U.S. patent application number 12/533801 was filed with the patent office on 2010-04-08 for optoinjection methods.
This patent application is currently assigned to CYNTELLECT, INC.. Invention is credited to Timothy M. Eisfeld, Elie G. Hanania, Manfred R. Koller, Bernhard O Palsson.
Application Number | 20100086984 12/533801 |
Document ID | / |
Family ID | 25504857 |
Filed Date | 2010-04-08 |
United States Patent
Application |
20100086984 |
Kind Code |
A1 |
Koller; Manfred R. ; et
al. |
April 8, 2010 |
OPTOINJECTION METHODS
Abstract
Optoinjection method for transiently permeabilizing a target
cell by (a) illuminating a population of cells contained in a
frame; (b) detecting at least one property of light directed from
the frame; (c) locating a target cell by the property of light; and
(d) irradiating the target cell with a pulse of radiation.
Inventors: |
Koller; Manfred R.; (San
Diego, CA) ; Hanania; Elie G.; (San Diego, CA)
; Eisfeld; Timothy M.; (San Diego, CA) ; Palsson;
Bernhard O; (La Jolla, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET, FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Assignee: |
CYNTELLECT, INC.
San Diego
CA
|
Family ID: |
25504857 |
Appl. No.: |
12/533801 |
Filed: |
July 31, 2009 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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11894720 |
Aug 20, 2007 |
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12533801 |
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10814966 |
Mar 30, 2004 |
7300795 |
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11894720 |
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09961691 |
Sep 21, 2001 |
6753161 |
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10814966 |
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09728281 |
Nov 30, 2000 |
6514722 |
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09961691 |
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09451659 |
Nov 30, 1999 |
6534308 |
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09728281 |
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09049677 |
Mar 27, 1998 |
6143535 |
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09451659 |
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08824968 |
Mar 27, 1997 |
5874266 |
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09049677 |
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Current U.S.
Class: |
435/173.5 |
Current CPC
Class: |
C12N 15/1034 20130101;
C12N 13/00 20130101; G01N 33/5005 20130101; G01N 33/56966 20130101;
C12M 35/02 20130101; C12N 15/87 20130101; C12N 5/0093 20130101;
G01N 33/5091 20130101; C12M 47/06 20130101; C12N 5/0087
20130101 |
Class at
Publication: |
435/173.5 |
International
Class: |
C12N 13/00 20060101
C12N013/00 |
Claims
1. A method for transiently permeabilizing a target cell,
comprising the steps of: (a) illuminating a population of
substantially stationary cells contained in a frame; (b) obtaining
a static representation of at least one property of light directed
simultaneously from the frame; (c) locating a target cell in the
population of cells, wherein the target cell is located with
reference to the static representation; and (d) irradiating the
target cell with a pulse of radiation, whereby the target cell is
transiently permeabilized.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/894,720, filed Aug. 20, 2007, which is a
continuation of U.S. patent application Ser. No. 10/814,966, filed
on Mar. 30, 2004, now U.S. Pat. No. 7,300,795; which is a
continuation of U.S. patent application Ser. No. 09/961,691, filed
on Sep. 21, 2001, now U.S. Pat. No. 6,753,161; which is a
continuation-in-part of U.S. patent application Ser. No.
09/728,281, filed Nov. 30, 2000, now U.S. Pat. No. 6,514,722; which
is a continuation-in-part of U.S. patent application Ser. No.
09/451,659, filed Nov. 30, 1999, now U.S. Pat. No. 6,534,308; which
is a continuation-in-part of U.S. patent application Ser. No.
09/049,677, filed Mar. 27, 1998, now U.S. Pat. No. 6,143,535; which
is a continuation-in-part of U.S. patent application Ser. No.
08/824,968, filed Mar. 27, 1997, now U.S. Pat. No. 5,874,266, each
of which is incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] This invention relates to methods for cell manipulation and
more specifically to methods for transiently permeabilizing a cell
so that a variety of exogenous materials, such as expressible
foreign DNA, can be loaded into the cell.
[0003] Previous loading methods have included chemical treatments,
microinjection, electroporation and particle bombardment. However,
these techniques can be time-consuming and suffer from low yields
or poor cell survival. Another technique termed "optoporation" has
used light directed toward cells and the surrounding media to
induce shock waves, thereby causing small holes to form temporarily
in the surface of nearby cells, allowing materials to
non-specifically enter cells in the area. Another technique termed
"optoinjection" also uses light, but directs the light to specific
cells. Nevertheless, previous light-based implementations
techniques have suffered from the same disadvantages as other
loading techniques.
[0004] Thus, there is a need for a method for rapid and efficient
loading of a variety of exogenous molecules into cells, with high
cell survival rates. The present invention satisfies this need and
provides related advantages as well.
SUMMARY OF THE INVENTION
[0005] The present invention provides optoinjection methods for
transiently permeabilizing a target cell. In the general method,
the steps are (a) illuminating a population of cells contained in a
frame; (b) detecting at least one property of light directed from
the frame; (c) locating a target cell by the property of light; and
(d) irradiating the target cell with a pulse of radiation.
[0006] In particular embodiments, a static representation is
obtained when the population of cells is substantially stationary;
the cells are illuminated through a lens having a numerical
aperture of at most 0.5; the pulse of radiation has a diameter of
at least 10 microns at the point of contact with the target cell;
or the resulting pulse of radiation delivers at most 1
.mu.J/.mu.m.sup.2. As a result, the method provides rapid and
efficient loading of a variety of exogenous molecules into cells,
with high cell survival rates.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 is a perspective view of one embodiment of a cell
treatment apparatus and illustrates the outer design of the housing
and display.
[0008] FIG. 2 is a perspective view of one embodiment of a cell
treatment apparatus with the outer housing removed and the inner
components illustrated.
[0009] FIG. 3 is a block diagram of the optical subassembly design
within one embodiment of a cell treatment apparatus.
[0010] FIG. 4 is a perspective view of one embodiment of an optical
subassembly within one embodiment of a cell treatment
apparatus.
[0011] FIG. 5 is a side view of one embodiment of an optical
subassembly that illustrates the arrangement of the scanning lens
and the movable stage.
[0012] FIG. 6 is a bottom perspective view of one embodiment of an
optical subassembly.
[0013] FIG. 7 is a top perspective view of the movable stage of the
cell treatment apparatus.
[0014] FIG. 8 shows cells under broad-spectrum light (8A), cells
showing loading of Texas-Red-Dextran (8B) and nonviable cells
(8C).
[0015] FIG. 9 illustrates that the efficiency of optoinjection is
energy dose-dependent
[0016] FIG. 10 compares expression of a plasmid in optoinjected
cells (10B) compared to control cells without optoinjection
(10A).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0017] A method and apparatus is described for selectively
identifying, and individually targeting with an energy beam,
specific cells within a cell population for the purpose of inducing
a response in the targeted cells. The population of cells can be a
mixed population or homogenous in origin. The responses of any of
the embodiments of the methods and apparatuses of the invention can
be lethal or non-lethal. Examples of such responses are set forth
above and throughout this disclosure. The cells targeted can be
labeled as is often the case when the specimen is a mixed
population. On the other hand, when the specimen is homogenous, the
targeted cells can be those individual cells that are being
interrogated or intersected by the illumination source or the
energy beam, in order to study the response of the cell. For
instance, such responses include the morphological or physiological
characteristics of the cell. Generally, the method first employs a
label that acts as a marker to identify and locate individual cells
of a first population of cells within a cell mixture that is
comprised of the first population of cells and a second population
of cells. The cells targeted by the apparatus and methods herein
are those that are selectively labeled, in the case of a mixed
population of cells, or the ones undergoing interrogation or
intersection by the illumination source or energy beam.
[0018] The chosen label can be any that substantially identifies
and distinguishes the first population of cells from the second
population of cells. For example, monoclonal antibodies that are
directly or indirectly tagged with a fluorochrome can be used as
specific labels. Other examples of cell surface binding labels
include non-antibody proteins, lectins, carbohydrates, or short
peptides with selective cell binding capacity. Membrane
intercalating dyes, such as PKH-2 and PKH-26, could also serve as a
useful distinguishing label indicating mitotic history of a cell.
