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WO2006037236A1 - Appareil laser et methode de manipulation de cellules - Google Patents

Appareil laser et methode de manipulation de cellules Download PDF

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Publication number
WO2006037236A1
WO2006037236A1 PCT/CA2005/001556 CA2005001556W WO2006037236A1 WO 2006037236 A1 WO2006037236 A1 WO 2006037236A1 CA 2005001556 W CA2005001556 W CA 2005001556W WO 2006037236 A1 WO2006037236 A1 WO 2006037236A1
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WIPO (PCT)
Prior art keywords
laser
cell
cells
pulses
femtosecond
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PCT/CA2005/001556
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English (en)
Inventor
Abdulhakem Elezzabi
Jason Acker
Vikram Kohli
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Canadian Blood Services
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Publication of WO2006037236A1 publication Critical patent/WO2006037236A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B18/00—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body
    • A61B18/18—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves
    • A61B18/20—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves using laser
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/0613—Apparatus adapted for a specific treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/067—Radiation therapy using light using laser light
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion

Definitions

  • the invention relates to the field of biophotonics. More specifically, the invention relates to the use of laser energy to manipulate cells and biological compositions or systems.- The present invention embodies apparatuses and methods relating thereto.
  • Photonic energy can be exploited to manipulate biological systems by interacting directly with biological structures or indirectly by activating molecules that will in turn affect the biological system. Corrective laser eye surgery is an example of the former and phot ⁇ dynamic therapy of the latter.
  • Optical breakdown is defined as the point where the material changes its conductive property, from non-conducting to conducting.
  • a shock wave is simply a pressure wave that propagates from the exposure point. The effects of a pressure wave induce disruptive forces, and for the case of cellular material, cause local morphological, and physical damage to the plasma membrane.
  • a plasma is defined as the local ionization of electrons from the material, which are no longer bound to the inert ions. A large density of ionized electrons is collectively called a plasma.
  • both the mechanical and thermal stresses induced by the interaction of the pulsed laser with the material should be minimized. It has been shown in recent literature that in ablation-based studies femtosecond pulses produce smoother ablation craters with less thermal and mechanical stresses as compared to picosecond and nanosecond, pulses. Stated another way, picosecond and nanosecond pulses induce large thermal and mechanical stresses that are not contained within the irradiation region (focal volume) . As a result, both the mechanical stresses and the induced temperature rise (due to the applied pulse) propagate outside the focal volume. This propagation is detrimental to cells, as the mechanical stresses not only disrupt the localized region, but also disrupt adjacent cells.
  • Femtosecond pulses have also been used to vaporize micrometer-sized regions of living cells. This ⁇ knockout" approach was also found to cause substantial cellular damage and cell death.
  • An apparatus and method for the manipulation of cellular structures and biological compositions employs laser energy, and preferably femtosecond laser pulses, to manipulate individual cells, cellular structures and/or biological compositions.
  • compositions are intended to include mammalian and non-mammalian cells, tissues and organs, for example.
  • laser energy may be employed to manipulate physiological and/or chemical properties of individual cells, cellular structures and/or biological compositions, both in vivo and in vitro.
  • laser energy may be employed in a variety of fashions, such as for example, in a manner whereby the parameters of pulse train, amplitude, polarization and frequency are pre ⁇ selected in accordance with. the objective of the manipulation.
  • laser energy may be delivered to a substrate of the present invention via single pulse or via multiple pulses.
  • an apparatus for modifying one or more properties of a cellular structure which comprises a pulsed laser source to create one or more femtosecond laser pulses of a predetermined duration and amplitude, means, optically coupled to said pulsed laser source, for focusing the one or more laser pulses with a predetermined amount of energy on the cellular structure, means for positioning the cellular structure relative to the focused laser beam, and wherein the properties are non- thermally modified.
  • non-thermally modified it is meant that the pulse, duration is sufficiently short to prevent thermally induced damage to adjacent cellular structures.
  • the apparatus may comprise means, such as a multi-channel microfluidic device for spatially and temporally controlling an environment surrounding the cellular structure.
  • a method for modifying a cellular structure which comprises providing one or more laser pulses having a predetermined amount of energy such that the energy produces non-thermal photodisruption in the cellular structure and focusing the laser pulses on the cellular structure for a time sufficient to produce the non-thermal photodisruption.
  • the method of the present invention is preferably employed to modify the physical -and/or chemical properties of a cellular structure or biological composition.
  • cellular manipulation may include cell loading with carbohydrate or nucleic acid-based compounds, sub ⁇ cellular loading, selective loading and targeting of embryos and human oocyte cells, single cell cutting, nerve welding, and cellular welding.
  • the method may advantageously be used to treat cells by affecting the formation of transient openings in cellular membranes enabling the insertion of material, such as nucleic acid- or carbohydrate-based molecules, within the cytoplasm or organelles.
  • FIG. 1 illustrates an apparatus and system according to an embodiment of the present invention.
  • FIG. 2 illustrates an alternate embodiment of the apparatus and system of the present invention.
  • Figure 3 illustrates addition embodiments of the apparatus and system of the present invention.
  • Figure 4 illustrates a light delivery system according to an embodiment of the present invention.
  • Figure 5 illustrates an alternate representation of • embodiment of Figure 4.
  • Figure 6 illustrates a waveguide delivery system according to an embodiment of the present invention.
  • Figure 7 is a representation of a cell and its intracellular components.
  • Figure 8 illustrates membrane targeting and transfection using a focused femtosecond laser system, according to an embodiment of the present invention.
  • Figure 9 illustrates a subcellular targeting and transfection representation using a focused femtosecond laser system.
  • Figure 10 provides a representation of membrane .targeting and transfection using a femtosecond fiber laser.
  • Figure 11 provides a representation of the coupling of a focused femtosecond laser system with a multi channel microfluidic device, according to an embodiment of the present invention.
  • Figure 12 provides a representation of the coupling of a focused femtosecond fiber laser with a multi channel microfluidic device, according to an embodiment of the present invention.
  • Figures 13 to 22 illustrate images of a nanoscale surgery protocol of live Madin-Darby Canine Kidney (MDCK) cells according to embodiments of the present invention.
  • Figure 23 illustrates a perforation-based study conducted on live Madin-Darby Canine Kidney (MDCK) cells, according to an embodiment of the present invention.
  • Figure 24 illustrates a pulse laser-mediated transfection of live Madin-Darby Canine Kidney (MDCK) cells, according to an embodiment of the present invention.
