Specific and Effective) Before

15. LEAP laser opto-injection of macromolecules into selected living cells Confocal images (right hand side) of optoinjected suspension cells (HeLa). Panels 1 and 2. All cells within the targeted square area (dashed square) were opto-injected with tetra methyl rhodamine-conjugated dextran; MW=10kD. Panels 3&4, Higher magnification (63x) images of the optoinjected area showed a visual difference between the dextran uptake of individual cells. In other experiments we successfully optoinjected dextrans up to 100kD. Ref: Szaniszlo, P., Rose, W.A., Wang, N., Reece, L.M., Tsulaia, T.V., Hanania, E.G., Elferink, C.J., Leary, J.F. "Scanning Cytometry with a LEAP: Laser-Enabled Analysis and Processing of Live Cells In Situ" Cytometry (accepted) 2005

16. High-throughput sorting of Human KG-1a stem progenitor cells from sparse mixture of T-lymphocytes by LEAP laser ablation Purification of suspension cells-low density. CD34-FITC labeled KG-1a cells (green) and CD4-PE labeled CEM cells (orange) were mixed then KG-1a cells were purified by LEAP ablation/ detachment of the CEM cells, Panel 1. KG-1a/CEM before; Panel 2. KG-1a/CEM after; Panel 3. KG-1a cells before/ after (green/black); Panel 4. CEM cells before/after (orange/ black). All CEM cells have been ablated (region 1) or detached (regions 2&3) while most KG-1a cells remain unaffected (region 4). Very few KG-1a cells were moved (region 5). Ref: Szaniszlo, P., Rose, W.A., Wang, N., Reece, L.M., Tsulaia, T.V., Hanania, E.G., Elferink, C.J., Leary, J.F. "Scanning Cytometry with a LEAP: Laser-Enabled Analysis and Processing of Live Cells In Situ" Cytometry (accepted) 2005

17. High-throughput sorting of Human KG-1a stem progenitor cells from dense mixture of T-lymphocytes by LEAP laser ablation Purification of suspension cells-high density. KG-1a cells (green) and CEM cells (orange) were mixed then KG-1a cells were purified by LEAP ablation/ detachment, Panel 1. KG-1a/CEM before; Panel 2. KG-1a/CEM after; Panel 3. KG-1a cells before/after (green/black); Panel 4. CEM cells before/after (orange/ black). Most CEM cells have been detached (region 1&5) but some were not affected (region 4). Although some KG-1a cells have been moved (region 3), most KG-1a cells were unaffected (region 2). Ref: Szaniszlo, P., Rose, W.A., Wang, N., Reece, L.M., Tsulaia, T.V., Hanania, E.G., Elferink, C.J., Leary, J.F. "Scanning Cytometry with a LEAP: Laser-Enabled Analysis and Processing of Live Cells In Situ" Cytometry (accepted) 2005

18. Table 1 LEAP-Mediated Purification of Adherent and Suspension Cells *Ablating defined regions from a confluent monolayer of cells. All other rows describe purification of cell samples from individual contaminating cells. Ref: Szaniszlo, P., Rose, W.A., Wang, N., Reece, L.M., Tsulaia, T.V., Hanania, E.G., Elferink, C.J., Leary, J.F. "Scanning Cytometry with a LEAP: Laser-Enabled Analysis and Processing of Live Cells In Situ" Cytometry (accepted) 2005.

19. Table 2 LEAP-Mediated Optoinjection of Adherent and Suspension Cells *Percent Optoinjection: Percentage of optoinjected cells out of all the cells that were targeted **Delivery Efficacy: Relative visual brightness of fluorescent dextran-optoinjected cells ***Indirect Optoinjection: Width of the annular zone of cells unintentionally optoinjected Ref: Szaniszlo, P., Rose, W.A., Wang, N., Reece, L.M., Tsulaia, T.V., Hanania, E.G., Elferink, C.J., Leary, J.F. "Scanning Cytometry with a LEAP: Laser-Enabled Analysis and Processing of Live Cells In Situ" Cytometry (accepted) 2005.

