U.S. patent application number 11/865677 was filed with the patent office on 2008-07-24 for systems and methods for identifying and disrupting cellular organelles.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Michael W. Berns, Thoru Pederson.
Application Number | 20080176332 11/865677 |
Document ID | / |
Family ID | 39641656 |
Filed Date | 2008-07-24 |
United States Patent
Application |
20080176332 |
Kind Code |
A1 |
Berns; Michael W. ; et
al. |
July 24, 2008 |
SYSTEMS AND METHODS FOR IDENTIFYING AND DISRUPTING CELLULAR
ORGANELLES
Abstract
This invention relates to optomechanical systems and methods for
altering, modifying or disrupting a target object. Such systems and
methods are used for, for example, ablating the endogenous nucleus
in a cell.
Inventors: |
Berns; Michael W.; (Irvine,
CA) ; Pederson; Thoru; (Worcester, MA) |
Correspondence
Address: |
PAUL, HASTINGS, JANOFSKY & WALKER LLP
875 15th Street, NW
Washington
DC
20005
US
|
Assignee: |
The Regents of the University of
California
Oakland
CA
|
Family ID: |
39641656 |
Appl. No.: |
11/865677 |
Filed: |
October 1, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60848513 |
Sep 29, 2006 |
|
|
|
Current U.S.
Class: |
436/55 ;
422/82.05 |
Current CPC
Class: |
C12M 35/02 20130101;
G01N 15/1475 20130101; Y10T 436/12 20150115; C12M 47/06 20130101;
G01N 2015/149 20130101 |
Class at
Publication: |
436/55 ;
422/82.05 |
International
Class: |
G01N 35/08 20060101
G01N035/08; H01S 3/00 20060101 H01S003/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] Procedures set forth in this application were supported in
part by Grant No. NIH RR 14892 awarded by the National Institute of
Health, and by Grant No. AFOSR No. F9620-00-1-0371 awarded by the
United States Air Force. The government may have certain rights in
any inventions derived therefrom.
Claims
1. An optomechanical system comprising: a) a platform configured to
accommodate at least one target object in a medium; b) a detector
assembly functionally associated with the platform and configured
to capture images of the target object or a portion thereof; c) at
least one emitter functionally associated with the platform and
configured to emit patterns of radiation suitable for: i) optically
trapping the target object; and ii) disrupting the target object or
portion thereof; d) a controller operatively associated with the
detector assembly and the emitter, wherein the controller is
configured to coordinate the pattern of radiation emission from the
emitter with the image of the target object, or portion thereof,
captured by the detector assembly.
2. The system of claim 1, wherein the target object is selected
from the group consisting of a cell, an organelle, a cell or
organelle complexed with a carrier, or any combination thereof.
3. The system of claim 1, wherein the platform is associated with a
flow path fluidly connected with a reservoir comprising one or more
target objects.
4. The system of claim 1, wherein the flow path is a microfluidic
flow path.
5. The system of claim 2, wherein the cell is a stem cell.
6. The system of claim 5, wherein the stem cell is an embryonic
stem cell.
7. The system of claim 2, wherein the organelle is a nucleus, a
mitochondria, a protoplast, a chloroplast or a centrosome.
8. The system of claim 1, wherein the controller is operated by a
user.
9. The system of claim 8, wherein the user is a remote user.
10. The system of claim 1, wherein the controller is configured to
determine a profile of the target object based on the images of the
target object.
11. The system of claim 3, wherein the controller is further
configured to synchronize entry of a target object in to the flow
path from the reservoir with image detection by the detector
assembly and radiation emission from the emitter.
12. The system of claim 1, wherein the emitter is a laser.
13. The system of claim 12, wherein the laser is a gas laser, a
solid-state laser, a tunable dye laser, or semiconductor laser.
14. The system of claim 1, wherein the detector assembly comprises
a complementary metal oxide semiconductor (CMOS) imager, a charge
coupled device (CCD) imager, a camera with photosensitive film, a
fluorescence imager, a Vidicon camera, or any combination
thereof.
15. A method comprising: a) directing a target object or portion
thereof to a platform; b) imaging the target object or portion
thereof; c) generating a profile of the target object or portion
thereof based upon the image; and d) contacting the target object
or portion thereof to radiation suitable for disrupting the target
object or portion thereof, wherein the contacting comprises: i)
emitting the radiation in a pattern determined by the profile of
the object; ii) translocating the emitted radiation in a pattern
determined by the profile of the object; or iii) maintaining the
emitted radiation substantially stationary while translocating the
object in a pattern determined by the profile of the object.
16. The method of claim 15, further comprising contacting the
target object or portion thereof with trapping radiation.
17. The system of claim 15, wherein the target object is selected
from the group consisting of a cell, an organelle, a cell or
organelle complexed with a carrier, or any combination thereof.
18. The method of claim 17, wherein the object is a cell.
19. The method of claim 18, wherein the cell is an embryonic stem
cell.
20. The method of claim 17, wherein the organelle is a nucleus.
21. The method of claim 15, wherein the radiation is emitted by a
laser.
22. The method of claim 21, wherein the laser is a gas laser, a
solid-state laser, a tunable dye laser, or semiconductor laser.
23. A method for identifying and disrupting a target object or
portion thereof, the method comprising: a) directing a target
object in to a platform; b) imaging a target object or portion
thereof; c) outlining the image of the target object or portion
thereof; d) generating a profile of the target object or portion
thereof by digitizing the outlined image; e) transmitting the
profile of the target object or portion thereof to an electronic
controller operatively associated with an emitter; f) contacting
the target object or portion thereof with radiation emission
suitable for: i) optically trapping the target object or portion
thereof; and ii) disrupting the target object or portion
thereof.
24. The method of claim 23, wherein contacting the target object or
portion thereof for disrupting the object or portion thereof
comprises: a) emitting the radiation in a pattern determined by the
profile of the object; b) translocating the emitted radiation in a
pattern determined by the profile of the object; or c) maintaining
the emitted radiation substantially stationary while translocating
the object in a pattern determined by the profile of the
object.
25. The method of claim 22, wherein translocating the emitted
radiation comprises scanning by a scanning mirror.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 60/848,513 filed Sep. 29, 2006, the disclosure
of which is incorporated herein by reference.
TECHNICAL FIELD
[0003] Provided herein are systems and methods for the alteration,
modification or disruption of target organelles and/or regions in a
cell or group of cells.
BACKGROUND
[0004] Systems and methods for disrupting cellular organelles have
been developed. Conventional systems generally rely on direct user
interface to perform the necessary capture and ablation procedure
on a targeted cell. Accordingly, conventional systems rely on the
presence of a technician or other end-user in order to design,
manipulate, sort, assemble, trap and disrupt a target object, such
as a cellular organelle.
SUMMARY
[0005] The application relates, in general, to the field of systems
and methods for altering, modifying or disrupting a target object
or portion thereof. A target object includes a cell or selected
region in a cell such as an organelle. The cell or portion thereof
can be associated with a carrier, such as a bead or other carrier
that facilitates movement and/or detection of the cell in an
optomechanical system.
[0006] In particular, the present application relates to
optomechanical systems that correlate image acquisition of a target
object with the controlled application of electromagnetic radiation
to the target. An optomechanical system provided herein can be used
in association with fluidic transport elements that transport and
position objects in an environment suitable for imaging and
electromagnetic radiation application. Embodiments of a system
include a remote controller for remotely controlling image
acquisition and radiation application. In some embodiments, a
system is adapted to identify a target object in the absence of
operator intervention. In other embodiments, a target object is
identified by an operator. According to various embodiments, a
system can be remotely controlled via a graphical user interface.
The user interface may be viewed from a host computer or from a
remote site.
