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
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
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
Cyntellect core technology (LEAP™)
F-theta lens approach permits high throughput
F-theta
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
12 mm
Microscope
1.5 mm
Small areas imaged by
slow sample
movements
Refocus after each
move
No laser or beam
steering
Major speed
advantages
Laser-based
manipulation
enabled
Ultra High-Throughput Imaging
1.5 mm
8.25 mm
Four wells per image at 2.5X
3 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
Legend
XGalvo
___ Laser Path
Mirror
Camera 2
Excitation Lamp
Beam
Expander
Filter Wheel
Filter
Wheel
Camera 1
ND Filter
Filter Wheel
Mirror
Beam
Objective
Expander Housing
Mirror
Objective
Bright
Field
Lamp
Filter Wheel
Stage
Prism
Housing for Focusing Lens
Filter
Wheel
___ Excitation Path
Filter Wheel
Mirror
YGalvo
___Bright Field Path
Laser
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.
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
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
(Ref: Clark et al., 2004)
532 nm laser beam
nanoparticle
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)
Laserablated cell
intact cell
nanoparticle
532 nm laser beam
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.
Laser-Mediated Purification
(Very Specific and Effective)
Before
After
Non-irradiated
control
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 optoinjected 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
High-throughput sorting of Human KG-1a stem progenitor cells from
sparse mixture of T-lymphocytes by LEAP laser ablation
Purification of suspension cellslow density. CD34-FITC labeled
KG-1a cells (green) and CD4PE 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
High-throughput sorting of Human KG-1a stem progenitor cells from
dense mixture of T-lymphocytes by LEAP laser ablation
Purification of suspension cellshigh 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
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.
Table 2
LEAP-Mediated Optoinjection of Adherent and Suspension Cells
Percent*
Optoinjection
Delivery
Efficacy
**
Indirect*
**
Optoinje
ction
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 Elimination
100
Photothermal
Cell Viability (%)
80
Photochemical
Photomechanical
60
40
20
0
1.E+07
1.E+08
1.E+09
Power Density
1.E+10
1.E+11
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
Optoinjection
(Cell Growth and Viability)
1000000
100
Optoinjected
Control
100000
Cell Viability
Cell Number
75
10000
1000
50
25
100
0
10
0
2
4
6
Day
8
10
0
2
4
6
Day
8
10
Shift from Passive to Interactive
Imaging
Passive
Interactive
Comparison of electroporation and
LEAP opto-injection
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
Benefits of LEAP
Reduced time and labor
Fewer cell manipulations
Higher cell yields
Combine primary/secondary
screening
Our MCF Team and Current Collaborators
Combinatorial
chemistry/aptamers
David Gorenstein (UTMB)
Xianbin Yang (UTMB)
Cagri Savran (Purdue)
DNA Repair
Stephen Lloyd (Oregon Health
Sciences Center)
Nanocrystal technology
Nick Kotov (Univ. Michigan)
Jo Davisson (Purdue)
Nanocapsule technology
Yuri Lvov (Louisiana Tech U)
Don Bergstrom (Purdue)
Kinam Park (Purdue)
Proteomics
Alex Kurosky (UTMB)
Jo Davisson (Purdue)
* Texas A&M University
** Johns Hopkins University
*** recently deceased
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
Mathematics/Statistics
James Hokanson*** (UTMB)
Judah Rosenblatt (UTMB)
Seza Orcun (Purdue)
Confocal Imaging
Massoud Motamedi (UTMB)
Gracie Vargas (UTMB)
Paul Robinson (Purdue)
In-vivo retinal imaging
Gerald Lutty group
(Johns Hopkins Univ.)
Bioinformatics
Bruce Luxon (UTMB)
Seza Orcun (Purdue)
LEAP technology
Fred Koller (Cyntellect, Inc.
San Diego, CA)
Microfluidics/engineering
Rashid Bashir (Purdue)
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.