20. Laser-Induced Cell Elimination

21. Laser-Based Cell Manipulation • Lethal effects (thermal, chemical, mechanical) • Optoinjection (selective cell transfection) • Photoactivation, uncaging, photochemistry • Chromophore-assisted laser inactivation (CALI) • Photobleaching • Interrogation (excitation of fluorescent reporter) • Tweezers/scissors

22. Optoinjection(Cell Growth and Viability)

23. Interactive Shift from Passive to Interactive Imaging Passive

24. Electroporation (3-6 days) LEAP opto-injection (1 day) Bulk cell culture trypsinize Transfect in electroporation cuvette Replate and culture 1-3 days trypsinize Sort by flow cytometry Replate and culture 1-3 days trypsinize Replate in assay plates Carry out cell-based assay Bulk cell culture trypsinize Replate in assay plates Opto-inject Select opto-injected cells by laser ablation of all others Comparison of electroporation and LEAP opto-injection Benefits of LEAP Reduced time and labor Fewer cell manipulations Higher cell yields Combine primary/secondary screening

25. Our MCF Team and Current Collaborators Molecular Cytometry Facility (MCF) Director: James Leary --------------------------------------------------UTMB Jacob Smith* – mathematics and scientific programming Tarl Prow** – nanotechnology; confocal microscopy; molecular biosensors for HCV Peter Szaniszlo – HHV6/HIV; stem cells; microgenomics (UTMB) Nan Wang – cell culture, molecular biology assays (UTMB) Bill Rose–nanocapsule design (UTMB) ------------------------------------------------ Purdue Lab Dir: Lisa Reece – flow cytometry/ cell-bead sorting for proteomics Christy Cooper- bioanalytical chemistry of nanocapsules Meggie Grafton (Purdue) -BioMEMS Emily Haglund (Purdue)-nanocapsules Mary-Margaret Seale (Purdue) -nanocapsules Michael Zordan (Purdue) - LEAP technology Combinatorial chemistry/aptamers David Gorenstein (UTMB) Xianbin Yang (UTMB) Cagri Savran (Purdue) Mathematics/Statistics James Hokanson*** (UTMB) Judah Rosenblatt (UTMB) Seza Orcun (Purdue) Confocal Imaging Massoud Motamedi (UTMB) Gracie Vargas (UTMB) Paul Robinson (Purdue) DNA Repair Stephen Lloyd (Oregon Health Sciences Center) Nanocrystal technology Nick Kotov (Univ. Michigan) Jo Davisson (Purdue) In-vivo retinal imaging Gerald Lutty group (Johns Hopkins Univ.) Nanocapsule technology Yuri Lvov (Louisiana Tech U) Don Bergstrom (Purdue) Kinam Park (Purdue) Bioinformatics Bruce Luxon (UTMB) Seza Orcun (Purdue) LEAP technology Fred Koller (Cyntellect, Inc. San Diego, CA) Proteomics Alex Kurosky (UTMB) Jo Davisson (Purdue) Microfluidics/engineering Rashid Bashir (Purdue) * Texas A&M University ** Johns Hopkins University *** recently deceased

26. LEAP Technology References & Patents • Koller MR, Hanania EG, Stevens J, Eisfeld TM, Sasaki GC, Fieck A, Palsson BO. High-throughput laser-mediated in situ cell purification with high purity and yield. Cytometry 2004;61A(2):153-61. • Clark IB, Hanania EG, Stevens J, Gallina M, Fieck A, Brandes R, Palsson BO, Koller MR. Optoinjection for efficient delivery of a broad range of compounds and macromolecules into diverse cell types with low toxicity. in press 2005. • Szaniszlo, P., Rose, W.A., Wang, N., Reece, L.M., Tsulaia, T.V., Hanania, E.G., Elferink, C.J., Leary, J.F. "Scanning Cytometry with a LEAP: Laser-Enabled Analysis and Processing of Live Cells In Situ" Cytometry (accepted) 2005. • Palsson B, Koller M, Eisfeld T; Method and apparatus for selectively targeting specific cells within a mixed cell population. USA patent 6,534,308. 2003. • Koller M, Hanania, EG., Eisfeld, TM., Palsson, BO.; Optoinjection methods. USA patent 6,753,161. 2004.