  • Figure 25 illustrates a Trypan Blue evaluation of the transfection process of cells according to an embodiment of the present invention.
  • Figure 26 illustrates a transfection process according to another embodiment of the present invention.
  • Figures 27 to 32 illustrates an evaluation of cells for the transfection of sucrose via a pulse laser technique according to an embodiment of the present invention.
  • Figures 33 to 35 illustrates images of live nanosurgery on live fibroblast cells according to embodiments of the present invention.
  • Figure 36 illustrates a pulse laser system according to another embodiment of the present invention.
  • Figure 37a-d illustrates images of nanosurgery performed on viable V79-4 fibroblast cells according to an embodiment of the present invention.
  • Figure 38a-d illustrate images of membrane surgery on a live MDCK cell according to an embodiment of the present invention.
  • Figure 39 illustrates a developing zebrafish (Brachydanio rerio) embryo at the Gastrula stage as exemplified for the purposes of an embodiment of the present invention.
  • Figure 40 illustrates an application of a focused femtosecond laser pulse apparatus and technique of embodiments of the present invention, in precision embryonic manipulation of zebrafish embryos.
  • Figures 41 to 46 illustrates further applications of embodiments of the present invention to the manipulation of zebrafish embryos.
  • biopreservation for example, is to protect the integrity and functionality of living cells, tissues, and organs, which has resulted in the development of techniques that can achieve biological stability and ensure a viable state following ex vivo storage.
  • the present invention provides a novel non-invasive tool for cell manipulation and surgery, including cell preservation as mentioned above.
  • a laser pulse technique and • apparatus for precise and relatively non-invasive cellular manipulation are provided.
  • a preferred embodiment of the present invention provides a high-intensity femtosecond laser pulse technique for cellular manipulation.
  • Cellular manipulation of the present invention may include any biological alteration of a sample, including but not limited to physical and chemical alterations.
  • the techniques of the present invention employ ultrashort or femtosecond laser pulses.
  • An apparatus and method of the present invention can be 'employed to manipulate cellular structures and biological compositions without inducing thermal pressure or shock to the biological sample, to which the present invention applied.
  • This • is in contrast to the thermal process of laser capture microdissection known in the art, where heat is required for thermoplastic activation and the selective procurement of targeted cells.
  • the absorption of high intensity ultrafast laser pulses by non-linear multiphoton absorption and ionization leads to multiphoton electronic excitation, whereby energy is transported to the liberated electrons without thermal diffusion to adjacent cellular material. Since femtosecond pulses are shorter than the thermal diffusion time (picoseconds to nanoseconds) , heat transport is minimized, and the biological sample remains unaffected by subsequent heat shock damage.
  • the present invention has wide application for a variety of cellular manipulations.
  • the preservation of germ cells will benefit from the pulse laser technique of the present invention, as oocyte and sperm preservation offers an attractive approach to alternative infertility treatments.
  • Current germ cell cryopreservation produces variable results, and is not widely used clinically.
  • the present invention can be employed to cryopreserve oocytes, sperm, embryos and other reproductive material.
  • reproductive materials such as sperm, oocytes and embryos such that genetic materials can be preserved well after the extinction of the animal, for example.
  • femtosecond laser pulses (10 ⁇ 15 seconds) for performing membrane surgery and nanosurgical cell isolation on live mammalian cells.
  • sub-10 to 500 femtosecond laser pulses were focused to an intensity of 10 10 - 10 13 W/cm 2 /pulse onto cells, sub-micron to micron dissection cuts were made on the biological membrane without morphologically compromising the cell.
  • femtosecond laser pulses can be accurately tailored for the creation of precise sub-micron optical pores without inducing thermal pressure or shock to the biological sample.
  • the pulses are so brief in duration that the biological lattice is unaffected, rendering the process non-thermal.
  • the pulse duration of the laser is less than 20 femtoseconds.
  • a laser of the present invention provides less cell damage in part due to the reduced pulse duration therein employed. Absorption of high intensity femtosecond pulses lead to multiphoton electronic excitation, whereby heat is transported to the liberated electrons without thermal diffusion to adjacent cellular material.
  • the techniques of the present invention have been shown to provide a tool for manipulating cells and embryos with improved precision and reduced invasiveness, whereby embryos are shown to survive • and prosper after treatment therewith.
  • the techniques and methods of the present invention provide a novel tool for application in micron and nanoscale surgeries and manipulations of cellular and biological systems.
  • a focused femtosecond laser pulse technique can create transient pores in an embryo that eventually seal, thereby providing a mechanism for manipulating and treating embryos, and other biological systems, as would be desired.
  • One such example of utilizing this tool is herein disclosed is for use cryopreserving embryos.
  • an embodiment of the present invention is described below for manipulating cellular material.
  • the pulse laser technique of the present invention is able to create tiny perforations in the biological plasma membrane without inducing irreparable damage.
  • cellular and sub-cellular manipulation is achieved, where individual cellular compartments can be targeted for a desired application.
  • the information presented below provides a brief summary of the components involved in pulse laser technique of an embodiment of the present invention.
  • a first design of a laser apparatus and method of an embodiment of the present invention involves a modified optical microscope used in conjunction with a femtosecond laser.
  • Dichroic mirrors are used to direct the ' femtosecond pulse train to the optical microscope, where the pulses are focused to a micron to sub-micron spot.
  • the femtosecond pulses are focused using two different microscope objectives.
  • the first type of microscope objective is an air, water, and oil immersion. It should be. noted that air, water, and oil-based microscope objectives contain a series of glass elements that focus the incident femtosecond beam.
  • a femtosecond laser with a pulse duration of 100 femtosecond can be broadened up to 200 femtosecond, or more, depending on the number of glass elements, employed in the objective.
  • a reflective objective is employed.
  • a reflective objective preferably contains no glass elements, and hence ⁇ the pulse is not broadened.
  • a pulse laser apparatus and system of the present invention can operate at 500 - sub-10 femtoseconds. According to a preferred embodiment of the present invention provides a pulse laser apparatus and method that provides a laser pulse ' duration of less than 20 femtoseconds.
  • stage housing the sample
  • stage can be accurately controlled using a computerized stage.
  • the stage is interfaced with software that controls the stage movement in the x-y-z direction.
  • the position of the sample can be adjusted with millimeter to nanometer precision. Interfacing a pulse selector and galvanometer, for selecting appropriate beam dwell times, with the stage, we can ablate the sample very easily.