[0007] In one embodiment, an optomechanical system is provided. The
system includes a platform configured to accommodate at least one
target object or portion thereof in a fluid medium. A target object
includes a cell, an organelle, a cell or organelle complexed with a
carrier, or any combination thereof. A system further includes a
detector assembly operationally associated with the platform and
configured to capture images associated with a target object
contained in the fluid medium; at least one radiation source
coupled to the detector assembly and operationally configured to
emit a pattern of radiation sufficient to specifically disrupt the
target object; and a controller operationally associated with the
detector assembly and the radiation source. In general, and in the
specific embodiments claimed, the controller is configured to
coordinate the pattern of radiation emission from the radiation
source with the image of the target object captured by the detector
assembly. In some aspects the platform is associated with a fluidic
flow path. The flow path can be connected to a reservoir that
includes a one or more target objects such as cells or portions
thereof. In one aspect the fluid flow path is a microfluidic flow
path.
[0008] In some aspects the object is a cell, such as an embryonic
stem cell. In other aspects, the object is an organelle. In still
other aspects, a portion of a target object, such as an organelle
is targeted for disruption. The portion of a cell can be a nucleus.
In other aspects the portion of a cell can be a mitochondrion,
chloroplast or any other nucleic acid-containing cellular structure
such as a ribosome, nucleolus, inter-chromatin granule cluster,
cytoplasmic P-body or any site of replication by a DNA or RNA
virus, viroid or other nucleic acid-containing entity.
[0009] In some embodiments, a controller associated with a system
of the invention can be operated by a user. In some aspects, the
user is a remote user.
[0010] In other aspects, the controller is configured to determine
a profile of the target object based on the images of the target
object. The controller can be further configured to synchronize
entry of an object in to a flow path associated with a platform.
The flow path may be connected to a reservoir. The platform may be
associated with image detection by the detector assembly and
radiation emission from the radiation source.
[0011] In some embodiments, the radiation source included in a
system provided herein is a laser. The laser can be a gas laser, a
solid-state laser, a tunable dye laser, or semiconductor laser.
[0012] In some aspects, the detector assembly includes a
complementary metal oxide semiconductor (CMOS) imager, a charge
coupled device (CCD) imager, a camera with photosensitive film, a
fluorescence imager, a Vidicon camera, or any combination
thereof.
[0013] In another embodiment, a method of disrupting an object is
provided. The method includes directing an object, such as a cell,
in to a flow path; imaging a target object or portion thereof;
generating a profile of the object based upon the image; and
exposing the target object to a radiation emission sufficient to
specifically disrupt the target object. In general the pattern of
radiation emission is determined by the profile of the object.
[0014] In another embodiment, a method for identifying and
disrupting a target object is provided. The method includes
directing an object in to a flow path; imaging a target object;
outlining the image of the object; generating a profile of the
object by digitizing the outlined image; transmitting the profile
of the object to a controller; contacting the target object or
portion thereof to radiation suitable for disrupting the target
object or portion thereof, wherein the contacting includes: i)
emitting radiation in a pattern determined by the profile of the
object; ii) translocating the emitted radiation in a pattern
determined by the profile of the object; or iii) maintaining the
emitted radiation substantially stationary while translocating the
object in a pattern determined by the profile of the object,
thereby exposing the target object to a pattern of radiation
emission sufficient to specifically disrupt the target object. In
some embodiments trapping radiation is applied to the object in
order to maintain the position of the object.
[0015] In another embodiment, an article of manufacture is
provided. The article includes a computer usable medium having
computer readable program code means embodied therein for causing
an optomechanical system to image a target organelle associated
with a cell; outline the image of the organelle; generate a profile
of the organelle; transmit the profile of the organelle to a
controller operationally associated with a movable reflective
surface; and expose the target organelle to a pattern of radiation
emission sufficient to specifically disrupt the target
organelle.
[0016] The details of one or more embodiments of the disclosure are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages will be apparent from the
description and drawings, and from the claims.
BRIEF DESCRIPTION OF DRAWINGS
[0017] FIG. 1 depicts exemplary external laser radiation paths of a
system.
[0018] FIG. 2 depicts exemplary internal radiation paths of a
system.
[0019] FIG. 3 depicts a flow diagram of various embodiments of a
system.
[0020] FIG. 4 depicts a control panel suitable for remote operation
of a system.
[0021] FIG. 5 depicts an exemplary remote session conducted via an
internet connection with a system.
[0022] FIG. 6 depicts an exemplary remote session demonstrating
remote fluorescence-guided subcellular surgery using a system.
[0023] FIG. 7 depicts two exemplary remote sessions demonstrating
remote laser ablation in phase contrast.
[0024] FIG. 8 depicts an exemplary remote session in which 10
micron diameter microspheres were remotely captured using trapping
lasers operatively associated with a system.
[0025] FIG. 9 depicts an embodiment of a system.
[0026] FIG. 10 depicts a graphical user interface suitable for
interacting with a system.
[0027] FIG. 11 depicts exemplary systems.
[0028] FIG. 12 depicts cells modified by a system.
[0029] FIG. 13 depicts an embodiment of an optomechanical system
that includes a Ti:sapphire laser emitting in the range of 100 to
300 femtosecond pulses at 72 MHz.
[0030] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0031] The application provides optomechanical systems and methods
for efficient inactivation of either a selected cellular organelle
or a specific region of the cell.
[0032] FIG. 1 depicts an exemplary embodiment of an optomechanical
system. In the embodiment of FIG. 1, a single emitter 105 includes
two laser lines designated trapping laser line 110 and ablation
laser line 115. An exemplary emitter is an ND:YAG pulsed laser
including a 75 MHz repetition rate. In other embodiments more than
one emitter can be used to supply a trapping laser line, ablation
laser line, or any other electromagenetic radiation line suitable
for use with an optomechanical system. For example, FIG. 2 depicts
an exemplary system that includes separate emitters for the
trapping laser and the ablation laser. Accordingly, in the context
of an optomechanical system it is understood that the term
"emitter" includes single as well as multiple emitters that provide
trapping, ablation and other electromagentic radiation to a
system.
[0033] Referring again to FIG. 1, trapping laser 110 comprises a
wavelength of between 750 nm and 1250 nm with between 50
femtosecond (fs) and 100 picosecond (ps) pulse durations. Ablation
laser 115 comprises a wavelength that is substantially double that
of trapping laser frequency (e.g., 375 nm to 625 nm) with between
50 fs and 100 ps pulse duration. With regard to pulse duration, it
is understood that emitters capable of emitting laser pulses in the
range of femto- to pico-second durations are suitable for use in an
optomechanical system. Exemplary emitters are set forth in "Laser
nanosurgery of single microtubules reveals location-dependent
depolymerization rates" (Journal of Biomedical Optics, Vol. 12(2),
024022-1 to 024022-8, March/April 2007) and in "Recruitment of DNA
damage recognition and repair pathway proteins following near-IR
femtosecond laser irradiation of cells" (Journal of Biomedical
Optics, Vol. 12(2), 020505-1 to 020505-3, March/April 2007), the
contents of which are incorporated herein by reference in their
entirety.
[0034] Referring again to FIG. 1, each laser line may be controlled
independently via polarizer 125. In some aspects, polarizer 125 may
be included in a motorized rotary mount. Power meters 130 may be
operatively associated with ablation laser line 115 and trapping
laser line 110. Ablation laser 115 and trapping laser 110 are
sampled and measured by mechanical shutters 135 to select laser
power before exposure. Beam expander 150 expands ablation laser
line 115 and beam expander 185 expands trapping laser line to
increase numerical aperture. In some aspects ablation laser line
115 and trapping laser line 110 are directed to the back aperture
of the objective lens mounted in an inverted microscope. In some
embodiments, beam splitters and shutters are used to combine
ablation laser line 115 and trapping laser line 110 with radiation
emitted from arc lamp 155 for simultaneous laser exposure and
fluorescence excitation through the epifluorescence port or through
bottom port 175. Tower 1 160 and tower 2 165 raise the laser lines
to the height of the microscope. An optional dual view imaging
system and a sensitive camera allow for low light level imaging,
FRAP, FRET and ratio imaging techniques. A microfluidic flow path
containing a target object or portion thereof and associated with
platform 180 is exposed to ablation laser line 115 and trapping
laser line 110, and optionally electromagnetic radiation from arc
lamp 155. It is understood that a target object includes a cell, an
organelle, a cell or organelle complexed with a carrier, or any
combination thereof. While the terms "cell" and "organelle" are
used in the examples provided below, it is understood that a target
object includes any object suitable for detection and disruption by
an optomechanical system provided herein.