Many membrane-permeable reagents are also available to distinguish
living cells from one another based upon selected criteria. For
example, phalloidin indicates membrane integrity, tetramethyl
rhodamine methyl ester (TMRM) indicates mitochondrial transmembrane
potential, monochlorobimane indicates glutathione reductive stage,
carboxymethyl fluorescein diacetate (CMFDA) indicates thiol
activity, carboxyfluorescein diacetate indicates intracellular pH,
fura-2 indicates intracellular Ca. sup.2+ level, and
5,5',6,6'-tetrachloro-1,1'1,3,3'-tetra-ethylbenzimidazolo
carbocyanine iodide (JC-1) indicates membrane potential. Cell
viability can be assessed by the use of fluorescent SYTO 13 or YO
PRO reagents. Similarly, a fluorescently-tagged genetic probe (DNA
or RNA) could be used to label cells which carry a gene of
interest, or express a gene of interest. Further, cell cycle status
could be assessed through the use of Hoechst 33342 dye to label
existing DNA combined with bromodeoxyuridine (BrdU) to label newly
synthesized DNA.
[0019] It should be noted that if no specific label is available
for cells of the first population, the method can be implemented in
an inverse fashion by utilizing a specific label for cells of the
second population. For example, in hematopoietic cell populations,
the CD34 or ACC-133 cell markers can be used to label only the
primitive hematopoietic cells, but not the other cells within the
mixture. In this embodiment, cells of the first population are
identified by the absence of the label, and are thereby targeted by
the energy beam.
[0020] After cells of the first population are identified, an
energy beam, such as from a laser, collimated or focused non-laser
light, RF energy, accelerated particle, focused ultrasonic energy,
electron beam, or other radiation beam, is used to deliver a
targeted dose of energy that induces the pre-determined response in
each of the cells of the first population, without substantially
affecting cells of the second population.
[0021] One such pre-determined response is photobleaching. In
photobleaching, a label in the form of a dye, such as rhodamine
123, GFP, fluorescein isothiocyanate (FITC), or phycoerythrin, is
added to the specimen before the instant methods are commenced.
After the population of cells has time to interact with the dye,
the energy beam is used to bleach a region of individual cells in
the population. Such photobleaching studies can be used to study
the motility, replenishment, dynamics and the like of cellular
components and processes.
[0022] Another response is internal molecular uncaging. In such a
process, the specimen is combined with a caged molecule prior to
the commencement of the instant methods. Such caged molecules
include the .beta.-2,6-dinitrobenzyl ester of L-aspartic acid or
the 1-(2-nitrophenyl)ethyl ether of
8-hydroxylpyrene-1,3,6-tris-sulfonic acid. Similarly, caging groups
including alphacarboxyl-2-nitrobenzyl (CNB) and
5-carboxylmethoxy-2-nitrobenzyl (CMNB) can be linked to
biologically active molecules as ethers, thioethers, esters,
amines, or similar functional groups. The term "internal molecular
uncaging" refers to the fact that the molecular uncaging takes
place on the surface or within the cell. Such uncaging experiments
study rapid molecular processes sucn as cell membrane permeability
and cellular signaling.
[0023] Yet another response is external molecular uncaging. This
uses approximately the same process as internal molecular caging.
However, in external molecular uncaging, the uncaged molecule is
not attached to or incorporated into the targeted cells. Instead,
the responses of the surrounding targeted cells to the caged and
uncaged variants of the molecule are imaged by the instant
apparatus and methods.
[0024] FIG. 1 is an illustration of one embodiment of a cell
treatment apparatus 10. The cell treatment apparatus 10 includes a
housing 15 that stores the inner components of the apparatus. The
housing includes laser safety interlocks to ensure safety of the
user, and also limits interference by external influences (e.g.,
ambient light, dust, etc.). Located on the upper portion of the
housing 15 is a display unit 20 for displaying captured images of
cell populations during treatment. These images are captured by a
camera, as will be discussed more specifically below. A keyboard 25
and mouse 30 are used to input data and control the apparatus 10.
An access door 35 provides access to a movable stage that holds a
specimen container of cells undergoing treatment.
[0025] An interior view of the apparatus 10 is provided in FIG. 2.
As illustrated, the apparatus 10 provides an upper tray 200 and
lower tray 210 that hold the interior components of the apparatus.
The upper tray 200 includes a pair of intake filters 215A,B that
filter ambient air being drawn into the interior of the apparatus
10. Below the access door 35 is the optical subassembly (not
shown). The optical subassembly is mounted to the upper tray 200
and is discussed in detail with regard to FIGS. 3 to 6.
[0026] On the lower tray 210 is a computer 225 which stores the
software programs, commands and instructions that run the apparatus
10. In addition, the computer 225 provides control signals to the
treatment apparatus through electrical signal connections for
steering the laser to the appropriate spot on the specimen in order
to treat the cells.
[0027] As illustrated, a series of power supplies 230A,B,C provide
power to the various electrical components within the apparatus 10.
In addition, an uninterruptable power supply 235 is incorporated to
allow the apparatus to continue functioning through short external
power interruptions.
[0028] FIG. 3 provides a layout of one embodiment of an optical
subassembly design 300 within an embodiment of a cell treatment
apparatus 10. As illustrated, an illumination laser 305 provides a
directed laser output that is used to excite a particular label
that is attached to targeted cells within the specimen. In this
embodiment, the illumination laser emits light at a wavelength of
532 nm. Once the illumination laser has generated a light beam, the
light passes into a shutter 310 which controls the pulse length of
the laser light.
[0029] After the illumination laser light passes through the
shutter 310, it enters a ball lens 315 where it is focused into a
SMA fiber optic connector 320. After the illumination laser beam
has entered the fiber optic connector 320, it is transmitted
through a fiber optic cable 325 to an outlet 330. By passing the
illumination beam through the fiber optic cable 325, the
illumination laser 305 can be positioned anywhere within the
treatment apparatus and thus is not limited to only being
positioned within a direct light pathway to the optical
components.
[0030] In one embodiment, the fiber optic cable 325 is connected to
a vibrating motor 327 for the purpose of mode scrambling and
generating a more uniform illumination spot.
[0031] After the light passes through the outlet 330, it is
directed into a series of condensing lenses in order to focus the
beam to the proper diameter for illuminating one frame of cells. As
used herein, one frame of cells is defined as the portion of the
biological specimen that is captured within one frame image
captured by the camera. This is described more specifically
below.
[0032] Accordingly, the illumination laser beam passes through a
first condenser lens 335. In one embodiment, this first lens has a
focal length of 4.6 mm. The light beam then passes through a second
condenser lens 340 which, in one embodiment, provides a 100 mm
focal length. Finally, the light beam passes into a third condenser
lens 345, which preferably provides a 200 mm focal length. While
the present invention has been described using specific condenser
lenses, it should be apparent that other similar lens
configurations that focus the illumination laser beam to an
advantageous diameter would function similarly. Thus, this
invention is not limited to the specific implementation of any
particular condenser lens system.
[0033] Once the illumination laser beam passes through the third
condenser lens 345, it enters a cube beam splitter 350 that is
designed to transmit the 532 nm wavelength of light emanating from
the illumination laser. Preferably, the cube beam splitter 350 is a
25.4 mm square cube (Melles-Griot, Irvine, Calif.). However, other
sizes are anticipated to function similarly. In addition, a number
of plate beam splitters or pellicle beam splitters could be used in
place of the cube beam splitter 350 with no appreciable change in
function.
[0034] Once the illumination laser light has been transmitted
through the cube beam splitter 350, it reaches a long wave pass
mirror 355 that reflects the 532 nm illumination laser light to a
set of galvanometer mirrors 360 that steer the illumination laser
light under computer control to a scanning lens (Special Optics,
Wharton, N.J.) 365, which directs the illumination laser light to
the specimen (not shown). The galvanometer mirrors are controlled
so that the illumination laser light is directed at the proper cell
population (i.e. frame of cells) for imaging. The "scanning lens"
described in this embodiment of the invention includes a refractive
lens. It should be noted that the term "scanning lens" as used in
the present invention includes, but is not limited to, a system of
one or more refractive or reflective optical elements used alone or
in combination. Further, the "scanning lens" may include a system
of one or more diffractive elements used in combination with one or
more refractive and/or reflective optical elements. One skilled in
the art will know how to design a "scanning lens" system in order
to illuminate the proper cell population.
[0035] The light from the illumination laser is of a wavelength
that is useful for illuminating the specimen. In this embodiment,
energy from a continuous wave 532 nm Nd:YAG frequency-doubled laser
(B&W Tek, Newark, Del.) reflects off the long wave pass mirror
(Custom Scientific, Phoenix, Ariz.) and excites fluorescent tags in
the specimen. In one embodiment, the fluorescent tag is
phycoerythrin. Alternatively, Alexa 532 (Molecular Probes, Eugene,
Oreg.) can be used. Phycoerythrin and Alexa 532 have emission
spectra with peaks near 580 nm, so that the emitted fluorescent
light from the specimen is transmitted via the long wave pass
mirror to be directed into the camera. The use of the filter in
front of the camera blocks light that is not within the wavelength
range of interest, thereby reducing the amount of background light
entering the camera.