  • micro- and nano-incisions are made in the ⁇ purely horizontal and/or vertical direction. More importantly, we are not limited by simple vertical or horizontal incisions. We can make diagonal incisions, circular incisions, and various ' odd shaped incisions.
  • our software can load an image file of the cell, and by directly drawing the incision that is required on the image; the software will control the stage in a manner such that the correct desired incision is made.
  • the stage's temperature can be dynamically controlled. This is advantageous since cells should be maintained at a specific temperature for biological activity. If the temperature is too low, the cell can freeze and cause irreversible injury and die. However, if the temperature is to high, the cell will dehydrate and die. Therefore, it is useful to anticipate temperature fluctuations, and the ability to control it dynamically is desirable.
  • UV fluorescence imaging and epi- fluorescence imaging are used when dye diffusion characterization is needed.
  • a unique feature of our imaging system is that we can focus both the cell and the focused laser spot simultaneously. This allows us to observe the induced changes as they occur. Live video can be captured, as well as still images. This is done by overlapping the images on a charge coupled device. The " charge coupled device then projects the image on a viewing screen, and also captures live video and still photography. We also have the ability to gate our video capture, such that we can evaluate subsequent changes that occur on a nanosecond or shorter time scale.
  • the focus of the two image sources is preferably decoupled.
  • our setup .we back illuminate the sample using white light, and deliver the focused femtosecond beam in the forward direction.
  • the femtosecond pulse image is focused using the z translation of the stage, and the white light image is focused using the mounted imaging system or condenser. Consequently, the imaging system consists of a series of lenses and filters.
  • the lenses magnify the white ' light image, and manual adjustments in lens separation achieve the desired focusing condition.
  • the filters function to attenuate the femtosecond pulse image, where both images are collected by a charged coupled device.
  • the focused images provide a controlled means for live evaluation of cell-laser interaction.
  • the femtosecond pulse image we mean the image produced by the focused femtosecond pulse.
  • the femtosecond pulse is generated by any of the laser systems mentioned in this application. Therefore, the image produced is the focused femtosecond pulse by any of these laser systems.
  • THG microscopy is a nonlinear process that is based on the absorption of incident light photons. The absorption causes the electrons to be excited thereby generating an electric field. If the intensity is measured by a detector, the acquired information provides accurate informational content of the sample being investigated.
  • the image produced is a 3D image that provides complete knowledge of the sample and its constituents. As stated, this technique is based on absorption, and any information contained in the scattered light is lost. However, combining THG with OCT provides greater image resolution.
  • OCT is based on the interference of the samples reflected light with a reference beam.
  • THG and OCT imaging are described D. Yelin and Y. Silberberg, Optics Express, vol. 5(8), (1999); Jeff A. Squier et al.. Optics Express, vol. 3(9), (1998); Dan Oron et al. Journal of Structural Biology (article in press); James G Fujimoto. Nature Biotechnology, vol. 21(11), (2003 November) the teachings of which are herein incorporated by reference.
  • illumination sources can be employ in connection with embodiments of the present invention.
  • control of the temperature of the cell can be advantageous.
  • conventional light sources i.e. standard white light sources coupled to optical microscopes, generate a large source of heat.
  • standard light sources generate 95% heat and 5% light. This heat, over a period of time, will dehydrate the sample and thereby induce cell death. Therefore, in conjunction with our dynamic temperature controlled stage, we preferably employ light sources that generate no heat and have superb light confinement.
  • Such sources are preferably fiber based light sources, and/or a photonic bandgap fiber light sources.
  • OCT and THG are also applied to intracellular components.
  • the ability to make odd shaped incisions is often desirable. Briefly, cells come in variety of shapes and sizes, and the ability to .selectively cut odd shaped organelles is desirable. Accordingly, the present invention has application in nerve and cellular welding. To perform such applications it is preferable to have the capability to maneuver a focused beam in an irregular manner. The ability to have control over any shape provides the laser technique of the present invention with dynamic means for accomplishing the aforementioned application.
  • FIG. 4 and 5 illustrate embodiments of the present invention where a multi-channel microfluidic device is coupled to a femtosecond fiber laser.
  • the microfluidic device can be multi-channeled.
  • a multi channel device allows, for example, for the analysis of various cell lineages, and the effects of different genetic material transfected into the cell.
  • the femtosecond fiber laser provides the mechanism for disruption, and due to the compact nature of the fluidic device and fiber laser, the integration yields a compact device that is easily transportable.
  • the femtosecond fiber laser is based on the delivery of short optical pulses through a fiber optic cable or waveguide. Placing the femtosecond fiber laser in close proximity to the irradiation zone of the fluidic channel accurately induces the desired effects. The effect in this case being the creation of a local perforation. As illustrated in Figures 4 and 5, multiple channels lead to the irradiation zone. The channels can be manually selected for study of different cell lineages. In addition, genetic material can be injected into the multiple ⁇ channels, where different material can be placed in each channel.
  • Being able to dynamically control the repetition rate adjusts the number of incident laser pulses irradiating the sample, and control over the emission wavelength alters the maximum absorption cross section. Since the incident absorbed energy is correlated to the repetition rate, controlling the repetition rate controls the amount of energy absorbed by the cells. Direct control provides precise manipulation of the cell. It should also be noted that the number of incident pulses also affects the survival rate of the cell.
  • the laser can be polarized.
  • the polarization state of 1 the laser beam used in creating the desired effects on targeted cells can be a linearly polarized Gaussian beam.
  • the polarized Gaussian beams can be selected from, but are not limited to, azimuthal, radial, circular, and elliptically polarized beams.
  • the focusing spot produced by a • circular and radial beam is 15% and 35% smaller than a linearly polarized beam. This is important since the perforation size is dependent on the focused spot size. More importantly, the size of the perforation plays a crucial role in cell survival, as large perforations induce increasing cell death rates.
  • An embodiment of our invention also include a laser trapping system.
  • ,0ur laser trapping system encompasses a laser beam that traps the cellular species within the focal volume. Trapping is achieved without inducing irreversible damage. With our laser trapping technique we can move cells to any desired location .by traversing the beam. Trapping the cell within the focal volume improves post-analysis efficiencies, as very often cells migrate after exposure.
  • a near field optics approach employs means to produced a near field focused beam, such as, for example, a femtosecond fiber laser with a fiber optic tip, and a apertured near field optical cantilever.
