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

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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_at_purdue.edu
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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.

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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.

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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
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Cyntellect core technology (LEAP)F-theta lens
approach permits high throughput
F-theta
12 mm
Microscope
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Ultra High-Throughput Imaging
1.5 mm
8.25 mm
12 mm
Four wells per image at 2.5X 3 min for 1536 wells
in 2 colors
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LEAP Interactive Laser for Laser Ablation or
Opto-injection
The interactive laser is a pulsed NdYAG laser at
1064nm that can be frequency doubled to 532nm or
frequency tripled to 355nm.
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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
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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.
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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
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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
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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
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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.
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Laser-Mediated Purification (Very Specific and
Effective)
Before
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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 MW10kD. Panels
34, 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
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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 23)
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
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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 15) 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
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Table 1 LEAP-Mediated Purification of Adherent
and Suspension Cells
Purity Yield Damage Laser 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.
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Table 2 LEAP-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.
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Laser-Induced Cell Elimination
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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

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Optoinjection(Cell Growth and Viability)
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Shift from Passive to Interactive Imaging
Passive
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Comparison of electroporation and LEAP
opto-injection
Benefits of LEAP Reduced time and labor Fewer
cell manipulations Higher cell yields Combine
primary/secondary screening
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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
Rosenanocapsule 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 AM University Johns Hopkins
University recently deceased
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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
200461A(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.