[0036] It is generally known that many other devices could be used
in this manner to illuminate the specimen, including, but not
limited to, an arc lamp (e.g., mercury, xenon, etc.) with or
without filters, a light-emitting diode (LED), other types of
lasers, etc. Advantages of this particular laser include high
intensity, relatively efficient use of energy, compact size, and
minimal heat generation. It is also generally known that other
fluorochromes with different excitation and emission spectra could
be used in such an apparatus with the appropriate selection of
illumination source, filters, and long and/or short wave pass
mirrors. For example, allophycocyanin (APC) could be excited with a
633 nm HeNe illumination laser, and fluoroisothiocyanate (FITC)
could be excited with a 488 nm Argon illumination laser. One
skilled in the art could propose many other optical layouts with
various components in order to achieve the objective of this
invention.
[0037] In addition to the illumination laser 305, an optional
treatment laser 400 is present to irradiate the targeted cells once
they have been identified by image analysis. Of course, in one
embodiment, the treatment induces necrosis of targeted cells within
the cell population. As shown, the treatment laser 400 outputs an
energy beam of 523 nm that passes through a shutter 410. Although
the exemplary laser outputs an energy beam having a 523 nm
wavelength, other sources that generate energy at other wavelengths
are also within the scope or the present invention.
[0038] Once the treatment laser energy beam passes through the
shutter 410, it enters a beam expander (Special Optics, Wharton,
N.J.) 415 which adjusts the diameter of the energy beam to an
appropriate size at the plane of the specimen. Following the beam
expander 415 is a half-wave plate 420 which controls the
polarization of the beam. The treatment laser energy beam is then
reflected off a mirror 425 and enters the cube beam splitter 350.
The treatment laser energy beam is reflected by 90 degrees in the
cube beam splitter 350, such that it is aligned with the exit
pathway of the illumination laser light beam. Thus, the treatment
laser energy beam and the illumination laser light beam both exit
the cube beam splitter 350 along the same light path. From the cube
beam splitter 350, the treatment laser beam reflects off the long
wave pass mirror 355, is steered by the galvanometers 360,
thereafter contacts the scanning lens 365, and finally is focused
upon a targeted cell within the specimen. Again, the "scanning
lens" described in this embodiment includes a refractive lens. As
previously mentioned, the term "scanning lens" includes, but is not
limited to, a system of one or more refractive or reflective
optical elements used alone or in combination. Further, the
"scanning lens" may include one or more diffractive elements used
in combination with one or more refractive and/or reflective
elements. One skilled in the art will know how to design a
"scanning lens" system in order to focus upon the targeted cell
within the specimen.
[0039] It should be noted that a small fraction of the illumination
laser light beam passes through the long wave pass mirror 355 and
enters a power meter sensor (Gentec, Palo Alto, Calif.) 445. The
fraction of the beam entering the power sensor 445 is used to
calculate the level of power emanating from the illumination laser
305. In an analogous fashion, a small fraction of the treatment
laser energy beam passes through the cube beam splitter 350 and
enters a second power meter sensor (Gentec, Palo Alto, Calif.) 446.
The fraction of the beam entering the power sensor 446 is used to
calculate the level of power emanating from the treatment laser
400. The power meter sensors are electrically linked to the
computer system so that instructions/commands within the computer
system capture the power measurement and determine the amount of
energy that was emitted.
[0040] The energy beam from the treatment laser is of a wavelength
that is useful for achieving a response in the cells. In the
example shown, a pulsed 523 nm Nd:YLF frequency-doubled laser is
used to heat a localized volume containing the targeted cell, such
that it is induced to die within a pre-determined period of time.
The mechanism of death is dependent upon the actual temperature
achieved in the cell, as reviewed by Niemz, M. H., Laser-tissue
interactions: Fundamentals and Applications (Springer-Verlag,
Berlin 1996).
[0041] A Nd:YLF frequency-doubled, solid-state laser
(Spectra-Physics, Mountain View, Calif.) is used because of its
stability, high repetition rate of firing, and long time of
maintenance-free service. However, most cell culture fluids and
cells are relatively transparent to light in this green wavelength,
and therefore a very high fluence of energy would be required to
achieve cell death. To significantly reduce the amount of energy
required, and therefore the cost and size of the treatment laser, a
dye is purposefully added to the specimen to efficiently absorb the
energy of the treatment laser in the specimen. In the example
shown, the non-toxic dye FD&C red #40 (allura red) is used to
absorb the 523 nm energy from the treatment laser, but one skilled
in the art could identify other laser/dye combinations that would
result in efficient absorption of energy by the specimen. For
example, a 633 nm HeNe laser's energy would be efficiently absorbed
by FD&C green #3 (fast green FCF), a 488 nm Argon laser's
energy would be efficiently absorbed by FD&C yellow #5 (sunset
yellow FCF), and a 1064 nm Nd:YAG laser's energy would be
efficiently absorbed by Filtron (Gentex, Zeeland, Mich.) infrared
absorbing dye. Through the use of an energy absorbing dye, the
amount of energy required to kill a targeted cell can be reduced
since more of the treatment laser energy is absorbed in the
presence of such a dye.
[0042] Another method of achieving thermal killing of cells without
the addition of a dye involves the use of an ultraviolet laser.
Energy from a 355 nm Nd:YAG frequency-tripled laser will be
absorbed by nucleic acids and proteins within the cell, resulting
in thermal heating and death. Yet another method of achieving
thermal killing of cells without the addition of a dye involves the
use of a near-infrared laser. Energy from a 2100 nm Ho:YAG laser or
a 2940 nm Er:YAG laser will be absorbed by water within the cell,
resulting in thermal heating and death.
[0043] Although this embodiment describes the killing of cells via
thermal heating by the energy beam, one skilled in the art would
recognize that other responses can also be induced in the cells by
an energy beam, including photomechanical disruption,
photodissociation, photoablation, and photochemical reactions, as
reviewed by Niemz (Niemz, supra). For example, a photosensitive
substance (e.g., hematoporphyrin derivative, tinetiopurpurin,
lutetium texaphyrin) (Oleinick and Evans, The photobiology of
photodynamic therapy: Cellular targets and mechanisms, Rad. Res.
150: S146-S156 (1998)) within the cell mixture could be
specifically activated in targeted cells by irradiation.
Additionally, a small, transient pore could be made in the cell
membrane (Palumbo et al., Targeted gene transfer in eukaryotic
cells by dye-assisted laser optoporation, J. Photochem. Photobiol.
36:41-46 (1996)) to allow the entry of genetic or other material.
Further, specific molecules in or on the cell, such as proteins or
genetic material, could be inactivated by the directed energy beam
(Grate and Wilson, Laser-mediated, site-specific inactivation of
RNA transcripts, PNAS 96:6131-6136 (1999); Jay, D. G., Selective
destruction of protein function by chromophore-assisted laser
inactivation, PNAS 85:5454-5458 (1988)). Also, photobleaching can
be utilized to measure intracellular movements such as the
diffusion of proteins in membranes and the movements of
microtubules during mitosis (Ladha et al., J. Cell Sci.,
110(9):1041 (1997); Centonze and Borisy, J. Cell Sci. 100 (part
1):205 (1991); White and Stelzer, Trends Cell Biol. 9(2):61-5
(1999); Meyvis, et al., Pharm. Res. 16(8):1153-62 (1999). Further,
photolysis or uncaging, including multiphoton uncaging, of caged
compounds can be utilized to control the release, with temporal and
spacial resolution, of biologically active products or other
products of interest (Theriot and Mitchison, J. Cell Biol. 119:367
(1992); Denk, PNAS 91(14):6629 (1994)). These mechanisms of
inducing a response in a targeted cell via the use of
electromagnetic radiation directed at specific targeted cells are
also intended to be incorporated into the present invention.
[0044] In addition to the illumination laser 305 and treatment
laser 400, the apparatus includes a camera 450 that captures images
(i.e. frames) of the cell populations. As illustrated in FIG. 3,
the camera 450 is focused through a lens 455 and filter 460 in
order to accurately record an image of the cells without capturing
stray background images. A stop 462 is positioned between the
filter 460 and mirror 355 in order to eliminate light that may
enter the camera from angles not associated with the image from the
specimen. The filter 460 is chosen to only allow passage of light
within a certain wavelength range. This wavelength range includes
light that is emitted from the targeted cells upon excitation by
the illumination laser 305, as well as light from a back-light
source 475.