  • the femtosecond pulses are coupled to the fiber tip and cantilever, and placed directly above the species of interest.
  • a near field optical technique using a fiber tip is provided.
  • a femtosecond fiber laser coupled to a fiber optic tip for performing photodisruption experiments.
  • tiny perforations in the biological membrane are achieved. This is accomplished by placing the fiber tip directly over the desired area.
  • imaging of the specific photodisruption 1 site is also achieved.
  • accurate positioning of the tip is obtained for direct targeting and manipulation. The benefits of using this technique is that the perforation size can be accurately controlled based on the size of the fiber tip.
  • a microscope objective In standard non-near field optics, a microscope objective has limited resolution. The limitation arises from the physics of diffraction theory where the resolution is limited to half the wavelength. For instance, if 800 nm light is used, ' , theoretically a resolution of .400 nm is achieved. However, actual resolution values are greater than half the wavelength due to aberration effects in focusing optics. However, with our setup, design, and procedure we are capable of obtaining a resolution of lOOnm or less. In one embodiment, the resolution is based on how small the fiber tip can be fabricated. With a resolution of 100 nm or less, high-resolution images of specific cellular sites can be achieved. More specifically, imaging subcelluar species that are nanometers in dimension can be accomplished. A benefit of our design is that the near field approach produces smaller perforations, and thus less damage to the cellular species. In fact, single proteins can be targeted on or inside subcellular species due to the small spot size created.
  • Another near field optical design approach is based on an apertured near field optical cantilever.
  • a femtosecond pulse is coupled to an optical cantilever for near field imaging and irradiation. Similar to the fiber tip approach, a 100 nm or less resolution is achievable with a 100 nm or less perforation size.
  • This novel technique we can manipulate single proteins in a controlled and precise fashion. Using our approach smaller perforation sizes are created, subcellular structures of a 100 nm or less can be targeted, and high-resolution images are obtained. Perforation size and image resolution can be dynamically adjusted by fabricating smaller apertures.
  • our design is versatile, and anticipates functional changes .
  • the Image capture device 1 can be a charged coupled device (CCD) , a mounted single-lens-reflex (SLR) camera, and/or any other camera device for processing of real time imaging,
  • Optical filters/attenuators 2 may consist of a variety of energy selectors/controllers, which are interchangeable, for choosing the appropriate wavelength image. These filters also function as attenuators, i.e. energy controllers, for selective enhancement of particular wavelengths.
  • Optical beamsplitter/spatial beamsplitter 3 comprise spatial beamsplitter filters for selective filtering of UV fluorescence, epifluorescence, and laser illumination imaging. The choice of reflection or transmission wavelength bandwidth is controlled by the appropriate spatial beamsplitter filter, coated for the desired wavelength. It should be noted that the filters are interchangeable, allowing for dynamic adjustments.
  • Focusing lens imaging system 4 is an imaging system containing aberration corrected lenses for simultaneous focusing of UV fluorescence, epi-fluorescence, standard white light, fibre white light, photonic bandgap fibre light, and/or laser light. Proper focusing is achieved by adjusting the lens separation, and viewing the image on the capture monitor. It should be noted that the entire imaging system may comprise elements 1, 2, 3, 4, 9, 14, and 15.
  • Illumination source and/or imaging source 5 provides an inlet source for epi-fluorescence, UV light, standard white light, fibre based light source, photonic band gap fibre source, and/or any other imaging source of interest.
  • Laser systems 6 include: solid-state diode pumped ultrafast laser, amplified femtosecond laser source, tunable femtosecond laser source, optical parametric amplifier, optical parametric oscillator, and solid-state diode pumped ultrasfast cavity dumped laser. It should be noted that the pulse controller 7 is synchronized with the pulse train for optimal pulse delivery via computer interface 15.
  • Pulse controller 7 can be an optical element, an electro-optic and/or an acousto-optic modulator, used to control the number of pulses irradiating the sample.
  • the laser system 6 and pulse controller are synchronized for optimal pulse delivery through a computer interface 15.
  • Microscope objective 8 is used for focusing the femtosecond pulses.
  • the microscope objective 8 is an air, water, or oil immersion type.
  • the glass objective can be replaced with a reflective objective, producing smaller focal volumes, with uncompromised pulse lengths.
  • a near-field optical probe, apertured hole, apertured near field optical cantilever, a hybrid atomic force microscopy near field optical cantilever, tapered and untapered fiber optic tip or waveguide can replace the microscope and reflective objective, thereby allowing for sub-wavelength imaging and manipulation.
  • Optical beamsplitter/spatial beamsplitter 9 is a spatial beamsplitter filter for selective filtering of UV fluorescence, ep-fluorescence, and laser illumination imaging. The choice of reflection or transmission wavelength bandwidth is easily controlled by the appropriate spatial filter.
  • Specialty designed grided/non-grided microscope slides 10 for proper sample holding, identification, targeting, and segregation. The slides are made of glass and/or non-glass based materials. The biological .
  • sample under investigation is shown at 11. It consists of any living or dead cells, tissue, organelles from mammalian or non-mammalian specimens which mammalian specimens can be of human or non-human origin, including subcellular organelles, mitochondria, nucleus, golgi apparatus, ribosomes, ' and lysosomes.
  • the biological sample is immersed in water, buffered salt solutions, culture media, nucleic acid based compounds, and/or any genetic material of interest.
  • a controlled x-y- z, or r, ⁇ , ⁇ , and/or tilt stage device for moving the biological sample is shown at 12. It should also be noted that in another configuration, the stage is computer- controlled for precise sample movement with millimeter to nanometer precision.
  • a dynamic temperature-controlling device 13 for temperature tuning may consist of either water, peltier, - 2 A - any fluid/solid/gas based system, and/or any other means of stabilizing and maintaining accurate temperature control.
  • a display device is shown at 14 for viewing in real time the different processes.
  • a computer/control device 15 interfaces with the laser system 6, pulse controller 7, and computer-controlled stage 12 & 20. Proper interfacing provides dynamic control in pulse delivery and sample movement.
  • Polarization converter/selector 16 is an optical element used for converting and selecting numerous polarization states.
  • Pulse compressor and shaper 17 is an optical element used for altering the shape and amplitude of the femtosecond pulses. The element amplifies and compresses the pulse, generating higher pulse energies and shorter pulse durations. The implementation of this element depends on the particular application being pursued.