[0035] Detector assembly 170 detects the image of an organelle
targeted for disruption. Bottom port 175 may serve to provide both
the trapping electromagnetic radiation to a microfluidic chamber
associated with a flow path 180 as well as to provide ablation
electromagnetic radiation to the chamber.
[0036] In some embodiments an optomechanical system further
includes a device for storing images detected by detector assembly.
In other embodiments, a single device can perform both control and
measurement functions (e.g., a device for storing images may be
incorporated in an electronic controller).
[0037] An optomechanical system optionally includes microfluidic
pathways for applications associated with moving single cells from
a reservoir to platform 180. Such applications can employ any
suitable type of microfluidic for a given application. Exemplary
microfluidics can include, without limitation, microfluidic
substrates, cells, tubes, ports and so forth and any combinations
thereof. Such microfluidics can also comprise, for example, wells,
channels, loading regions, loading ports, flow control channels,
nutrient channels, mixing and reaction zones, recovery wells,
arrays and combinations thereof. Exemplary microfluidics can also
comprise silicon or other semiconductor materials such that a first
emitter of a system of the invention can form an optomechanical
trap through or substantially proximate to the microfluidic or a
plurality of microfluidics, which can include, for example, wells,
channels, loading regions, loading ports, flow control channels,
nutrient channels, mixing and reaction zones, recovery wells,
arrays and combinations thereof.
[0038] Referring again to FIG. 1, trapping laser line 110 and
ablation laser line 115 traverse optomechanical system via
reflective elements 145 and other conventional mirrors,
amplifications, lenses, and so forth, as well as any combinations
thereof, such as is shown in the figures by way of example only.
These elements can be used with a system or method of the invention
as well as any other suitable optical means, equipment components,
devices and so forth as would be appreciated by one of ordinary
skill within the art. In particular, it is understood that one or
more trapping lasers from separate laser devices may be combined
with one or more ablation lasers from separate laser devices to
achieve an optomechanical system.
[0039] Labeling of target and non-target organelles can also permit
additional optimization of organelle disruption using an
optomechanical system or method provided herein. As described
throughout the application, an optomechanical system can be used in
automated configurations that minimize user intervention.
Automation can be carried out by any suitable means such as, for
example, computer control comprising control of a series of
shutters, flipper mirrors, motorized stages, acousto-optic devices,
analog-to-digital signal conversion (as well as combinations
thereof) and incorporated in an optomechanical system to permit
rapid data acquisition and position correlation.
[0040] Referring to FIG. 2, exemplary external electromagnetic
radiation paths for trapping laser and ablation laser lines are
provided. A device that emits electromagnetic radiation (EMR)
includes any device capable of generating energy in the
electromagnetic spectrum. Accordingly, the term "electromagnetic
radiation" includes cosmic rays, gamma rays, x-rays, ultraviolet
light, visible light, infrared light, radar, microwaves, TV, radio,
cell phones and all electronic transmission systems. In this
document, a device that emits electromagnetic radiation includes a
"laser" device. Such devices are electromagnetic radiation emitting
devices using light amplification by stimulated emission of
radiation at wavelengths from 180 nanometers (nm) to 1 millimeter
(mm). "Ablation lasers" and "trapping lasers" are examples of laser
devices provided herein. As used herein, a "trapping laser" refers
to a device that emits electromagnetic radiation suitable for
trapping objects or particles by exploiting the properties of
momentum associated with light. When light passes through a fluid
medium, the optical path is bent by refraction in the fluid
material. The bending of the light path corresponds to a transfer
of momentum from the light to the refracting object or particle.
The transfer of momentum exerts a force, which is capable of
holding or manipulating the motion of the object or particle. This
process is also designated "optical trapping." The term "laser
ablation" refers to the process of using electromagnetic radiation
to disrupt or modify a target object. An "ablation laser" refers to
a device that emits electromagnetic radiation suitable for
disrupting or modifying a target object.
[0041] Referring again to FIG. 2, the left panel shows the path of
the ablation laser line. The laser line passes through a shutter,
is optionally expanded, and directed onto a fast scanning mirror
for beam steering. In this embodiment, the scanning mirror "steers"
the laser across a target object while the target object remains
substantially stationary. It is understood that the same or similar
effect can be achieved by keeping the laser beam substantially
stationary while moving a platform comprising a target object such
that the object to be trapped or ablated can be moved in a pattern
consistent with the targeted object. In this aspect, the object
rather than the beam is moved such that the laser beam ablates the
desired pattern consistent with the target object. In other
aspects, controlled shields or masks can be used such that the
desired target object can be ablated while other parts of, for
example, a cell are shielded from ablation.
[0042] Referring again to FIG. 2, a beam may be directed to an open
port associated with a microscope where a mirror reflects the beam
up through the microscope stand and into the microscope objective.
The right panel shows the radiation path of the trapping laser line
and the externalized epi-fluorescence radiation path. The beam is
optionally expanded by one or more biconvex lenses and mixed with
the arc lamp emission with a short band-pass dichroic mirror.
Custom dichroic mirrors in filter cubes housed in the microscope's
reflector turret reflect the laser and part of the arc lamp's
visible spectrum up into the microscope objective.
[0043] FIG. 3 depicts exemplary internal radiation paths of a
system provided herein. In this example, optics were chosen to
passively mix and separate visible and near infra-red (NIR)
radiation. Green laser light enters from underneath the microscope
stand through a port (sometimes referred to as a "Keller" or
"Keller-Berns" port) from below the microscope, the tube lens, one
of the reflector turret's filter cubes, and the microscope
objective. NIR radiation from a trapping laser and visible light
from the arc lamp (both shown as circles in the filter cube) enter
the back of the microscope stand, normal to the plane of the
specimen in this figure, and are reflected upwards by one of the
reflector turret's filter cubes. Long-red radiation from the
halogen lamp may be selected by a filter in front of the condenser
for phase contrast imaging. Optionally, visible emission from the
specimen enters a dual video adaptor device modified to pass the
long-red phase contrast light to the video camera and to reflect
the shorter wavelength fluorescent emission up through the dual
view system and onto the ORCA-AG CCD camera.
[0044] Various exemplary embodiments of an optomechanical system
are described in the flow diagram of FIG. 4. The flow diagram
depicts connectivity between hardware associated with a system and
software suitable for operating the system from a remote
location.
[0045] FIG. 5 depicts exemplary control panel that may be used to
monitor and control an optomechanical system. The gray box at the
top of the screenshot is a drop-down menu to configure the session
during operation of an optomechanical system. Shown are controls
for stage movement, laser ablation, and image acquisition/display.
The intensity histogram of each image may be displayed to aid in
determining the proper setting for camera acquisition and image
display.
[0046] FIG. 6 depicts an exemplary remote session in which PTK2
(rat kangaroo kidney) cells were imaged with a 63.times.PH3 1.4 na
oil immersion objective. The user may remotely adjust the camera
gain, acquisition time, and region of interest to optimize contrast
and reduce image data size. Subfigure panels demonstrate remote
operation of the X-Y stage and objective focus during a remote
experiment. Note resolution of the nuclear envelope.
[0047] FIG. 7 depicts an exemplary remote session demonstrating
remote fluorescence-guided subcellular surgery using an
optomechanical system. The remote user searched for an appropriate
cell and interactively focused and positioned the microtubule
organizing center (a.k.a. centrosome) below the laser crosshair
(first image). The next images show: post-firing of a 3 ms
macropulse, the remaining region of the microtubule organizing
center interactively positioned below the laser scissors crosshair,
and a second 3 ms macropulse fired to delete the remaining
microtubule organizing center.