[0045] The back-light source 475 is located above the specimen to
provide back-illumination of the specimen at a wavelength different
from that provided by the illumination laser 303. This LED
generates light at 590 nm, such that it can be transmitted through
the long wave pass mirror to be directed into the camera. This
back-illumination is useful for imaging cells when there are no
fluorescent targets within the frame being imaged. An example of
the utility of this back-light is its use in attaining proper focus
of the system, even when there are only unstained, non-fluorescent
cells in the frame. In one embodiment, the back-light is mounted on
the underside of the access door 35 (FIG. 2).
[0046] Thus, as discussed above, the only light returned to the
camera is from wavelengths that are of interest in the specimen.
Other wavelengths of light do not pass through the filter 460, and
thus do not become recorded by the camera 450. This provides a more
reliable mechanism for capturing images of only those cells of
interest. It is readily apparent to one skilled in the art that the
single filter 460 could be replaced by a movable filter wheel that
would allow different filters to be moved in and out of the optical
pathway. In such an embodiment, images of different wavelengths of
light could be captured at different times during cell processing,
allowing the use of multiple cell labels.
[0047] It should be noted that in this embodiment, the camera is a
charge-coupled device (CCD) and transmits images back to the
computer system for processing. As will be described below, the
computer system determines the coordinates of the targeted cells in
the specimen by reference to the image captured by the CCD
camera.
[0048] Referring now to FIG. 4, a perspective view of an embodiment
of an optical subassembly is illustrated. As illustrated, the
illumination laser 305 sends a light beam through the shutter 310
and ball lens 315 to the SMA fiber optic connector 320. The light
passes through the fiber optic cable 325 and through the output 330
into the condenser lenses 335, 340 and 345. The light then enters
the cube beam splitter 350 and is transmitted to the long wave pass
mirror 355. From the long wave pass mirror 355, the light beam
enters the computer-controlled galvanometers 360 and is then
steered to the proper frame of cells in the specimen from the
scanning lens 365.
[0049] As also illustrated in the perspective drawing of FIG. 4,
the treatment laser 400 transmits energy through the shutter 410
and into the beam expander 415. Energy from the treatment laser 400
passes through the beam expander 415 and passes through the
half-wave plate 420 before hitting the fold mirror 425, entering
the cube beam splitter 350 where it is reflected 90 degrees to the
long wave pass mirror 355, from which it is reflected into the
computer controlled galvanometer mirrors 360. After being steered
by the galvanometer mirrors 360 to the scanning lens 365, the laser
energy beam strikes the proper location within the cell population
in order to induce a response in a particular targeted cell.
[0050] In order to accommodate a very large surface area of
specimen to treat, the apparatus includes a movable stage that
mechanically moves the specimen container with respect to the
scanning lens. Thus, once a specific sub-population (i.e. field) of
cells within the scanning lens field-of-view has been treated, the
movable stage brings another sub-population of cells within the
scanning lens field-of-view. As illustrated in FIG. 5, a
computer-controlled movable stage 500 holds a specimen container
(not shown) to be processed. The movable stage 500 is moved by
computer-controlled servo motors along two axes so that the
specimen container can be moved relative to the optical components
of the instrument. The stage movement along a defined path is
coordinated with other operations of the apparatus. In addition,
specific coordinates can be saved and recalled to allow return of
the movable stage to positions of interest. Encoders on the x and y
movement provide closed-loop feedback control on stage
position.
[0051] The flat-field (F-theta) scanning lens 365 is mounted below
the movable stage. The scanning lens field-of-view comprises the
portion of the specimen that is presently positioned above the
scanning lens by the movable stage 500. The lens 365 is mounted to
a stepper motor that allows the lens 365 to be automatically raised
and lowered (along the z-axis) for the purpose of focusing the
system.
[0052] As illustrated in FIGS. 4 to 6, below the scanning lens 365
are the galvanometer-controlled steering mirrors 360 that deflect
electromagnetic energy along two perpendicular axes. Behind the
steering mirrors is the long wave pass mirror 355 that reflects
electromagnetic energy of a wavelength shorter than 545 nm.
Wavelengths longer than 545 nm are passed through the long wave
pass mirror, directed through the filter 460, coupling lens 455,
and into the CCD camera, thereby producing an image of the
appropriate size on the CCD sensor of the camera 450 (see FIGS. 3
and 4). The magnification defined by the combination of the
scanning lens 365 and coupling lens 455 is chosen to reliably
detect single cells while maximizing the area viewed in one frame
by the camera. Although a CCD camera (DVC, Austin, Tex.) is
illustrated in this embodiment, the camera can be any type of
detector or image gathering equipment known to those skilled in the
art. The optical subassembly of the apparatus is preferably mounted
on a vibration-isolated platform to provide stability during
operation as illustrated in FIGS. 2 and 5.
[0053] Referring now to FIG. 7, a top view of the movable stage 500
is illustrated. As shown, a specimen container is mounted in the
movable stage 500. The specimen container 505 rests on an upper
axis nest plate 510 that is designed to move in the
forward/backward direction with respect to the movable stage 500. A
stepper motor (not shown) is connected to the upper axis nest plate
510 and computer system so that commands from the computer cause
forward/backward movement of the specimen container 505.
[0054] The movable stage 500 is also connected to a timing belt 515
that provides side-to-side movement of the movable stage 500 along
a pair of bearing tracks 525A,B. The timing belt 515 attaches to a
pulley (not shown) housed under a pulley cover 530. The pulley is
connected to a stepper motor 535 that drives the timing belt 515 to
result in side-to-side movement of the movable stage 500. The
stepper motor 535 is electrically connected to the computer system
so that commands within the computer system result in side-to-side
movement of the movable stage 500. A travel limit sensor 540
connects to the computer system and causes an alert if the movable
stage travels beyond a predetermined lateral distance.
[0055] A pair of accelerometers 545A,B is preferably incorporated
on this platform to register any excessive bumps or vibrations that
may interfere with the apparatus operation. In addition, a two-axis
inclinometer 550 is preferably incorporated on the movable stage to
ensure that the specimen container is level, thereby reducing the
possibility of gravity-induced motion in the specimen
container.
[0056] The specimen chamber has a fan with ductwork to eliminate
condensation on the specimen container, and a thermocouple to
determine whether the specimen chamber is within an acceptable
temperature range. Additional fans are provided to expel the heat
generated by the electronic components, and appropriate filters are
used on the air intakes 215A,B.
[0057] The computer system 225 controls the operation and
synchronization of the various pieces of electronic hardware
described above. The computer system can be any commercially
available computer that can interface with the hardware. One
example of such a computer system is an Intel Pentium II, III or
IV-based computer running the Microsoft WINDOWS NT operating
system. Software is used to communicate with the various devices,
and control the operation in the manner that is described
below.
[0058] When the apparatus is first initialized, the computer loads
files from the hard drive into RAM for proper initialization of the
apparatus. A number of built-in tests are automatically performed
to ensure the apparatus is operating properly, and calibration
routines are executed to calibrate the apparatus. Upon successful
completion of these routines, the user is prompted to enter
information via the keyboard and mouse regarding the procedure that
is to be performed. Once the required information is entered, the
user is prompted to open the access door 35 and load a specimen
onto the movable stage.
[0059] Once a specimen is in place on the movable stage and the
door is closed, the computer passes a signal to the stage to move
into a home position. The fan is initialized to begin warming and
defogging of the specimen. During this time, cells within the
specimen are allowed to settle to the bottom surface. In addition,
during this time, the apparatus may run commands that ensure that
the specimen is properly loaded, and is within the focal range of
the system optics. For example, specific markings on the specimen
container can be located and focused on by the system to ensure
that the scanning lens has been properly focused on the bottom of
the specimen container. Such markings could also be used by the
instrument to identify the container, its contents, and even the
procedure to be performed. After a suitable time, the computer
turns off the fan to prevent excess vibrations during treatment,
and cell processing begins.
[0060] First, the computer instructs the movable stage to be
positioned over the scanning lens so that the first area (i.e.
field) of the specimen to be treated is directly in the scanning
lens field-of-view. The galvanometer mirrors are instructed to move
such that the center frame within the field-of-view is imaged in
the camera. As discussed below, the field imaged by the scanning
lens is separated into a plurality of frames. Each frame is the
proper size so that the cells within the frame are effectively
imaged by the camera.