  • Energy selectors/controllers 18 are optical devices used for selecting the desired pulse energy, thereby allowing dynamic tailoring of energy delivery for each of the pursued applications. This is important since each application requires varying pulse energies.
  • Emitted laser pulse 19 can be emitted with specific frequency, amplitude, wavelength, pulse energy, pulse duration, and multiple or single pulse delivery.
  • Stage controller 20 is used for dynamic adjustments in the x-y-z stage, or r, ⁇ , ⁇ , and/or tilt stage. The controller interfaces with the stage, and the computer generates the movement inputs. Therefore, changes to the control software, implementing stage movements, translates into direct movements in the x-y-z stage.
  • a Laser trapping system & Microscopy is shown at 21. This laser system can be used specifically for trapping the biological specimen contained within the focused volume.
  • the femtosecond pulses generated through sequence 6, 7, 16-18, and 22, are collinearly coupled with the femtosecond pulses of 21. While element 21 provides the trapping mechanism, the sequence provides the photodisruption process.
  • This system is also used for Second and Third Harmonic Generation Microscopy (SHG & THG) , for deep tissue imaging of the biological specimen under investigation.
  • standard Confocal Microscopy is also applied, where the laser system 6 provides the excitation source, while 12, 15 and 20 provide sample movement. The scanned image is captured by element 1.
  • the excitation sources operate in a non-photodisruption manner, namely, delivering pulse energies less than a nanojoule (10 ⁇ 9 J) .
  • a typical pulse energy used in microscopy studies is a picojoule (ICT 12 J) with femtoseconds in duration.
  • X-Y Galvanometer 22 is used as a, scanning mirror for selective laser beam irradiation.
  • the combination of element 7 with 22 provides a dynamic irradiation rate that is otherwise limited through the use of either element individually.
  • the pulse gating time, with this configuration, is based on the product of the ⁇ on-off time of elements 7 and 22.
  • An alternative path for light source emission for imaging is shown in Figure 2 at 23.
  • This method of imaging provides an alternative approach to that shown at 5, in Figure 1,- According to this embodiment, epi-fluorescence, UV light, standard white light, fibre based white light, and/or photonic band gap fibre light is coupled to the microscope base entrance aperture and focusing objective (element 25) .
  • This approach is desirable if laser trapping, photodisruption, white light imaging, and epi or UV imaging are required simultaneously. Consequently, this is accomplished by using both elements 5 and 23 simultaneously.
  • the microscope is supported by microscope base 24.
  • the Focusing light objective for imaging 25 is used for focusing epi-fluorescence, UV light, standard white light, fibre based white light, and/or a photonic band gap fibre light source, for backward illumination of the biological sample.
  • a scanning reflective mirror 26 for back reflecting the transmitted excitation source is shown.
  • the transmitted intensity and the reflective intensity .28, interfere at the spatial beamsplitter, element 3, producing the resulting optical coherence tomography image (OCT) 29.
  • OCT optical coherence tomography image
  • This image is captured by element 1.
  • the excitation source 27 can be any of the sources listed for element 6. Short pulsed lasers are desirable since the wavelength bandwidth is large. Large bandwidth means shorter coherence length, and thus higher spatial resolution.
  • Light back reflected from the biological sample is shown at 28.
  • An OCT image 29 results from the interference of light from element 26 and 28.
  • a detection unit 30 detects the OCT image captured by element 1.
  • a Signal processing unit for processing, altering, shaping, and amplifying the detected OCT signal is shown at 31.
  • a light delivery system 32 such as a fibre optic tip, can be used «, to deliver the femtosecond pulses for photodisruption studies as illustrated in Figures 4 and 5.
  • the fibre optic tip plus the laser system can be replaced with a diode pumped femtosecond fibre laser.
  • the fibre optic tip is replaced with a tapered waveguide, apertured hole, or a near field optical cantilever.
  • Microfluidic device 33 is a device with numerous fluidic channels for investigating different cell types simultaneously. Each channel may contain different protein, carbohydrate or nucleic acid material for intracellular delivery. Zone 34 indicates the irradiation zone where photodisruption occurs.
  • Figure 6 illustrates an example of a Tapered waveguide delivery for delivering femtosecond pulses, for photodisruption studies.
  • femtosecond pulses are coupled to the tapered waveguide.
  • Fiber 35 may also serve as a means for delivering material to the cell thereby affecting spatial resolution of the delivery.
  • the delivered material could be for example a solution comprising DNA molecules.
  • a Near field optical cantilever 36 can be used for near field studies. As the aperture size decreases the image resolution increases. Using the near field approach, an image resolution of sub-wavelength is achieved.
  • An alternative approach involves using a hybrid atomic force microscope near field optical cantilever. This device can be used both for imaging and topography mapping. In the atomic force microscope, the device provides detail information of the sample structure based on deflection measurements. Aperture holes 37 are used to focus the femtosecond pulses.
  • Figures 7 'to 12 are provided for the purpose of further illustration of embodiments of the present invention.
  • Figure 7, depicts a cell and its subcellular components.
  • Figure 8 illustrates a process of membrane targeting and transfection by a focused femtosecond laser system, according to an embodiment of the invention whereby any of the laser systems of the present invention may be used.
  • Figure 9 depicts subcellular targeting and transfection using any of the femtosecond laser systems of the invention.
  • Figure 10 illustrates transfection delivery by a femtosecond fiber laser according to an embodiment of the invention.
  • Figure 11 illustrates a femtosecond laser system coupled to a microfluidic device.
  • Figure 12 depicts the coupling of a femtosecond fiber laser to a microfluidic device according to an embodiment of the invention.
  • a microfluid device as exemplied in Figures 11 and 12 may be coupled with a pulse laser apparatus of the present invention and employed for use in the manipulation of cellular material, and , more specifically, for use in drug delivery, gene therapy or targeted transfection to a biological composition.
  • the parameters of pulse train, amplitude, polarization, and frequency of the laser apparatus and system of the present invention may vary.
  • Single pulses are delivered as well as multiple pulses, according to embodiments of the present invention. These pulses are preferably delivered individually, as illustrated in the Figures 8-12, as (a) , (b) , (c) , and (d) .
  • Embodiments of the present invention are herein further exemplified by way of the following examples.
  • Nanosurgery was viewed with a CCD mounted on the modified optical microscope, and images were captured using video software. Nanosurgery is accomplished using a 60X 0.7 high numerical aperture (NA) microscope objective lens in conjunction with a sublO femtosecond laser system operating at 800nm. A Pulse energy of a 0.5 joules to few tens of microjoules, focused to an intensity of 10 10 - 10 13 W/cm 2 is used.