[0048] FIG. 8 depicts two exemplary remote sessions demonstrating
remote laser ablation in phase contrast. Erythrocytes served as a
convenient test specimen and targeting guide. Panel A shows an
exemplary first session where a remote user has selected a region
of interest of the CCD chip to speed image transfer for focusing
the specimen. Shown is pre and post-focusing. Panel B shows a
second exemplary session where a region of cells was brought into
focus remotely. Panel C demonstrates optomechanical system beam
steering capabilities by ablating at the center of the field (see
crosshairs shown in FIG. 4) (see "i"), ablating single shots by
beam steering (see "ii"), ablating along a line (see "iii"), and
ablating within a remote user-defined rectangle (see "iv"). For
examples iii and iv, the user defined the pixel radius of a single
ablation, and the optomechanical system calculated the number of
laser exposures necessary to fill the region in.
[0049] FIG. 9 depicts an exemplary remote session in which 10
micron diameter microspheres were remotely captured in the trapping
laser line. The microspheres can be composed of any suitable
material, such as polystyrene. Every fifth frame of an 8.2
frame/sec time series is shown. In frame 1, the two downward-left
arrows (three arrows in subsequent frames) indicate reference
microspheres moving through fluid flow in a 35-mm Petri dish. The
upward-right arrow indicates a microsphere captured in the laser
tweezers. A slight axial displacement of this microsphere (due to
the laser tweezers) as compared with the free-floating microspheres
can be observed, as the center dark region of the microsphere has
transitioned to white. Frames 2-5 show the displacement of the
reference microspheres relative to the trapped microsphere. Just
prior to frames 6-10, the remote user released the microsphere from
the trap through an optomechanical system control (note the
reversal of the axial displacement), and the microsphere is carried
away by the fluid flow. Note the second microsphere attracted to
the laser focus but held away by the trapped one.
[0050] FIG. 10 depicts an optomechanical system graphical user
interface (GUI). The exemplary user interface of an optomechanical
system is shown as it may be viewed on the host computer or from a
remote site. The interface offers control of the microscope stage,
laser power and exposure, arc lamp excitation, image acquisition
and focus, or any combination thereof. The interface and its
underlying functionality can be configured to automate a vast array
of laser microbeam experiments, with appropriate user feedback and
control.
[0051] FIG. 11 provides images of an embodiment of an
optomechanical system. In this embodiment, three distinct elements
are shown: 1) a laser microscope; 2) control hardware; and 3) a
control software-web server. The exemplary optomechanical system
includes hardware and software that allows the system to be
controlled by remote users via the internet.
[0052] Referring to FIG. 13, a schematic diagram of an embodiment
of an optomechanical system is provided. In this embodiment a
Ti:sapphire laser beam is used to provide an ablation laser line.
As shown in FIG. 13, the line may be expanded and passed through a
shutter and neutral density wheel before being directed through the
microscope and objective. The beam may be focused at the imaging
plane. A dichroic mirror in the laser path reflects the beam and
allows for fluorescence excitation light to transmit into the
objective and illuminate the specimen.
[0053] The exemplary Ti:sapphire femtosecond laser is a Coherent
Mira 900 Ti:sapphire laser (Coherent Incorporated, Santa Clara,
Calif.) emitting at about 200 femtosecond pulses at about 76 MHz
was used for ablation at 800 nm. Laser power was controlled
manually by a neutral density wheel, which allowed regulation of
beam power from 0 to 700 mW. Laser power was measured by a
FieldMaxII TOP power meter coupled to a PM3 probe (Coherent
Incorporated, Santa Clara, Calif.) with a 19-mm-diameter sensor. In
this example the probe was placed directly after the objective
where the diameter of the laser beam was smaller than the sensor
diameter. This allowed for collection of all light exiting the
objective, which resulted in an estimate of the power level at the
focal point of the specimen. The diameter of the laser beam at the
focal point was calculated from the equation d=1.22.lamda./NA,
where .lamda. is the wavelength and NA is a numerical aperture of
the objective. Laser exposure time was controlled by a motorized
Oriel (Stratford, Conn.) electronic shutter controller, which
allowed a minimum of 5-ms exposure time. Laser exposure times were
set at about 150 ms. The beam was directed into the
epi-illumination port of a Zeiss Axiovert S100 2TV microscope (Carl
Zeiss Incorporated, Thornwood, N.Y.) by a dichroic filter designed
to pass visible arc lamp emission and reflect 800-nm laser light.
The beam was focused by a Zeiss 63.times.PH3 oil immersion
apochromat objective lens (numerical aperture 1.40).
[0054] Referring again to FIG. 13, targeting was accomplished by
moving the stage (Ludl Incorporated, Hawthorne, N.Y.) by joystick
control until the region of interest was positioned at the
pre-aligned laser focal point. Laser ablation was accomplished by
stage movement, such that the target region was exposed to the
focused laser. Stage movement was controlled by a PCI-7344 motion
controller and a MID-7604, 4 Axis Integrated Stepper Driver Power
Unit (National Instruments), all controlled by software described
in the present application. In this embodiment the optomechanical
system included a 532-nm Spectra-Physics Vanguard laser with 76 MHz
repetition rate and 80-ps pulse duration. Average power was
attenuated by rotation of a neutral density wheel.
[0055] In this embodiment image acquisition occurred immediately
before laser exposure, after laser exposure, and at pre-selected
sequential time points following ablation. Twelve time series
images were acquired at 5- to 7-s intervals. All controls for laser
ablation and imaging may be located on a control panel described in
the application
[0056] A 75-W mercury/xenon arc lamp was used to excite
fluorescence. Exposure of the sample to excitation illumination was
controlled by a motorized Oriel mechanical shutter and varied from
0.5 to 2 s. An external excitation interference filter (CFP bp 426
to 446 nm; YFP bp 490 to 510 nm) was used to select the excitation
wavelengths. The microscope filter cube included a beamsplitter and
emission interference filter (CFP dichroic long-pass 455
nm/emission 460 to 500 nm; YFP dichroic long-pass 515 nm/emission
520 to 550 nm). Images were acquired using a Hamamatsu Orca
C4742-95-12HR digital charge-coupled device (CCD) camera (Hamamatsu
Corporation, Bridgewater, N.J.0. Images were stored as 16-bit tiff
images.
[0057] In some embodiments an optomechanical system includes a
fluorescence excitation source for YFP experiments. An exemplary
excitation source includes an X-Cite 120 Fluorescence Illumination
System (Exfo Photonics Solution, Incorporated, Ontario, Canada).
Exposure of the excitation source was controlled by Zeiss Axiovert
200M. Images were acquired using a Hamamatsu Orca-ER C4742-80
digital CCD camera.
[0058] In another embodiment an optomechanical system may be used
in methods related to nuclear inactivation of cells in a
microfluidic flow path or cells growing on a flat surface. With
regard to cells growing on a flat surface, the cells may be
observed under a microscope using either alone or in combination
under phase contrast, single or multiphoton fluorescence (or some
other form of microscopy). This can be done in cells prior to
fusion or after fusion. For example, the nuclei of a stem cell
fused with a somatic cell may be identified by any number of
methods: phase contrast microscopy, interference microscopy,
morphology, fluorescence, location etc. Once identified, either by
computer recognition or by a user, a laser of appropriate
wavelength and energy parameters, will be focused on the nucleus or
any other structure that is to be disrupted, eliminated, or
inactivated. The laser may be scanned (i.e., translocated across
the target object) to expose the entire nucleus, or the beam may be
geometrically shaped to encompass the entire nucleus such that
scanning becomes unnecessary, or the platform comprising a target
object is translocated while the beam remains substantailly focused
and stationary, or any combination thereof. The result will be
inactivation of the unwanted nucleus. Using a computer-controlled
automated microscope, thousands of cell nuclei can be irradiated in
several minutes, thus providing a high number of cells that contain
the cytoplasm of the stem cell but only the programmed or
to-be-reprogrammed nucleus of the somatic cell.