[0061] The back-light 475 is then activated in order to illuminate
the field-of-view so that it can be brought into focus by the
scanning lens. Once the scanning lens has been properly focused
upon the specimen, the computer system divides the field-of-view
into a plurality of frames so that each frame is analyzed
separately by the camera. This methodology allows the apparatus to
process a plurality of frames within a large field-of-view without
moving the mechanical stage. Because the galvanometers can move
from one frame to the next very rapidly compared to the mechanical
steps involved in moving the stage, this method results is an
extremely fast and efficient apparatus.
[0062] Other means of ensuring that the specimen is in focus are
also available. For example, a laser proximeter (Cooke Corp.,
Auburn, Mich.) could rapidly determine the distance between the
scanning lens and the sample, and adjust the scanning lens position
accordingly. Ultrasonic proximeters are also available, and would
achieve the same objective. One skilled in the art could propose
other means of ensuring that the specimen is in focus above the
scanning lens. in one preferred embodiment, the apparatus described
herein processes at least 1, 2, 3, 4, 5, 6, 7, or 14 square
centimeters of a biological specimen per minute. In another
embodiment, the apparatus described herein processes at least 0.25,
0.5, 1, 2, 3, 4 or 8 million cells of a biological specimen per
minute. In one other embodiment, the apparatus can preferably
induce a response in targeted cells at a rate of 50, 100, 150, 200,
250, 300, 350, 400 or 800 cells per second.
[0063] Initially, an image of the frame at the center of the
field-of-view is captured by the camera and stored to a memory in
the computer. Instructions in the computer analyze the focus of the
specimen by looking at the size of, number of, and other object
features in the image. If necessary, the computer instructs the
z-axis motor attached to the scanning lens to raise or lower in
order to achieve the best focus. The apparatus may iteratively
analyze the image at several z-positions until the best focus is
achieved. The galvanometer-controlled mirrors are then instructed
to image a first frame, within the field-of-view, in the camera.
For example, the entire field-of-view might be divided into 4, 9,
12, 18, 24 or more separate frames that will be individually
captured by the camera. Once the galvanometer mirrors are pointed
to the first frame in the field-of-view, the shutter in front of
the illumination laser is opened to illuminate the first frame
through the galvanometer mirrors and scanning lens. The camera
captures an image of any fluorescent emission from the specimen in
the first frame of cells. Once the image has been acquired, the
shutter in front of the illumination laser is closed and a software
program (Epic, Buffalo Grove, Ill.) within the computer processes
the image.
[0064] The power sensor 445 discussed above detects the level of
light that was emitted by the illumination laser, thereby allowing
the computer to calculate if it was adequate to illuminate the
frame of cells. If not, another illumination and image capture
sequence is performed. Repeated failure to sufficiently illuminate
the specimen will result in an error condition that is communicated
to the operator.
[0065] Shuttering of illumination light reduces undesirable heating
and photobleaching of the specimen and provides a more repeatable
fluorescent signal. An image analysis algorithm is run to locate
the x-y centroid coordinates of all targeted cells in the frame by
reference to features in the captured image. If there are targets
in the image, the computer calculates the two-dimensional
coordinates of all target locations in relation to the movable
stage position and field-of-view, and then positions the
galvanometer-controlled mirrors to point to the location of the
first target in the first frame of cells. It should be noted that
only a single frame of cells within the field-of-view has been
captured and analyzed at this point. Thus, there should be a
relatively small number of identified targets within this
sub-population of the specimen. Moreover, because the camera is
pointed to a smaller population of cells, a higher magnification is
used so that each target is imaged by many pixels within the CCD
camera.
[0066] Once the computer system has positioned the galvanometer
controlled mirrors to point to the location of the first targeted
cell within the first frame of cells, the treatment laser is fired
for a brief interval so that the first targeted cell is given an
appropriate dose of energy. The power sensor 446 discussed above
detects the level of energy that was emitted by the treatment
laser, thereby allowing the computer to calculate if it was
adequate to induce a response in the targeted cell. If not
sufficient, the treatment laser is fired at the same target again.
If repeated shots do not deliver the required energy dose, an error
condition is communicated to the operator. These targeting, firing,
and sensing steps are repeated by the computer for all targets
identified in the captured frame.
[0067] Once all of the targets have been irradiated with the
treatment laser in the first frame of cells, the mirrors are then
positioned to the second frame of cells in the field-of-view, and
the processing repeats at the point of frame illumination and
camera imaging. This processing continues for all frames within the
field-of-view above the scanning lens. When all of these frames
have been processed, the computer instructs the movable stage to
move to the next field-of-view in the specimen, and the process
repeats at the back-light illumination and auto-focus step. Frames
and fields-of-view are appropriately overlapped to reduce the
possibility of inadvertently missing areas of the specimen. Once
the specimen has been fully processed, the operator is signaled to
remove the specimen, and the apparatus is immediately ready for the
next specimen.
[0068] Although the text above describes the analysis of
fluorescent images for locating targets, one can easily imagine
that the non-fluorescent back-light LED illumination imaces will be
useful for locating other types of targets as well, even if they
are unlabeled.
[0069] The advantage of using the galvanometer mirrors to control
the imaging of successive frames and the irradiation of successive
targets is significant. One brand of galvanometer is the Cambridge
Technology, Inc. model number 6860 (Cambridge, Mass.). This
galvanometer can reposition very accurately within a few
milliseconds, making the processing of large areas and many targets
possible within a reasonable amount of time. In contrast, the
movable stage is relatively slow, and is therefore used only to
move specified areas of the specimen into the scanning lens
field-of-view. Error signals continuously generated by the
galvanometer control boards are monitored by the computer to ensure
that the mirrors are in position and stable before an image is
captured, or before a target is fired upon, in a closed-loop
fashion.
[0070] In the context of the present invention, the term "specimen"
has a broad meaning. It is intended to encompass any type of
biological sample placed within the apparatus. The specimen may be
enclosed by, or associated with, a container to maintain the
sterility and viability of the cells. Further, the specimen may
incorporate, or be associated with, a cooling apparatus to keep it
above or below ambient temperature during operation of the methods
described herein. The specimen container, if one is used, must be
compatible with the use of the illumination laser, back-light
illuminator, and treatment laser, such that it transmits adequate
energy without being substantially damaged itself.
[0071] Of course, many variations of the above-described embodiment
are possible, including alternative methods for illuminating,
imaging, and targeting the cells. For example, movement of the
specimen relative to the scanning lens could be achieved by keeping
the specimen substantially stationary while the scanning lens is
moved. Steering of the illumination beam, images, and energy beam
could be achieved through any controllable reflective or
diffractive device, including prisms, piezo-electric tilt
platforms, or acousto-optic deflectors. Additionally, the apparatus
can image/process from either below or above the specimen. Because
the apparatus is focused through a movable scanning lens, the
illumination and energy beams can be directed to different focal
planes along the z-axis. Thus, portions of the specimen that are
located at different vertical heights can be specifically imaged
and processed by the apparatus in a three-dimensional manner. The
sequence of the steps could also be altered without changing the
process. For example, one might locate and store the coordinates of
all targets in the specimen, and then return to the targets to
irradiate them with energy one or more times over a period of
time.
[0072] To optimally process the specimen, it should be placed on a
substantially flat surface so that a large portion of the specimen
appears within a narrow range of focus, thereby reducing the need
for repeated auto-focus steps. The density of cells on this surface
can, in principle, be at any value. However, the cell density
should be as high as possible to minimize the total surface area
required for the procedure.
[0073] A further embodiment of the invention provides optoinjection
methods for transiently permeabilizing a target cell. In the
general method, the steps are (a) illuminating a population of
cells contained in a frame; (b) detecting at least one property of
light directed from the frame; (c) locating a target cell by the
property of light; and (d) irradiating the target cell with a pulse
of radiation.
[0074] The "cells" used in the method can be any, biological cells,
including procaryotic and eucaryotic cells, such as animal cells,
plant cells, yeast cells, human cells and non-human primate cells.
The cells can be taken from organisms or harvested from cell
cultures. The method can also be applied to permeabilize
subcellular organelles.
[0075] It follows that the term "population" of cells means a group
of more than one of such cells. While performing the method, the
population of cells can be presented in a specimen container such
as 505.
[0076] The cells can also be associated with an exogenous label
such as a fluorophore. Other labels useful in the invention have
been described in detail above.
[0077] The population of cells can be "illuminated" by any source
that can provide light energy, including a laser and an arc lamp.
The light energy can be of any wavelength, such as visible,
ultraviolet and infrared light. When the light is from a laser,
such as 400, useful wavelengths can range from 100 nm to 1000 nm,
200 nm to 800 nm, 320 nm to 695 nm, and 330 nm to 605 nm.