  • NA numerical aperture
  • Figure 14 exemplifies precise surgery using nanojoule pulses focused to 10 13 W/cm 2 .
  • the femtosecond pulses are focused by a microscope objective lens to a micron-sub-micron spot size .
  • Figure 13 refers to a spot size, as defined by the width of the cut, of about 800 nm.
  • the focused femtosecond beam is scanned across the live cells, thereby performing nanoscaled surgery.
  • the pulse laser technique is highly localized, and the nanosurgical procedure is contained entirely within the focal volume of the focused femtosecond beam. Adjacent material is undisturbed, and no cell collapse or cell morphology is seen.
  • the present invention provides a selective tool that allows for the manipulation of live cells through non-invasive means.
  • Figures 15-19 illustrate progressive sequential imaging of a pulse laser technique of the present invention. As shown, surgical line cuts were performed using the pulse laser apparatus of the present invention. Figures 15 to 19 are an extension of Figure 13 above.
  • a IOOX 0.95 high numerical aperture (NA) microscope objective lens is used for performing live nanosurgery of Madin-Darby Canine Kidney (MDCK) cells.
  • MDCK Madin-Darby Canine Kidney
  • the advantage of using a IOOX 0.95 NA microscope objective, over 6OX 0.7 NA, is that the 0.95 NA 'produces a, focal spot that is smaller than a 0.7 NA. Theoretically, a focal spot as small as 0.5 ⁇ m is achievable with a IOOX 0.95 NA microscope objective lens.
  • femtosecond pulses are coupled to a high NA microscope objective lens, and focused by the IOOX 0.95 NA microscope objective.
  • the focused beam is scanned across the cell numerous times, both vertically and horizontally, illustrating the non ⁇ invasive procedure to nanosurgery.
  • Nanosurgery is performed using a wavelength of 800 ran, with a pulse energy of 0.5 joules to few tens of microjoules, focused to an intensity of 10 10 - 10 13 W/cm 2 .
  • numerous nanoscale cuts are made without inducing ' cell disassociation and cell morphology. If cell disassociation and cell morphology were seen, then this would indicate cell death.
  • Figure 20 and 21 the cell's shape remains intact, and no changes in cell morphology are seen.
  • Figure 21 depicts dimensional analysis, and for a 12 ⁇ m cell a cut size of 800 ran is achieved.
  • Figure 22 depicts a nanosurgery technique of the present invention wherein a cut size of 0.625 urn is achieved on a 4.75 urn cell. Our novel technique produces highly localized, well-defined, and controlled nanosurgery without disturbing adjacent material.
  • the lack of cell disassociation is due to an additional process that occurs when nanosurgery is performed.
  • This process is called cellular welding, namely the welding of the upper and lower plasma membrane.
  • the region that is irradiated fuses the upper and lower membrane.
  • the fusing causes the upper and lower membrane to attach, thereby circumventing cell collapse and cell disassociation. From the images, one would expect after numerous cuts that the cell would collapse. However, the welding procedure maintains the structure of the cell without inducing irreparable cell damage.
  • Figure 23 refers to a perforation-based study conducted on live Madin-Darby Canine Kidney (MDCK) cells. Perforations in the biological plasma membrane are made using a 6OX 0.7 high numerical aperture (NA) microscope objective lens. Femtosecond pulses, with pulse energy of 0.5 joules to few tens of microjoules centered at 800 nm are focused using a 0.7NA microscope objective lens to an intensity of 10 10 - 10 13 W/cm 2 . As seen in Figure 23, perforations are created when femtosecond pulses are focused onto live mammalian cells. Figure 23 illustrates a 0.75 urn perforation (hole) created with the pulse laser apparatus of the present invention in a 7 um cell. It is important to note that an additional perforation is created in close proximity to the previous. This illustrates the localization feature of our technique. Namely, the ability to create multiple perforations without inducing changes to adjacent perforations.
  • a 6OX 0.7 high numerical aperture (NA) microscope objective lens
  • a IOOX 0.95 high numerical aperture (NA) objective lens is used in conjunction with a sub-10 femtosecond laser system operating at 800 nm to evaluate laser-mediated transfection of live Madin-Darby Canine Kidney (MDCK) cells ( Figure 24) .
  • the femtosecond pulses are focused using a 0.95 NA microscope objective to a focusing intensity of 10 10 - 10 13 W/cm 2 .
  • the focused pulses are gated using a mechanical shutter, where pulse selection is varied based on the gating time of the shutter.
  • the cells were surrounded by two biological dyes, Syto 13 a green fluorescent nucleic acid stain,- and ethidium bromide (EB) a red fluorescent nucleic acid stain.
  • EB ethidium bromide
  • Syto 13 is a permeable dye that readily diffuses across the lipid membrane, whereas ethidium bromide is impermeable to the lipid membrane. Therefore, to verify transfection a local perforation is created and the diffusion of ethidium bromide is evaluated. Note that a perforation is usually required in the biological membrane for ethidium bromide diffusion.
  • Figure .24 illustrates verification of the transfection process.
  • all cells that have been perforated have been done with 100% efficiency.
  • the benefits of our study are evident.
  • the drug or gene needs only to be placed outside the cell. By creating a local perforation in the biological membrane, the drug, gene or biopreserving-based compound will readily diffuse in.
  • a IOOX 0.95 high numerical aperture (NA) microscope objective lens is used in conjunction with a sub-10 femtosecond laser system operating at 800 nm.
  • the femtosecond pulses are focused using the 0.95 NA, and selective pulse irradiation is controlled using a mechanical shutter.
  • Live Madin-Darby Canine Kidney (MDCK) cells were surrounded by Syto 13 and ethidium bromide (EB) in the first study. In the second study the live cells were surrounded by trypan blue (TB) . Two studies are presented to illustrate the repeatability of our technigue.
  • trypan blue is ' a biological dye that is impermeable to the lipid membrane. Trypan blue is not a fluorescent-based dye, so evaluation of transfection is done under standard white light illumination with a color charge coupled device (CCD) .
  • CCD color charge coupled device
  • cells are surrounded by Syto 13 and ethidium bromide.
  • a local perforation in the biological membrane was created, resulting in the diffusion of ethidium bromide.
  • ethidium bromide is a fluorescent- based nucleic acid stain, evaluation requires fluorescence imaging.