[0059] In another embodiment, inactivation of a target organelle in
a cell is provided. The term "organelles" includes such cellular
structures as the mitochondria, nucleus, endoplasmic reticulum, and
microtubules of a target cell. In this embodiment, a target
organelle associated with a cell in suspension or attached to a
solid can be selectively disrupted or inactivated. For example, a
stem cell nucleus can be inactivated as it passes through the
focused laser beam in a flowing situation. The method is suitable
for use in situations including: (a) inactivation of the stem cell
nucleus prior to fusion, and (b) inactivation of the stem cells
nucleus in the fused hybrid cell. With regard to example (a), the
stem cell nucleus will be inactivated as it flows through the
focused laser beam because of specific fluorescent molecules that
are either being or have been expressed by the nucleus (thus making
it detectable as well as sensitive to the laser light), or by some
exogenous molecule that has been added to the cell suspension or
microinjected, electroporated or introduced into the cell in any
other way such that it is incorporated into the nucleus and makes
the nucleus sensitive to the light. Thus, "photosensitization" of
the target object using selective dyes is encompassed by the
methods provided herein. The cell nucleus is inactivated as it
passes through the laser beam. This may be accomplished in a
microfluidic chamber or in a larger vessel more commonly used in
FACS cell sorting machines. The cells with inactivated nuclei are
then collected and used for the cell fusion process. With regard to
example (b), the cells that are passed through the system are fused
stem cells and somatic cells. The stem cell nuclei are
destroyed/inactivated as they pass through the focused laser beam
due to their having been pre-tagged with a dye or some other
light-absorbing molecule (as described above) so that when these
cells pass through the laser beam the light-absorbing molecule
absorbs the light which in turn inactivates the nucleus of the stem
cell. This can be performed in a microfluidic device or in a larger
vessel used in standard FACS cell sorting machines.
[0060] A system provided herein may include a motorized inverted or
upright microscope stand, external optics to direct the ablation
and trapping lasers into the microscope, a CCD digital camera, a
hardware-software suite for the control of laser power, the
specimen stage, and microscope stand focus and illumination. The
system is equipped with objectives of numerical aperture and
working distance. In some embodiments, the system includes a fast
scanning mirror that allows the laser beam that comes into the
microscope to move in the microscope field by altering the position
of the mirror. The control of the fast scanning mirror is
accomplished by a computer which is interfaced with the computer
keyboard's mouse and the scanning mirror hardware. The object can
be outlined and excised (or exposed to the laser beam) by outlining
it on the computer monitor (which projects an image of what is
under the microscope). The outline is then converted into digital
signals that than controls the movement of the scanning mirror.
Thus the mirror moves in such a way that the laser beam follows the
outline that the mouse made and destroys/dissects/exposes the
specific target region that was outlined on the monitor screen
using the mouse (or any other device such as a touch-screen
pen).
[0061] In general, research microscopes have been developed to
include motorized versions of their inverted microscopes. In
exemplary embodiments, an optomechanical system utilizes a Zeiss
Axiovert 200M with motorized objective turret, reflector turret
(for fluorescence filter cubes), condenser turret, halogen lamp
shuttering with intensity control, mercury arc lamp shuttering,
camera port selection, objective focus, and parfocality adjustments
for switching between objective lenses. The microscope also has a
motorized optivar turret to increase the system magnification by
1.63 or 2.53. For laser ablation experiments, a 63.times. oil NA
1.4 plan-apochromat PH3 oil objective is used (though 100.times.,
40.times. and others can be used). The microscope stand has a
built-in computer, which uses a controller area network (CAN) to
communicate with motors and encoders within the microscope stand.
The CAN receives commands through a serial interface typically
attached to a computer running an image acquisition/microscope
control program. Rather than using the software provided with the
microscope, which was found to be cumbersome and slow for our
purposes, custom control software capable of communicating with the
CAN as described below was developed.
[0062] Features of the motorized microscope which are especially
relevant to remote operation of a laser microscope are the
shift-free reflector turret, microscope light path selection,
illumination control, and objective focus. The shift-free reflector
turret allows the user to repeatedly switch between any of five
fluorescence filter cubes in the turret without a detectable pixel
shift in the image. This is of great importance when performing
resolution-limited targeting for laser ablation, as it ensures that
the laser will always focus at the expected pixel location.
Likewise, the Axiovert microscope can switch between camera ports
repeatedly with no detectable pixel shifts when initiating an
ablation sequence. One skilled in the art will recognize that other
commercial microscopes provide similar or modified automated
features that are compatible with this invention. In addition, such
microscopes can be constructed to suit particular requirements
under specific laboratory situations.
[0063] A Zeiss dual video adaptor is mounted on the left-hand
camera port to allow simultaneous imaging of transmitted light and
fluorescence. The 50/50 beam splitter shipped with the video
adaptor was replaced with a long-pass dichroic mirror (640 DCLP
Chroma Technologies, Rockingham, Vt.), which transmits longer red
light and reflects the shorter visible spectrum upwards to the
second camera. A band-pass filter centered at 680 nm (d680/603
Chroma Technologies) is placed in front of the condenser lens to
limit the transmission light wavelengths. Fluorescence emission is
reflected upwards by the dichroic mirror into a chromatic image
splitter (Dual View, Optical Insights, Albuquerque, N. Mex.) that
forms two images of the specimen simultaneously on the camera,
representing two bands in the visible spectrum. The Dual-View image
splitter also has a straight-through option mode with no image
splitting. A closed circuit television camera may be mounted on the
transmission port of the dual video adaptor for imaging
bright-field or phase-contrast images from the long-red light path.
Phase-contrast images can be captured with the high-sensitivity
camera by removing the 680 nm band pass filter.
[0064] Referring again to FIG. 1, specimens may be mounted on
platform 180 and platform may be associated with microfluidic
devices suitable for presenting a cell to the platform. In one
embodiment platform 180 is an X-Y stepper stage (Ludl Electronic
Products Hawthorne, N.Y.) controlled with a National Instruments
"flexmotion" PXI-7344 stepper motor controller and an MID-7604
power drive (National Instruments, Austin, Tex.). The flexmotion
board may be mounted in a PXI electronic controller chassis for
implementing software suitable for controlling system 100.
Exemplary software includes the LabVIEW Realtime operating system,
which is a graphics-free computing environment designed to maximize
performance of control hardware. The optomechanical system host
electronic controller communicates with the PXI chassis through a
local area network (100 Mbps) running TCP/IP protocols. Optionally,
an on-board program is included to run on the motion controller
allowing local joystick control independent of both the host and
the PXI electronic controller's CPUs. Motorized objective focus
control may be achieved through the CAN by Zeiss' Harmonic Drive DC
motor, providing 25 nm steps with 10 mm travel for precise focus
control over multiple objectives' working distances. To achieve
stable temperature control for specimens imaged by an oil-immersion
objective lens, both the specimen and the objective lens may be
heated. Specimens in 35-mm Petri dishes are heated with a stage
heater (heater: DH-35; controller: TC-324B; Warner Instruments
Corporation, Hamden, Conn.) while the objective is heated with a
collar-type objective heater (heater: OBJSTD with controller,
Bioptechs, Inc., Butler, Pa.). Other methods of temperature control
are also compatible with embodiments of the optomechanical
system.
[0065] The epi-illumination system was removed from the microscope
stand for direct access by the trapping beam to the back aperture
of the microscope objective. The epi-illumination system was
mounted distal to the microscope and coupled through two 400 mm
positive achromatic doublets (Newport Corp., Newport, Calif.) into
the microscope. The motorized Axiovert is commercially supplied
with a motorized shutter for the fluorescence light path which we
removed because of inherent delays between computer commands to
open the shutter and the opening event. Instead, an electronic
shutter (Vincent Associates, Rochester, N.Y.) was mounted between
the arc lamp and the epi-fluorescence lens system, with a notable
decrease in delay time. Other methods of epi-illumination beam
entry into the microscope are also possible, including normal entry
through a fiber optic cable.
[0066] Connectivity of key controllers and actuators in an
optomechanical system is demonstrated in FIG. 5. The unique
identifiers in each block of the diagram (e.g., 610C) are for
reference to an online reference manual available on the world-wide
web at robolase.ucsd.edu. In the diagram the electronic controller
is designated "Main PC." The electronic controller includes
software suitable for operating an optomechanical system.