Particular wavelengths include 349 nm, 355 nm, 488 nm, 523 nm, 532
nm, 580 nm, 590 nm, 633 nm, 1064 nm, 2100 nm and 2940 nm. Other
illumination sources include any source for an energy beam, as
described in detail above. The light can then be directed by any
conventional means, such as mirrors, lenses and beam-splitters, to
the population of cells.
[0078] Once the cells are illuminated, they can be observed in a
"frame." As previously defined, one "frame" of cells is the portion
of the biological specimen that is captured within one frame image
captured by the camera. A particularly useful frame can have an
area of at least 50, 70, 85, 95 or 115 mm.sup.2. A useful
magnification range for the camera is between 2.times. and
40.times. and more particularly between 2.5.times. and 25.times.
and still more particularly between 5.times. and 10.times..
[0079] When the frame is illuminated, one or more properties of
light can then be detected from the frame. The detectable
properties include light having visible, ultraviolet and infrared
wavelengths, the intensity of transmittance and reflectance,
fluorescence, linear and circular polarization, and phase-contrast
illumination. These properties can be detected by conventional
optical devices such as the devices already described in detail
above.
[0080] The target cell can then be located based on its size, shape
and other preselected visual properties, and then irradiated with a
pulse of radiation. The radiation then causes a temporary
permeabilization of the surface of the target cell. While not
limiting the method to a particular mechanism, it is believed that
the light causes localized melting or other disruption of the cell
membrane's continuity, allowing small pores to form without killing
the cell.
[0081] As a result of transiently permeabilizing the cells,
exogenous molecules in the presence of the cell can then enter the
cell, whether by diffusion or other mechanism. The term "presence
of the cell" herein as applied to an exogenous molecule means in
the area near the cell, such as the surrounding medium, so that if
the cell were permeabilized, the exogenous molecule could then
enter the cell.
[0082] The term "exogenous molecule" herein means any molecule or
material that does not naturally occur in the cells of the
population. It also includes molecules or materials that may occur
naturally in the cell, but in significantly higher concentrations
than occur naturally in the cell. Exogenous molecules include
nucleic acids, polypeotides, carbohydrates, lipids and small
molecules. Particular nucleic acids include RNAs, expression
plasmids, expression cassettes and other expressible DNA.
Particular polypeptides include antibodies and other proteins,
which can be introduced into cells to explore interactions between
exogenous and endogenous proteins for applications in proteomics.
Other polypeptides include peptides for introduction into
antigen-displaying dendritic cells. Particular carbohydrates
include non-naturally occurring metabolites, such as isotopically
labeled sugars, and polysaccharides, such as labeled dextrans.
Particular lipids include preselected lipids for incorporation into
the cell membrane or other organelles, as well as liposomes and
liposomes containing other exogenous molecules of interest.
Particular small molecules include ligands for endogenous receptors
to study ligand-receotor binding. Similarly, drugs can be
introduced into cells, which, in turn, can be introduced as a
delivery device into a patient for therapeutic purposes. The term
also encompasses dyes capable of absorbing visible, ultraviolet or
infrared light.
[0083] Exogenous molecules can have a size of greater than 0.1,
0.2, 0.3, 0.5, 1, 2, 3, 5, 10, 20, 30, 50, 70, 100 or even 200
kiloDaltons. Although the efficiency rate of cells that are loaded
with at least one exogenous molecule will vary depending on the
size and nature of the exogenous molecule, loading efficiencies can
be as high as 5%, 10%, 20%, 50%, 75% or even 90% of the population
of cells. It should also be emphasized that the method encompasses
techniques where two or more exogenous molecules are loaded into
cells simultaneously or sequentially.
[0084] Significantly, as result of using the method, greater than
50%, 60%, 70%, 80%, 90%, 95% or even 98% of the irradiated target
cells can be viable after completion of the method. Methods for
measuring cell survival rates are well known in the art and
membrane-permeable reagents for distinguishing living cells have
been described above. For example, preselected reagents can be
added to the media before, during or after performing the method.
Specific examples of useful reagents include Calcein AM as an
indicator of viability and Sytox Blue as an indicator for dead
cells. Other well-known methods include trypan blue exclusion,
propidium iodide and .sup.51Cr-release assay.
[0085] It should be noted that the general method presented above
has several alternate embodiments that are particularly useful.
[0086] First, the general method can be used when the population of
cells is substantially stationary. The term "substantially
stationary" herein means that the cells are relatively immobile
with respect to the medium and are not in flowing medium, and the
cells are not subjected to gross movement of a container; but, they
can be subject to vibrations and slight movements that normally
occur in a typical laboratory. While the term encompasses cells
that are immobilized to a surface or within the medium,
substantially stationary cells need not be immobilized or otherwise
bound to a surface to be considered substantially stationary. Thus;
the term includes cells that have settled to the bottom of a
specimen container.
[0087] When a population of cells is substantially stationary, it
becomes useful to obtain a static representation of the cells in
the frame. The term "static representation" herein means a
substantially complete image of the cells taken during a fixed and
discrete time period, rather than as a continuous image, as in a
"live" monitor.
[0088] Because the cells are substantially stationary, the static
representation can then be used as a reliable indicator of the
location of one or more cells at subsequent points in time.
Moreover, a static representation can be obtained under one set of
conditions and another static representation obtained under a
different set of conditions so that the two representations can be
compared usefully without undue concern for movement of the cells.
For example, an image of the cells under visible light can be
compared with a corresponding fluorescence image to identify
fluorescently tagged cells of interest among a general population
of cells. The static representation can also be used as the basis
for computer-aided identification and determination of the location
of a target cell of interest, based on any of the light properties
discussed above.
[0089] Second, the general method can be performed where the
population of cells is illuminated through a lens having numerical
aperture of at most 0.5, 0.4 or 0.3. The term "numerical aperture"
or "N.A." used herein is defined N.A.=n(sin .mu.), where n is the
refractive index of the imaging medium between the lens and the
cells, and .mu. is one-half of the angular aperture.
[0090] As a consequence of using a lens having such a low numerical
aperture, the lens can have a greater working distance, such as at
least 5, 7 or 10 mm. The term "working distance" herein means the
distance between the front of the lens to the object, meaning the
nearest surface of the population of cells. A particularly useful
lens is a flat-field (F-theta) lens, as exemplified by lens 365,
described above. It should be noted that confocal microscopy is not
possible under such lens parameters.
[0091] Third, the pulse of radiation can have a diameter of at
least 2, 5, 7, 10, 15, 20, 25 or 30 microns at the point of contact
with the target cell. In most cases, the breadth of the radiation
will be much wider than any individual cell. Consequently, the beam
of radiation need not be separately targeted to a particular point
on a cell or cell surface to be effective, but can be directed to
the general area of a cell population without losing effectiveness.
As a result, sensitivity to beam steering accuracy is reduced and
throughput is dramatically increased.
[0092] Fourth, the energy delivered by the pulse of radiation can
be limited to at most 2, 1.5, 1, 0.7, 0.5, 0.3, 0.2, 0.1, 0.05,
0.02, 0.01 or even 0.005 .mu.J/.mu.m.sup.2. This has the advantage
of increasing the survival rate while maintaining efficient loading
rates. Moreover, unlike previous methods, the effective energy
levels are low enough to allow the use of common plastic specimen
containers without damaging the container.
[0093] The general method can also be modified to increase
throughput. At the most basic level, the direction of the pulse of
radiation can be adjusted to irradiate a second target cell in the
population in a given frame. Similarly, subsequent fields of view
of the population of cells can be processed as described above.
This is especially useful when the population of cells remains in a
substantially stationary location relative to the lens.
Alternatively, the cells can be moved relative to the lens between
applications of the method for further steps of detecting, locating
and irradiating cells.
[0094] To maximize throughput of the cells, one or more of the
steps of the method can be automated, as exemplified by the
apparatus described in detail above. For example, each of the steps
can be controlled by a microprocessor. Similarly, a static
representation can be processed as an image or a data set stored in
computer memory. By automating each of the steps, the optoinjection
method can irradiate at least 5,000, 10,000, 20,000, 50,000,
70,000, 100,000 or even 150,000 cells per minute
[0095] The following examples illustrate the use of the described
method and apparatus in different applications.