  • the two cells that were exposed were readily transfected with ethidium bromide.
  • unexposed cells remained green. This indicated that no mutual effects occur between exposed cells and unexposed cells when using our novel approach. Clearly, a selective approach to transfection was thereby demonstrated.
  • Trypan Blue is used to evaluate the transfection process. As depicted in Figure 25, four cells are transfected with Trypan Blue: Figure 26 illustrates the transfection process where clearly all four cells have been transfected. This is indicated by the cells being blue. It is noted that one of the cells migrated post-portion.
  • the study presented in this section is tailored to the biopreservation of live mammalian cells .
  • Madin-Darby Canine Kidney (MDCK) cells are investigated, and the transfection of sugar is reported.
  • the sugar used in this experiment is sucrose.
  • Sucrose is impermeable to the lipid membrane, and therefore, for transfection, a local perforation in the membrane is required.
  • a IOOX 0.95 high numerical aperture (NA) microscope objective lens is used to create a local perforation in the biological membrane.
  • Femotosecond pulses with a pulse energy of 0.5 joules to few tens of microjoules are focused using a 0.95 NA microscope objective.
  • the pulses are dynamically selected using a mechanical shutter.
  • the live cells were surrounded by 1.0 molar concentration of sucrose,, and upon creating the perforation, sucrose diffused into the cell.
  • a cavitation bubble is seen with a bright spot centered about a dark background. This cavitation is caused by the instantaneous onset of a plasma resulting in a sub-micro explosion.
  • the plasma is contained within the focal volume of the focused spot, and localized changes occur on the biological membrane.
  • the size of the cavitation is controlled by adjusting the pulse energy of the focused femtosecond pulse train, and the number of pulses irradiating the live cells.
  • Figure 30 illustrates our non-invasive, highly localized approach to transfection.
  • three cells are joined together and only one cell has been targeted for carbohydrate loading. Notice how the adjacent cells remain attached to the exposed cell without cell disassociation or cell collapse. This illustrates the localized nature of our transfection process, and the ability to select the cell of interest. Therefore, we show here a controlled process for precise targeting producing 100% transfection.
  • the benefits of transfecting carbohydrates into live mammalian cells have overwhelming consequences for the biopreservation, aquaculture and reproduction industry.
  • One of the objectives of transfection of carbohydrates is to provide the aquaculture and infertility industries with novel methods and tools for biopreserving embryos and human oocyte cells. Accordingly the laser technique of the present invention has wide application in the field of life sciences.
  • cryopreservation with intracytoplasmic sperm injection has lead to an overall success rate of 1%
  • ICSI human oocytes cryopreserved using a slow cooling protocol yielded an efficiency of 5.6% (A. Eroglu et al. Fertility and Sterility, vol. 77(1), pp. 152-158m, (January 2002)) .
  • Carbohydrates that can be used for biopreservation applications are known in the art.
  • sugar has been shown to act as a glassy matrix that protects the cell from forming ice crystals when cryopreserved. They can form hydrogen bonds to polar groups, and thus replace the binding site of intracellular water.
  • sugars can form glassy states, which can change the properties of the cytoplasm. This is important since intracellular water forms ice crystals which eventually compromise the biological membrane thereby leading to complete loss in cell viability.
  • cell viability is dependent on extra cellular sucrose concentration, as well as laser input power (or alternatively pulse energy) . More specifically, if the extracellular sucrose concentration is to high, sucrose diffusion will induce stresses on the biological membrane. Such stresses cause the cell to rupture, resulting in cell death. Alternatively, if the input power is to high, and the pulses are not gated in a precise manner, the cell will absorb more energy than is required for creating the perforation. This excess energy prevents the lipid membrane from sealing, and thereby compromises the plasmalemma leading to cell death. Therefore, properly controlling both the input power and sucrose concentration is of vital importance.
  • the transfection process is identical to the previous section; a IOOX 0.95 high numerical (NA) microscope objective lens is used in creating a perforation. Femtosecond pulses from a sub-10 femtosecond laser system are focused using a 0.95 NA microscope objective lens. The pulses are gated using a mechanical shutter, thereby controlling the amount of energy absorbed by the cell. An input power of 275 mW was used for 0.5 and 0.2 molar sucrose samples, and 270 mW for the 0.4 and 0.3 molar sucrose samples.
  • NA numerical
  • 0.5 molar sucrose was investigated using an input power of 275 mW. A group of fifteen cells were chosen, and eight of the fifteen cells were transfected. All cells that were transfected lost complete cell viability. However, all unexposed cells were completely intact with 100% cell survival.
  • 0.2 molar concentration was investigated. A similar input power and shutter time was used. In data set one, eight cells were investigated and six cells were transfected. On the other hand in data set two, thirteen cells were investigated and six were transfected. The first data set resulted in all cells but one remaining intact and having membranes that were uncompromised. In the second set, out of six cells that were transfected all of the cells remained intact, and only cell five detached from the substrate.
  • cells exposed to an input power of 270-275 mW, surrounded by 0.5 molar sucrose lead to complete loss in cell viability and eventual cell death.
  • 0.4 molar sucrose concentration approximately 30-40% of the cells were viable and alive, for 0.3 molar concentration approximately 80% of the cells were viable and alive, and for 0.2 molar approximately 92-100% of the cells were viable and alive.
  • the present invention provides a novel, non-invasive tool for cell preservation.
  • Applications of our novel process for the biopreservation industry will be of great advantage to the field of human embryo and oocyte preservation, and provide alternative approaches to infertility treatments.
  • Nanosurgery is accomplished by using a IOOX 0.95 high numerical aperture (NA) microscope objective lens, in conjunction with a sub-10 femtosecond laser system.
  • the femtosecond pulses are focused using a 0.95 NA microscope (air) objective lens .
  • Figure 33 two fibroblast cells are shown tethered together, where the width of the tethered region is ⁇ 1 ⁇ m. It should be noted that the cells are spread out as epithelial sheets and not rounded up in spherical form.
  • Figure 33 depicts the location of the focused femtosecond laser spot. With an energy per pulse of approximately 4 nJ, an intensity of 10 13 W/cm 2 is generated at the focal volume. With this intensity, ionization of the media occurs through a nonlinear process as depicted. Accompanying the ionization is a ⁇ cavitation bubble, see Figures 33 and 34, which eventually subside.
  • Figures 33 - 35 show that the adjacent fibroblast cell is unaffected by the focused femtosecond beam. No morphological changes are observed in the adjacent cell, and cell disassociation as well as cell collapse are not observed on either fibroblast cell.