Controller connects to the ablation laser and the microscope
through two serial communications: to the ORCA camera controller
through a firewire connection and to a PXI chassis through the
optomechanical system local network. The PXI chassis contains a
motion control card that connects to a stepper motor driver that
responds to joystick commands, controls two shutter drivers, and
drives the platform (e.g., XY microscope stage). The chassis
optionally includes a data acquisition card that receives data from
a power meter and communicates with the ablation laser's beam
steering controller through analog voltage outputs. In this
embodiment the optomechanical system can also operate with other
chassis such as an MXI chassis.
[0067] Optics outside the microscope stand guide the ablation and
trapping lasers into the microscope and supplies an optomechanical
system with automated laser power control, laser shuttering, and
laser power monitoring (see FIG. 1). An exemplary laser ablation
radiation source includes a diode-pumped Spectra-Physics Vanguard
with a second harmonic generator (SHG) providing TEM00 mode 532 nm
laser light linearly polarized with 100:1 purity with a 76 MHz
repetition rate, 12 ps pulse duration, and 2 W average power. The
un-attenuated laser power is far in excess of that necessary for
resolution-limited subcellular laser ablation and left
un-attenuated is well above the plasma threshold causing
catastrophic damage to cells in the vicinity of the laser. The
laser beam polarization purity is considerably increased from 100:1
through the first glan linear polarizer (CLPA-12.0-425-675, CVI
Laser, LLC, Albuquerque, N. Mex.) with a 5.times.10.sup.5
extinction ratio rotated for maximum transmission (95%). Laser
power is controlled by rotating an identical glan linear polarizer
placed in series to the first and mounted in a motorized rotational
mount driven by an open loop 2-phase stepper motor with
0.05.degree. accuracy (PR50PP, Newport Corp.). The stepper motor
rotates the polarizer from its vertical orientation with maximum
transmission (95%) to its horizontal orientation with minimum
transmission well below the damage threshold of biological samples.
The stepper motor is controlled via the flexmotion board in the PXI
chassis. Light exiting the second polarizer is partially reflected
by a laser-line beam sampler, with dual antireflection-coated
surfaces. The sampled beam may be measured by a photodiode (2032
photoreceiver, NewFocus, San Jose, Calif.) and converted to a
voltage. A calibrated photometer (1825-C, Newport Corp.) may be
used to determine the relationship between the photodiode voltage
and average laser power in the main beam. A mechanical shutter
(Vincent Associates) with a 3-ms duty cycle gates the main laser
beam to provide "short" bursts of pulses to the microscope.
[0068] The laser beam may be expanded using an adjustable beam
expander (2-8.times., 633/780/803 nm correction, Rodenstock,
Germany) and lowered to a height just above the optical table by
two additional mirrors. Telecentric beam steering is achieved by
placing a single dual-axis fast scanning mirror (Newport Corp.) at
an image plane conjugate to the back focal plane of the microscope
objectives. This image plane is formed by a 250 mm biconvex lens
positioned with its front focal plane at the image plane of the
microscope Keller port (below the microscope stand) and with its
back focal plane at the fast scanning mirror surface. To access the
sub-microscope Keller port, the microscope is raised 70 mm above
the table via custom-machined metal alloy posts to leave room for a
45.degree. mirror, which vertically redirects incident laser light
running parallel to the table through the Keller port (FIG. 3).
Once inside the microscope stand, the laser light passes through
the tube lens and one of the five fluorescence filter cubes of the
reflector turret before entering the back of the objective lens.
The reflector turret can be set up either with one filter slot
blank or since the turret is automated, the system can position a
fluorescence filter cube into place, with appropriate laser
transmission characteristics. It is understood that the present
invention encompasses microscope designs that do not bring the
laser in through a Keller port.
[0069] All external mirrors in the ablation laser light path are
virtually loss-less dielectric mirrors optimized for 450
reflections of 532 nm S-polarized light (Y2-1025-45-S, CVI Laser
LLC, Albuquerque, N. Mex.).
[0070] In some embodiments the trapping laser light source is an
ytterbium continuous wave fiber laser with a 5-mm collimator
providing randomly polarized TEM.sub.00 mode 1,064 nm laser output
with 10 W maximum power (IPG Photonics Corp., Oxford, Mass.). Laser
power is controlled programmatically through serial port
communication. Laser light is reflected off two mirrors and into a
custom beam expander comprised of two anti-reflection coated
bi-convex lenses (f=100 mm, 400 mm) placed telescopically to expand
and collimate the beam. The 100 mm lens is placed so that its back
focal plane lies on the surface of the second mirror, which is
mounted in a second dual-axis fast scanning mirror (Newport Corp.)
to achieve telecentric beam steering in the specimen plane. The
laser then reflects off a 2 inch diameter short pass dichroic beam
splitter placed behind the microscope in the arc lamp illumination
light path to merge the two light paths. Laser light is then
reflected upwards by a dichroic mirror mounted in the reflector
turret, and into the back of the objective lens where it is focused
in the specimen plane. The 100 mm lens mount can be adjusted
axially to move the laser trap depth relative to the image plane. A
mechanical shutter is placed in the beam path and is controlled by
the flexmotion controller. All external mirrors in the trapping
laser light path are virtually loss-less dielectric mirrors
optimized for 450 reflections of 1064 nm S-polarized light
(Y1-1025-45-S, CVI Laser).
[0071] A detector assembly may include a high quantum efficiency
digital camera to capture transmitted and fluorescent images. In
some embodiments, an optomechanical system implements a Hamamatsu
Orca-AG deep-cooled 1,344.times.1,024 pixel 12-bit digital CCD
camera with digital (fire wire) output. The ORCA can read out sub
regions of the chip for increased frame rates, bin pixels for
increased signal-to-noise, and adjust gain and exposure time to
trade off between signal-to-noise characteristics and arc lamp
exposure times. An optomechanical system may use Hamamatsu's Video
Capture Library for LabVIEW (ver 1.0) plug-in to communicate with
the ORCA camera controller through its DCAMAPI driver (FIG. 4).
[0072] Various software may be engaged during operation of an
optomechanical system. In one example, a hardware control suite and
web server software are used for sharing an optomechanical system
with remote users. Exemplary control software programmed in the
LabVIEW 7.1 (National Instruments) programming language may be used
to control the microscope, cameras, and external light paths
associated with an optomechanical system. The control software also
manages image and measurement file storage. It communicates with
the user through the graphical user interface or the control panel
in LabVIEW. The control panel receives user input and displays
images and measurements. The control software interprets commands
sent by the user into appropriate hardware calls and returns the
results of that action to the front panel and/or computer's hard
drive.
[0073] As noted above, FIG. 5 provides a snapshot of the front
panel. The upper-left panel contains laser parameter controls. This
panel contains two tabs: one in green to control the ablation laser
and one in blue to control the trapping laser. Ablation laser
controls include a slider to select power in the focal plane, two
buttons to fire the laser either at the center of the field or at
the green crosshairs which are positioned with the mouse, and
selection of the filter cube turret position during ablation. Once
either fire button is pressed, the control software calls the
microscope CAN to select the Keller port and the appropriate filter
cube. The control software then continuously quarries the CAN to
ensure the completion of both actions before opening the shutter
for a single 3 ms laser burst. Beam steering is sufficiently faster
than the camera port and filter turrets, such that a quarry of its
position prior to opening the shutter is unnecessary. Laser
tweezers controls (not shown) include laser power selection,
shutter state and beam positioning controls.
[0074] Referring to FIG. 5, the center panel on the left contains
stage and ablation laser steering controls. The "stage control" tab
contains left/right and up/down rockers to move the microscope
stage with position feedback. A slider selects either step or servo
mode to move the stage either in increments specified in the "X/Y
Step Size" control, or continuously while the rockers are pressed.