Example 1
Autologous HSC Transplantation
[0096] A patient with a B cell-derived metastatic tumor in need of
an autologous HSC transplant is identified by a physician. As a
first step in the treatment, the patient undergoes a standard HSC
harvest procedure, resulting in collection of approximately
1.times.10.sup.10 hematopoietic cells with an unknown number of
contaminating tumor cells. The harvested cells are enriched for HSC
by a commercial immunoaffinity column (ISOLEX 300, Nexell
Therapeutics, Irvine, Calif.) that selects for cells bearing the
CD34 surface antigen, resulting in a population of approximately
3.times.10.sup.8 hematopoietic cells, with an unknown number of
tumor cells. The mixed population is thereafter contacted with
anti-B cell antibodies (directed against CD20 and CD22) that are
conjugated to phycoerythrin. The labeled antibodies specifically
bind to the B cell-derived tumor cells.
[0097] The mixed cell population is then placed in a sterile
specimen container on a substantially flat surface near confluence,
at approximately 500,000 cells per square centimeter. The specimen
is placed on the movable stage of the apparatus described above,
and all detectable tumor cells are identified by reference to
phycoerythrin and targeted with a lethal dose of energy from a
treatment laser. The design of the apparatus allows the processing
of a clinical-scale transplant specimen in under 4 hours. The cells
are recovered from the specimen container, washed, and then
cryopreserved. Before the cells are reinfused, the patient is given
high-dose chemotherapy to destroy the tumor cells in the patient's
body. Following this treatment, the processed cells are thawed at
37.degree. C. and are given to the patient intravenously. The
patient subsequently recovers with no remission of the original
cancer.
Example 2
Allogeneic HSC Transplantation
[0098] In another embodiment, the significant risk and severity of
graft-versus-host disease in the allogeneic HSC transplant setting
can be combated. A patient is selected for an allogeneic transplant
once a suitable donor is found. Cells are harvested from the
selected donor as described in the above example. In this case, the
cell mixture is contacted with phycoerythrin-labeled anti-CD3
T-cell antibodies. Alternatively, specific allo-reactive T-cell
subsets could be labeled using an activated T-cell marker (e.g.
CD69) in the presence of allo-antigen. The cell population is
processed by the apparatus described herein, thereby precisely
defining and controlling the number of T-cells given to the
patient. This type of control is advantageous, because
administration of too many T-cells increases the risk of
graft-versus-host disease, whereas too few T-cells increases the
risk of graft failure and the risk of losing of the known
beneficial graft-versus-leukemia effect. The present invention and
methods are capable of precisely controlling the number of T-cells
in an allogeneic transplant.
Example 3
Tissue Engineering
[0099] In another application, the present apparatus is used to
remove contaminating cells in inocula for tissue engineering
applications. Cell contamination problems exist in the
establishment of primary cell cultures required for implementation
of tissue engineering applications, as described by Langer and
Vacanti, Tissue engineering: The challenges ahead, Sci. Am.
280:86-89 (1999). In particular, chondrocyte therapies for
cartilage defects are hampered by impurities in the cell
populations derived from cartilage biopsies. Accordingly, the
present invention is used to specifically remove these types of
cells from the inocula.
[0100] For example, a cartilage biopsy is taken from a patient in
need of cartilage replacement. The specimen is then grown under
conventional conditions (Brittberg et al., Treatment of deep
cartilage defects in the knee with autologous chondrocyte
transplantation, N.E. J. Med. 331:889-895 (1994)). The culture is
then stained with a specific label for any contaminating cells,
such as fast-growing fibroblasts. The cell mixture is then placed
within the apparatus described and the labeled, contaminating cells
are targeted by the treatment laser, thereby allowing the slower
growing chondrocytes to fully develop in culture.
Example 4
Stem Cell Therapy
[0101] Yet another embodiment involves the use of embryonic stem
cells to treat a wide variety of diseases. Since embryonic stem
cells are undifferentiated, they can be used to generate many types
of tissue that would find use in transplantation, such as
cardiomyocytes and neurons. However, undifferentiated embryonic
stem cells that are implanted can also lead to a jumble of cell
types which form a type of tumor known as a teratoma (Pedersen, R.
A., Embryonic stem cells for medicine, Sci. Amer. 280:68-73
(1999)). Therefore, therapeutic use of tissues derived from
embryonic stem cells must include rigorous purification of cells to
ensure that only sufficiently differentiated cells are implanted.
The apparatus described herein is used to eliminate
undifferentiated stem cells prior to implantation of embryonic stem
cell-derived tissue in the patient.
Example 5
Generation of Human Tumor Cell Cultures
[0102] In another embodiment, a tumor biopsy is removed from a
cancer patient for the purpose of initiating a culture of human
tumor cells. However, the in vitro establishment of primary human
tumor cell cultures from many tumor types is complicated by the
presence of contaminating primary cell populations that have
superior in vitro growth characteristics over tumor cells. For
example, contaminating fibroblasts represent a major challenge in
establishing many cancer cell cultures. The disclosed apparatus is
used to particularly label and destroy the contaminating cells,
while leaving the biopsied tumor cells intact. Accordingly, the
more aggressive primary cells will not overtake and destroy the
cancer cell line.
Example 6
Generation of a Specific mRNA Expression Library
[0103] The specific expression pattern of genes within different
cell populations is of great interest to many researchers, and many
studies have been performed to isolate and create libraries of
expressed genes for different cell types. For example, knowing
which genes are expressed in tumor cells versus normal cells is of
great potential value (Cossman, et al., Reed-Sternberg cell genome
expression supports a B-cell lineage, Blood 94:411-416 (1999)). Due
to the amplification methods used to generate such libraries (e.g.
PCR), even a small number of contaminating cells will result in an
inaccurate expression library (Cossman et al., supra; Schutze and
Lahr, Identification of expressed genes by laser-mediated
manipulation of single cells, Nature Biotechnol. 16:737-742
(1998)). One approach to overcome this problem is the use of laser
capture microdissection (LCM), in which a single cell is used to
provide the starting genetic material for amplification (Schutze
and Lahr, supra). Unfortunately, gene expression in single cells is
somewhat stochastic, and may be biased by the specific state of
that individual cell at the time of analysis (Cossman et al.,
supra). Therefore, accurate purification of a significant cell
number prior to extraction of mRNA would enable the generation of a
highly accurate expression library, one that is representative of
the cell population being studied, without biases due to single
cell expression or expression by contaminating cells. The methods
and apparatus described in this invention can be used to purify
cell populations so that no contaminating cells are present during
an RNA extraction procedure.
Example 7
Transfection of a Specific Cell Population
[0104] Many research and clinical gene therapy applications are
hampered by the inability to transfect an adecuate number of a
desired cell type without transfecting other cells that are
present. The method of the present invention would allow selective
targeting of cells to be transfected within a mixture of cells. By
generating a photomechanical shock wave at or near a cell membrane
with a targeted energy source, a transient pore can be formed,
through which genetic (or other) material can enter the cell. This
method of gene transfer has been called optoporation (Palumbo et
al. supra). The apparatus described above can achieve selective
optoporation on only the cells of interest in a rapid, automated,
targeted manner.
[0105] For example, white blood cells are plated in a specimen
container having a solution containing DNA to be transfected.
Fluorescently-labeled antibodies having specificity for stem cells
are added into the medium and bind to the stem cells. The specimen
container is placed within the cell processing apparatus and a
treatment laser is targeted to any cells that become fluorescent
under the illumination laser light. The treatment laser facilitates
transfection of DNA specifically into the targeted cells.
Example 8
Selection of Desirable Clones in a Biotechnology Application
[0106] In many biotechnology processes where cell lines are used to
generate a valuable product, it is desirable to derive clones that
are very efficient in producing the product. This selection of
clones is often carried out manually, by inspecting a large number
of clones that have been isolated in some manner. The present
invention would allow rapid, automated inspection and selection of
desirable clones for production of a particular product. For
example, hybridoma cells that are producing the greatest amounts of
antibody can be identified by a fluorescent label directed against
the Fc region. Cells with no or dim fluorescent labeling are
targeted by the treatment laser for killing, leaving behind the
best producing clones for use in antibody production.
Example 9
Automated Monitoring of Cellular Responses
[0107] Automated monitoring of cellular responses to specific
stimuli is of great interest in high-throughput drug screening.
Often, a cell population in one well of a well-plate is exposed to
a stimulus, and a fluorescent signal is then captured over time
from the cell population as a whole. Using the methods and
apparatus described herein, more detailed monitoring could be done
at the single cell level. For example, a cell population can be
labeled to identify a characteristic of a subpopulation of cells
that are of interest. This label is then excited by the
illumination laser to identify those cells. Thereafter, the
treatment laser is targeted at the individual cells identified by
the first label, for the purpose of exciting a second label,
thereby providing information about each cell's response. Since the
cells are substantially stationary on a surface, each cell could be
evaluated multiple times, thereby providing temporal information
about the kinetics of each cell's response. Also, through the use
of the large area scanning lens and galvanometer mirrors, a
relatively large number of wells could be quickly monitored over a
short period of time.
