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

OSC Engineering in Cancer: Imaging and Diagnostics Workshop

November 29, 2005

James F. Leary, Ph.D.

SVM Professor of NanomedicineProfessor of Basic Medical Sciences and Biomedical EngineeringMember: Purdue Cancer Center; Oncological Sciences Center;

Bindley Biosciences Center; Birck Nanotechnology CenterPurdue University, W. Lafayette, IN 47907

Email: jfleary@purdue.edu

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.

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.

High-throughput Cell Separation for Delivery of Highly Enriched Cell Subpopulations for Gene Expression Microarray Analysis of Nanoparticle-Treated Cells

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

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 .

Cyntellect core technology (LEAP™)F-theta lens approach permits high throughput Cyntellect core technology (LEAP™)F-theta lens approach permits high throughput

12 mm

1.5 mm

Major speed advantages

Laser-based manipulation enabled

F-theta

Microscope

Large areas imaged by rapid mirror deflectionsF-theta lens is flat field corrected (in focus) over large areaLaser steering to hit specific cells via rapid mirror deflections

Small areas imaged by slow sample movementsRefocus after each moveNo laser or beam steering

Ultra High-Throughput Imaging

8.25 mm

1.5 mm

Four wells per image at 2.5X3 min for 1536 wells in 2 colors

12 mm

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.

LEAP: Optical Path

LaserBeam Expander

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Bright Field Lamp

Excitation Lamp

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LEAP: Robotic Sample LoadingThe 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.

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

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

High-Speed laser Opto-Injection of Nanomaterials into Selected Single Cells

human cell

nanoparticle532 nm laser beam

(Ref: Clark et al., 2004)

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)

High-Speed Laser Ablation of Non Opto-Injected Cells

intact cell

nanoparticle 532 nm laser beam

Laser-ablated cell

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.

Before

Laser-Mediated Purification

(Very Specific and Effective)

Laser-Mediated Purification

(Very Specific and Effective)

AfterNon-irradiated

control

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.

LEAP laser opto-injection of macromolecules into selected living 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

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).

High-throughput sorting of Human KG-1a stem progenitor cells from sparse mixture of T-lymphocytes by LEAP laser ablation

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

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

High-throughput sorting of Human KG-1a stem progenitor cells from dense mixture of T-lymphocytes by LEAP laser ablation

Table 1LEAP-Mediated Purification of Adherent and Suspension Cells

Purity Yield DamageLaser Power

Adherent Cells - Confluent (Region)*

100% 90% Some 50-100%

Adherent Cells - Confluent (Individual up to 5%)

100% 80% Some 50-100%

Adherent Cells - Low Density (Individual)

90% 90% Some 25-75%

Suspension Cells - High Density (Individual)

90-95% 50-75% None 25-50%

Suspension Cells - Low Density (Individual)

95-100% 80% None 25-75%

*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.

Table 2LEAP-Mediated Optoinjection of Adherent and Suspension Cells

Percent* Optoinjection

Delivery Efficacy

**

Indirect***

Optoinjection

Visible Damage

Laser Power

Adherent Cells

100 High 30-60µm None 25-50%

Suspension Cells

100 Low 5-30µm None 10-20%

*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.

Laser-Induced Cell EliminationLaser-Induced Cell Elimination

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1.E+07 1.E+08 1.E+09 1.E+10 1.E+11

Power Density

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Photothermal

Photochemical

Photomechanical

Laser-Based Cell ManipulationLaser-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

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Optoinjection(Cell Growth and Viability)

Optoinjection(Cell Growth and Viability)

Interactive

Shift from Passive to Interactive Imaging

Shift from Passive to Interactive Imaging

Passive

Comparison of electroporation and LEAP opto-injection

Bulk cell culture trypsinize

Transfect in electroporation cuvette

Replate and culture 1-3 days trypsinizeSort by flow cytometry

Replate and culture 1-3 days trypsinize

Replate in assay platesCarry out cell-based assay

Electroporation (3-6 days)

Bulk cell culture

trypsinize

Replate in assay plates

Opto-inject

Select opto-injected cells by laser ablation of all others

LEAP opto-injection (1 day)

Benefits of LEAPReduced time and laborFewer cell manipulations

Higher cell yieldsCombine primary/secondary

screening

Our MCF Team and Current CollaboratorsMolecular Cytometry Facility

(MCF)

Director: James Leary--------------------------------------------------

UTMBJacob Smith* – mathematics and scientific programming Tarl Prow** – nanotechnology; confocal microscopy; molecular biosensors for HCVPeter Szaniszlo – HHV6/HIV; stem cells; microgenomics (UTMB)Nan Wang – cell culture, molecular biology assays (UTMB)Bill Rose–nanocapsule design (UTMB)------------------------------------------------

PurdueLab Dir: Lisa Reece – flow cytometry/ cell-bead sorting for proteomicsChristy Cooper- bioanalytical chemistry of nanocapsulesMeggie Grafton (Purdue) -BioMEMSEmily Haglund (Purdue)-nanocapsulesMary-Margaret Seale (Purdue) -nanocapsulesMichael Zordan (Purdue) - LEAP technology

Mathematics/StatisticsJames Hokanson*** (UTMB)Judah Rosenblatt (UTMB)Seza Orcun (Purdue)

Combinatorial chemistry/aptamersDavid Gorenstein (UTMB)Xianbin Yang (UTMB)Cagri Savran (Purdue)

BioinformaticsBruce Luxon (UTMB)Seza Orcun (Purdue)

Confocal ImagingMassoud Motamedi (UTMB)Gracie Vargas (UTMB)Paul Robinson (Purdue)

DNA RepairStephen Lloyd (Oregon Health Sciences Center)

ProteomicsAlex Kurosky (UTMB)Jo Davisson (Purdue)

Nanocrystal technologyNick Kotov (Univ. Michigan)Jo Davisson (Purdue)

Nanocapsule technologyYuri Lvov (Louisiana Tech U)Don Bergstrom (Purdue)Kinam Park (Purdue)

Microfluidics/engineeringRashid Bashir (Purdue)

LEAP technologyFred Koller (Cyntellect, Inc.San Diego, CA)

In-vivo retinal imaging Gerald Lutty group(Johns Hopkins Univ.)

* Texas A&M University

** Johns Hopkins University

*** recently deceased

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.