A similar pair of rockers moves the microscope objective for focus
control. The "Click and Move" control is a novel control designed
to minimize exposure of the cells to the arc lamp light during
stage movements. The user simply chooses the crosshair tool from
the toolbar to the left of the image and clicks on an object of
interest in the image. The program then calculates that pixel's
displacement from the field of view center and moves the stage to
center the object. The "Expose?" check box provides the option to
follow the move with an exposure. The "Coordinate List" tab allows
the user to store the current position in a list or to return to
any stored coordinate. The "Coordinate Utility" tab allows the user
to load an old list of coordinates, to clear the current list, or
to save the current list to the hard drive. The "Special Moves" tab
contains controls for beam steering and for laser ablation through
a series of z-coordinates. The user can select the rectangle tool
from the tool bar and draw a rectangle around a region in the
image. There is a control in this tab to carve out that rectangle
by firing single macropulses (one opening of the mechanical shutter
which will pass multiple individual laser pulses) at evenly spaced
locations in the rectangle. Since the laser causes nearly
diffraction-limited ablation, the program calculates the number of
macropulses necessary to fill in the rectangle-based on the pixel
dimensions of the rectangle and the pixel extent of a single
diffraction-limited ablation. It is understood that the control
panels described above and elsewhere in this document are exemplary
only. The skilled artisan will recognize that a system provided
herein can be controlled by any type of panel suitable for
monitoring and managing the activities of the system. Such panels
can be modified to accommodate additional functions according to a
users specific requirements.
[0075] Referring again to FIG. 5, the lower panel on the left
contains image acquisition controls. The "Image Acquisition" tab
contains controls for exposing single images, continuous
acquisition (Focus), image storage, and image printing. The user
can select the filter cube to place during the acquisition, whether
to gate the arc lamp during the exposure, and controls to calculate
a ratio image when used with the chromatic image. The "Root
Directory" control specifies the top directory for file saving
using our automated file naming system, and an indicator displaying
the full path and name of the last saved image. The file path and
name are designed to prevent accidental overwriting of data during
successive operations of the program, coding the file name with the
current time. The "Time Series" tab contains controls for acquiring
a time series of images. The time series uses setting from the
"Image Acquisition" tab and contains controls for the number of
images and the duration between images as well as an indicator of
the last image saved in the time series. The "Raster Scan" tab has
a control to raster through user-selected stage coordinates and
acquire images at those locations at time durations set through a
control.
[0076] Referring to FIG. 5, the lower panel on the right contains
camera and microscope controls. The "Image Display" tab displays
the last acquired image plus the toolbar for selecting ablation and
click-and-move coordinates. The "Microscope Control" tab contains
controls for the microscope stand to select the objective, filter
cube, condenser filter, optovar, and image port. The "Camera
Controls" tab contains controls for camera gain, digitization
offset, exposure time, and binning. It also contains an
area-of-interest control to only transfer image data from an area
of interest defined with the rectangle tool in the image display.
Lastly, this tab has controls for click and move parameters
including pixel coordinates of the field of view center and the
pixel/microscope step gain.
[0077] The upper panel on the right contains a message box and the
image histogram. The message box displays important messages, such
as error notifications or equipment status, and draws user
attention by pulsing the large green digital LED to the left of the
message box when a new message arrives. The gray box controls the
image display lookup table for mapping 12-bit images to the 8-bit
display. This control uses four modes of look-up table: (1)
Full-dynamic, in which the range of nonzero intensities are divided
into 256 equally spaced bins, (2) 90%-dynamic, in which the dynamic
range containing the middle 90% of the cumulated histogram of the
image is divided into 256 equally spaced bins, (3) Given-range, in
which the range of grayscale values specified by the "Maximum
Value" and "Minimum Value" slider controls are equally divided into
256 bins, and (4) Down-shift, in which the grayscale values are
shifted to the right in 8-bit increments, as specified by a
control. An image histogram displays the pixel intensity histogram
of the last acquired image to aid in the selection of an
appropriate lookup table and to quantify separation between the
background noise mode and the pixels of interest.
[0078] Two web server packages were compared. The first is the
"remote panel" feature provided by National Instruments as a
feature of LabVIEW. The LabVIEW web server publishes the control
panel as an html document to which multiple users can log on during
runtime. Once users connect to the an optomechanical system
webpage, the front panel of An optomechanical system will appear in
their web browser window with all the functionality available to a
user operating from the host computer. Those logged on can either
participate as an observer, or request control of the control panel
to perform an experiment. It is not necessary for the remote user
to have LabVIEW installed. To operate the An optomechanical system
remote panel, it is only necessary to install the free LabVIEW
run-time engine installed automatically at the first connection to
any remote panel. The server can be configured to allow browser
access for viewing, viewing and controlling, or to deny access to a
programmable list of IP address. The LabVIEW protocol works by only
transmitting changes to the control panel as they occur, as opposed
to continuously transmitting the entire control panel as well as
the states of the buttons and controls.
[0079] The second web server is a web-based protocol "Log me in"
available on the world wide web at "logmein.com" (3 am Labs, Inc.,
Woburn, Mass.). This exemplary protocol belongs to a family of
software that allows remote control of a PC through a live window
that functionally duplicates the host PC from anywhere with an
Internet connection. The protocol uses a peer-to-peer session
handoff to provide high-speed remote control by eliminating the
gateway, thereby allowing the two PCs to communicate directly. The
logmein.com host computer maintains a constant secure sockets layer
(SSL)-secured connection with one of the logmein.com gateways. This
link is initiated by an optomechanical system and the firewall
treats it as an outgoing connection. The client browser operated by
a remote user establishes a connection to Logmein.com and
authenticates itself after which the gateway forwards the
subsequent encrypted traffic between the client and the host. The
remote user is not required to download additional programs to
connect to an optomechanical system; however, there is an optional
ActiveX control download to improve image quality. The user can
switch the host's display between "low quality" 8-bit color images
and "high quality" 16-bit color images during the session to trade
off between color resolution and frame transfer rates.
[0080] It is known that reprogramming of a stem cell is possible by
fusion of a somatic cell nuclei with human embryonic stem cells
(Science, 309:1369-1372, 2005). By placing somatic cell nuclei
(specifically from skin fibroblasts) into the cytoplasm of stem
cells via standard cell fusion methods, it has been possible to
activate genes that had long been silent in the somatic cell nuclei
taken from the adult donor. These may be genes that cause the
descendants of the fused cell to differentiate into any number of
cell/tissue types. It has been suggested that the cytoplasm of the
stem cell contains biochemical factors/signals that cause the
somatic cell chromosomes/genes to be activated and/or re-programmed
thus resulting in a cascade of biochemical signaling ultimately
resulting in the production of molecules and structure of virtually
any tissue type. How the above steps will be understood and
eventually harnessed to produce new tissues and organs, and to help
in the understanding of disease processes that affect these
tissues, will be a major area of research and development for many
decades to come.
[0081] In somatic cell nuclear transfer, or any other mode of
generating stem cells that involves placing a somatic or other
(zygote, embryo, fetus, transplant or implant) nucleus into an
ovum, early embryo, fetus or other cell type at any other stage of
embryonic or post-natal stage of development, it may be desirable
to remove, destruct or biologically inactivate the DNA or RNA
genomes of the host cell, i.e. the fused or pre-fused cell, whether
these genomes be nuclear, mitochondrial, chloroplast, viral, viroid
or any other endosymbiotic or infectious agent with a genome
composed of DNA, RNA or both.
[0082] As early as 1969, Berns used a microscope-focused laser beam
to excise small regions of chromosomes in living cells (Berns et
al., Nature 221: 74-75, 1969; Berns et al., Exp. Cell Res. 56:
292-298, 1969; Berns et al., J. Cell Biol. 43: 621-626, 1969). This
was followed by the demonstration that the laser beam could be used
to inactivate genes (reviewed in Berns and Rounds, Sci. Am. 222:
98-103, 1970) and more recently, that genes could be inactivated on
chromosomes by using multiphoton absorption (Berns et al., PNAS 97:
9504-9507, 2000). The contents of these publications are
incorporated herein by reference in their entirety.