[0108] As a specific example, consider the case of alloreactive
T-cells as presented in Example 2, above. In the presence of
allo-antigen, activated donor T-cells could be identified by CD69.
Instead of using the treatment laser to target and kill these
cells, the treatment laser could be used to examine the
intracellular pH of every activated T-cell through the excitation
and emitted fluorescence of carboxyfluorescein diacetate. The
targeted laser allows the examination of only cells that are
activated, whereas most screening methods evaluate the response of
an entire cell population. If a series of such wells are being
monitored in parallel, various agents could be added to individual
wells, and the specific activated T-cell response to each agent
could be monitored over time. Such an apparatus would provide a
high-throughput screening method for agents that ameliorate the
alloreactive T-cell response in graft-versus-host disease. Based on
this example, one skilled in the art could imagine many other
examples in which a cellular response to a stimulus is monitored on
an individual cell basis, focusing only on cells of interest
identified by the first label.
Example 10
Photobleaching Studies
[0109] Photobleaching, and/or photobleach recovery, of a specific
area of a fluorescently-stained biological sample is a common
method that is used to assess various biological processes. For
example, a cell suspension is labeled with rhodamine 123, which
fluorescently stains mitochondria within the cells. Using the
instant illumination laser, the mitochondria within one or more
cells are visualized due to rhodamine 123 fluorescence. The
treatment laser is then used to deliver a focused beam of light
that results in photobleaching of the rhodamine 123 in a small area
within one or more cells. The photobleached area(s) then appear
dark immediately thereafter, whereas adjacent areas are unaffected.
A series of images are then taken using the illumination laser,
providing a time-lapse series of images that document the migration
of unbleached mitochondria into the area that was photobleached
with the treatment laser. This approach can be used to assess the
motion, turnover, or replenishment of many biological structures
within cells.
[0110] Thus, in cultured rat neurites, the photobleach recovery of
mitochondria is a measure of the size of the mobile pool of
mitochondria within each cell (Chute, et al., Analysis of the
steady-state dynamics organelle motion in cultured neurites, Clin.
Exp. Pharmco. Physiol. 22:360 (1995)). The rate of photobleach
recovery in these cells is dependent on intracellular calcium and
magnesium concentrations, energy status, and microtubule integrity.
Neurotoxic substances, such as taxol or vinblastine, will affect
the rate of photobleach recovery. Therefore, an assay for
neurotoxic substances could be based on the measurement of
photobleach recovery of mitochondria within a statistically
significant number of neurites that had been exposed to various
agents in the wells of a multi-well plate. In such an application,
the apparatus described herein and used as described above, would
provide a rapid automated method to assess neurotoxicity of many
substances on a large number of cells. Based on this example, one
skilled in the art could imagine many other examples in which
photobleaching is induced and photobleach recovery is monitored in
order to obtain useful information from a biological specimen.
Example 11
Uncaging Studies
[0111] Use of caged compounds to study rapid biological processes
involves the binding (i.e. caging) of a biologically relevant
substance in an inactive state, allowing the caged substance to
diffuse into the biological specimen (a relatively slow process),
and then using a laser to induce a photolysis reaction (a
relatively fast process) which liberates (i.e. uncages) the
substance in situ over microsecond time scales. The biological
specimen is then observed in short time-lapse microscopy in order
to determine the effect of the uncaged substance on some biological
process. Cages for many important substances have been described,
including Dioxygen, cyclic ADP ribose (cADPR), nicotinic acid
adenine dinucleotide phosphate (NAADP), nitric oxide (NO), calcium,
L-aspartate, and adenosine triphosphate (ATP). Chemotaxis is one
example of a physiological characteristic that can be studied by
uncaging compounds.
[0112] Uncaging studies involve the irradiation of a portion of a
biological specimen with laser light followed by examination of the
specimen with time-lapse microscopy. The apparatus of the current
invention has clear utility in such studies. As a specific example,
consider the study of E. coli chemotaxis towards L-aspartate
(Jasuja et al., Chemotactic responses of Escherichia coli to small
jumps of photoreleased L-aspartate, Biophys. J. 76:1706 (1999)).
The beta-2,6-dinitrobenzyl ester of L-aspartic acid and the
1-(2-nitrophenyl)ethyl ether of
8-hydroxylpyrene-1,3,6-tris-sulfonic acid are added to the wells of
a well plate containing E. coli. Upon irradiation with the
treatment laser, a localized uncaging of L-aspartate and the
fluorophore 8-hydroxylpyrene-1,3,6-tris-sulfonic acid (pyranine) is
induced. The L-aspartate acts as a chemoattractant for E. coli.,
and in subsequent fluorescent images (using the illumination laser)
the pyranine fluorophore acts as an indicator of the degree of
uncaging that has occurred in the local area of irradiation.
Time-lapse images of the E. coli. in the vicinity illuminated by
visible wavelength light, such as from the back-light, of the
uncaging event are used to measure the chemotactic response of the
microorganisms to the locally uncaged L-aspartate. Due to the
nature of the present invention, a large number of wells, each with
a potential anti-microbial agent added, are screened in rapid order
to determine the chemotactic response of microorganisms. Based on
this example, one skilled in the art could imagine many other
examples in which uncaging is induced by the treatment laser,
followed by time-lapse microscopy in order to obtain useful
information on a large number of samples in an automated
fashion.
Example 12
Optoinjection of NIH-3T3 Cells with 70 kD Dextran
[0113] This example illustrates an optoinjection method for
transiently permeabilizing a target cell. NIH-3T3 cells were grown
in a 96-well plate. The growth medium was removed and replaced with
PBS containing 1% BSA and 0.1 mM Texas-Red-Dextran (70 kDa)
(Molecular Probes, Eugene, Oreg.). Upon illumination of the cells
under broad-spectrum light, a static image (FIG. 8A) was obtained
to determine which cells to target.
[0114] A 30 micron energy beam having a wavelength of 523 nm was
directed sequentially to the target cells through a flat-field lens
having a magnification of 2.5.times., a numerical aperture (N.A.)
of 0.25, and a working distance of greater than 10 mm. Over 500
cells were targeted per second.
[0115] After irradiating the target cells, the wells were washed,
and Sytox Blue (10 mM, Molecular Probes) was added to stain
non-viable cells. As shown in FIG. 8B, about 70% of the cells
showed loading of the Texas-Red-Dextran as an exogenous molecule.
Moreover, only one cell was non-viable (FIG. 8C), equivalent to
about a 95% survival rate.
Example 13
Optoinjection of SU-DHL-4 Cells with Sytox Green
[0116] Using the same hardware apparatus as in Example 12, SU-DHL-4
cells were placed in 96-well plates in PBS with 1% HSA. The
membrane-impermeable dye Sytox Green (Molecular Probes) was added
at 0.05 mM, the cells were allowed to settle, and then were imaged
and targeted with a 30 micron laser beam. Different energy levels
ranging from 2 to 15.mu.J per pulse of the laser were used in each
of five wells, with each target cell receiving one pulse. As show
in FIG. 9, the efficiency of optoinjection was energy
dose-dependent, ranging from 58% at 4.mu.J/cell (0.0057.
mu.J/.mu.m.sup.2) to 92% at 15 .mu.J/cell (0.021.mu.J/.mu.m.sup.2).
In all cases, cell viability was greater than 95%.
Example 14
Optoinjection of 293T Cells with pEGFP-N1 Plasmid
[0117] In this experiment, the exogenous molecule was a DNA plasmid
of 4.3 kb encoding the fluorescent EGFP protein (pEGFP-N1). The
same hardware apparatus was used as in Example 12. The cells were
in a medium of PBS and 1% HSA, and then 0.1 microgram of plasmid
was added to each well. The cells were imaged, located and targeted
with the laser beam such that each cell received 1 to 8 pulses of
15.mu.J each (0.021.mu.J/.mu.m.sup.2). The cells were washed,
placed in growth medium, and then cultured for 48 to 96 hours.
After culturing, cells were evaluated for the expression of the
fluorescent EGFP protein. As shown in FIG. 10, a number of cells
displayed the fluorescent phenotype in the treated wells (FIG.
10A), whereas no fluorescence was observed in the control well
(FIG. 10B), which were treated identically with the exception of
delivering the laser pulses.
[0118] Although aspects of the present invention have been
described by particular embodiments exemplified herein, the present
invention is not so limited. The present invention is only limited
by the claims appended below.
* * * * *