[0083] Provided herein are systems and methods for high throughput
imaging and modification of a target cellular organelle in a cell,
such as a stem cell nucleus. The cell may be growing flat in a
dish, or unattached in suspension. A system may be engineered to
include a robotic laser ablation and tweezers microscope. The
system may be configured to be operated via the internet using most
internet accessible devices, including laptops, desktop computers,
and personal data assistants (PDAs). The system affords individual
users the ability to conduct micromanipulation experiments (cell
surgery or trapping) from remote locations. A system described
herein greatly expands the availability of complex and expensive
research technologies via investigator-networking over the
internet, or via direct high speed optical connections between
institutions.
[0084] The system offers three unique features: (1) the freedom to
operate the system from any internet-capable computer, (2) the
ability to image, ablate, and/or trap cells and their organelles by
"remote-control," and (3) the security and convenience of
controlling the system in the laboratory on the user's own personal
computer and not on the host machine.
[0085] Time delays between commands received on the host computer
and the completion of actuation were characterized by
programmatically placing timers in the system control software.
Time delays during switching between ablation and imaging were
maximized during measurement by imaging through the binocular port
with filter cube 2 and ablating from the Keller port through filter
cube 5. When switching from imaging to ablation, the system takes
610, 19, and 20 ms (mean, standard deviation, and N) for the
imaging port transition and 688, 6, and 10 ms for the filter cube
transition, with a total duration of 1400, 41, and 10 ms from the
press of the button to the completion of the ablation, with a 3 ms
laser exposure time. A t-test showed no significant increase in
total ablation time when the laser is steered before the ablation
(P>0.05, N=10 for both samples). The imaging port transition
required switching both the base port slider and the side port
turret. When switching from ablation to imaging, the system takes a
total 1387, 103, and 10 ms to transition the imaging port and the
filter wheel followed by 677, 34, and 10 ms or 702, 34, and 10 ms
for the subsequent image with or without operating the arc lamp
shutter, respectively. The images were acquired in snap mode with a
1 ms exposure time to measure the latency of the camera
digitization and readout. Computational latency during continuous
image acquisition was quantified by measuring the total time to
acquire a set of images (1 ms exposure time each) in which the size
of the sets ranged from 23 to 36 individual images. The total times
of ten sets were recorded and averaged within the sets with a
between-set average 133, 3, and 10 ms per image delay time.
[0086] In one example, a connection between San Diego, Calif., USA
and Boca Raton, Fla., USA using a hotel administered T1 connection
operating at a maximum of 10 Mbps was established. This experiment
tested remote control of the microscope stage movement and control
of objective focus (see FIG. 6). High resolution images were
acquired with a 63.times.PH3 phase contrast NA1.4 oil immersion
objective. This experiment implemented the LabVIEW web server for
remote operation. Note the resolution of the double-membrane
nuclear envelope. Images transfer times were 2-3 s per image for a
256.times.256 sub-region of the CCD.
[0087] A second experiment was conducted from Boca Raton, Fla.
(USA), again implementing the LabVIEW web server. In this
experiment, cells with green fluorescent protein labeled
microtubules were observed and manipulated under phase contrast and
epi-fluorescence illumination (FIG. 7). In these cells, the
centrosome microtubule organizing center (MTOC) was irradiated with
the laser scissors. The MTOC region was irradiated at three
different time points, with a progressive loss of fluorescence with
each laser exposure. In this experiment, the following remote
procedures occurred: (1) the microscope stage was moved until an
appropriate cell was located, (2) the microscope was focused at
different z-axis optical planes in the cells to determine the
desired optical plane for laser exposure, (3) laser parameters
(wavelength, power/energy, and number of exposures) were selected,
(4) the laser was targeted to a specific region (the MOTC) in the
cell, and (5) the result was digitally recorded and the cell was
followed for a desired time period. In this particular cell, two
laser exposures were performed until the desired ablation was
observed.
[0088] a third experiment, which was conducted from Brisbane,
Queensland, Australia implemented the Logmein.com web server. In
this experiment beam steering for remote laser targeting and
ablation over a long-distance internet connection was demonstrated
(FIG. 8). Nucleated red blood cells were deposited on a microscope
cover glass by the smear method and mounted in a Rose chamber in
San Diego Calif. Remote control of the system from Australia
successfully demonstrated (1) all of the manipulation capabilities
described in the previous experiment, (2) that the ablation laser
beam could be moved to different locations in the same cell, with a
spatial resolution of less than a micron for both the lesion
diameter as well as the distance between individual lesions, and
(3) that multiple discrete visible lesions could be placed in the
same cell. The time from initial remote command (pressing of the
fire button) in Australia to actual observation of the event on the
host system was determined by measuring delay times between oral
communication of the command over a telephone connection and
actuation of the command on system. Any delay time was
unperceivable. Full-frame transfer rates were measured by counting
the number of screen refreshes per 10 s interval. An 8.2 frames/s
rate measured from the system host computer corresponded to 1.5
frames/s in low quality mode and 0.7 frames/s in high quality
mode.
[0089] A fourth experiment, conducted from Atlanta, Ga.,
implemented the Logmein.com web server. In this experiment, 10
.mu.m diameter fluorospheres (Coulter Corp., Hialeah, Fla.) were
suspended in water in a 35 mm glass-bottom Petri dish in which
thermal flow in the water was induced by heating the objective lens
using the objective heater set at 37.degree. C. The remote user
selected "focus" mode on the control panel to stream images across
the internet. The remote user was able to open and close a
mechanical shutter, allowing the trapping beam to be focused on the
target microsphere through a push-button control in the laser
tweezers tab of the control panel. A time series was recorded after
a microsphere was trapped and then released to demonstrate the
ability of a remote user to manipulate objects with the laser
tweezers (FIG. 9). A 40.times. oil 1.3NA PH3 objective lens was
used.
[0090] Provided herein are various optomechanical systems that can
be operated via the internet using most internet accessible devices
including laptops, desktop computers, etc. These systems afford
investigators the ability to conduct micromanipulation experiments
(cell surgery or trapping) from remote locations. These systems
greatly expand the availability of complex and expensive research
technologies via investigators networking over the internet or
other high speed dedicated communication lines. It serves as a
model for other "internet-friendly" technologies leading to
large-scale networking and data-sharing between investigators,
groups, and institutions on a global scale. In various embodiments,
an optomechanical system provides: (1) the freedom to operate the
system from any internet capable computer, (2) the ability to
image, ablate, and/or trap cells and their organelles by
"remote-control," and (3) the security of operating the system in
house from the researcher's own laptop without the risk of leaving
data on the host computer. Provided herein are examples of: (1)
precise control of microscope movement and live cell visualization,
(2) subcellular microsurgery on the microtubule organizing center
of live cells viewed under phase contrast and fluorescence
microscopy, (3) precise targeting of multiple sites within single
red blood cells, and (4) laser trapping of an individual cell.
[0091] An optomechanical system provided herein is applicable in a
wide array of live cell experiments and commercial applications
relative to medicine. The exemplary ablation laser wavelength of
532 nm lies within the excitation band of many common fluorophores,
including GFP, YFP, and FITC so that in some embodiments ablation
would only require changing the imaging path and not the reflector
position. In this case, image acquisition could begin about one and
one-half seconds after initiation by the user. Researchers studying
changes immediately following the ablation (in the first 4 s), for
example cytoskeletal dynamics, could interface the ablation laser
into the epi-fluorescent light path.
[0092] The examples set forth above are provided to give those of
ordinary skill in the art a complete disclosure and description of
how to make and use the embodiments of the apparatus, systems and
methods of the invention, and are not intended to limit the scope
of what the inventors regard as their invention. Modifications of
the above-described modes for carrying out the invention that are
obvious to persons of skill in the art are intended to be within
the scope of the following claims. All patents and publications
mentioned in the specification are indicative of the levels of
skill of those skilled in the art to which the invention pertains.
All references cited in this disclosure are incorporated by
reference to the same extent as if each reference had been
incorporated by reference in its entirety individually.
[0093] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
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