  • a non-invasive nanosurgery procedure that allows live cells to be manipulated and controlled in a precise fashion with inducing irreparable cell damage.
  • V79-4 American Type Culture Collection (ATCC) CCL-93 deposited July 1988, passage 7 and Madin-Darby Canine Kidney cells (MDCK; ATCC CCL-34, isolated September 1958) were cultured at 37 0 C in an atmosphere of 95% air plus 5% carbon dioxide in supplemented medium consisting of minimum essential media with Hanks salts, 16 mmol/L sodium bicarbonate, 2 mmol/L L- glutamine, and 10% fetal bovine serum (all components from Hyclone Laboratories).
  • Cells in exponential growth phase were harvested by exposure to a ' 0.25% trypsin solution at 37 0 C, washed twice with supplemented medium, plated onto sterile untreated glass coverslips (12 mm 2 Fisher Brand) , and cultured at 37 0 C for 12 hours to allow the cells to attach.
  • Membrane surgery and nanosurgical isolation of MDCK and fibroblast cells was achieved using a Kerr lens modelocked titanium sapphire laser oscillator, producing sub-10 femtoseconds laser pulses, with a center wavelength of 800 nm and a repetition rate of 80 MHz.
  • the ultrashort pulses were coupled to a modified optical microscope and directed towards the biological sample, as shown in Figure 36.
  • a 0.95 high numerical aperture microscope objective was used, producing a spot size of -800 nm.
  • an intensity of 10 13 W/cm 2 /pulse was generated at the focal spot.
  • Fibroblast and MDCK cells were placed on an x-y-z translation stage for precise sample movement and translated at a speed of 1 mm/second. A small volume of media was placed over the cells and the stage was temperature controlled to 4 0 C to minimize cell dehydration. The nanosurgical procedure was viewed with a charged coupled device (CCD) mounted on the modified optical microscope and captured using video software.
  • CCD charged coupled device
  • Figure 37a through 37d illustrate nanosurgery on viable V79-4 cells.
  • the cultured fibroblast cells are spread out and attached by focal integrin-base surface junctions. These surface junctions bind to the secreted extracellular matrix containing a meshwork of polysaccharides permeated by fibrous proteins.
  • FIG 37a two fibroblast cells are shown tethered together by focal adhesions, where the width of the tethered region is ⁇ 1 ⁇ m.
  • the dissection interface is precisely ablated by traversing the cells relative to the focused femtosecond laser spot.
  • Disruption of focal adhesions detaches the fibroblast cell from the adjacent cell, and the cell responds by folding, thereby isolating the single mammalian cell, as shown in Figure 37c.
  • Ablation entails the removal of cellular material contained within the focal volume, and is achieved with nanometer precision without compromising membrane structure. Such precision is evident in Figure 37c where the adjacent cell remains morphologically intact.
  • the two-fibroblast cells are clearly isolated and detached, as shown in Figure 37c,d.
  • folding of the isolated fibroblast occurs, as shown in Figure 37d.
  • Figure 37d depicts a fibroblast cell, post-laser surgery, nanosurgically liberated from the substrate, and neighboring cell. Post-manipulation assessments of long-term viability were not performed.
  • Figure 38 depicts membrane surgery on a live MDCK cell.
  • the cell was traversed relative to the focused femtosecond laser spot, precise nanosurgical cuts were made on the biological membrane.
  • Figure 38a illustrates the nano-surgery where three nano-incisions have been made, each with an incision width of -800 run.
  • Figure 38b the plasma membrane of the mammalian cell is dissected along the long axis of the 12 ⁇ m cell, followed by two additional sub-micron incisions, Figure 38c. Similar to the isolation of fibroblast cells, membrane surgery arises from the precise ablation of cellular material contained within the laser focal volume. Only morphological assessments of cell viability were performed.
  • MDCK cells Unlike fibroblasts, MDCK cells have a permeating mesh- work of polysaccharides and proteins surrounding the entire exterior membrane. MDCK cells are devoid of focal adhesion, where cell-substrate bonds mediate cell adhesion. As illustrated in Figure 38, the arrows indicate the photoablated regions of the extracellular matrix surround ⁇ ing the MDCK cell.
  • the adhesive matrix can be completely ablated when the laser traces the exterior contour of the cell membrane. Therefore, single cell isolation of MDCK cells is realizable, with a precision determined by the laser spot size and laser .scanning.
  • femtosecond lasers as a nanosurgical tool has far reaching implications for several biological disciplines. Since a sub-diffraction laser spot size can be achieved, histochemically prepared proteins both on the cellular membrane 'and intramembrane can be precisely ablated to identify functional changes in cell behavior.
  • the technique of the present invention can be employed to deliver therapeutic agents to cells of interest, or otherwise manipulate cells to achieve a desired result.
  • the laser pulse technique of the present invention provides new insight for a wide domain of biological disciplines, with consequential impact on present and future research.
  • a femtosecond laser pulse technique of the present invention was employed as a tool in the cellular manipulation of zebrafish (Brachydanio rerio) embryos.
  • Brachydanio rerio embryos serve .as a model system for studying vertebrate development and genetics. They are a closer model system to humans than the common invertebrate systems of Drosophila and Caenorhabditis elegans.
  • the ability to cryopreserve fish embryos provides the facility for long- term storage of several fish species, and the establishment of genome resource banking and . re- population of exotic species. This would have profound influence on medical research, aquaculture, and conservation biology.
  • Zebrafish embryos are multicompartmental (i.e. yolk and blastoderm) biological systems with permeability barriers provided by the non-cellular membranes and the syncytial layer surrounding the developing embryo and yolk.
  • the low permeability barrier to water and cryoprotectants of the multinucleated yolk syncytial layer, the changing permeability coefficient as a function of developmental stages, the different osmotic p'roperties of each membrane compartment, and the chilling sensitivity have been the main factors hindering the successful cryopreservation of zebrafish embryos.
  • the yolk syncytial layer has been reported as the major permeability barrier preventing the permeation of cryoprotectants, and insufficient permeation throughout the embryo yields unsuccessful vitrification and intraembryonic freezing.
  • the application of femtosecond pulses of the present invention can be applied to any embryo stage without a change in cyroprotectant permeation. Moreover, intact embryos can be accurately permeabilized without compromising the chorion.
  • Figure 39 depicts a developing zebrafish (Brachydanio rerio) embryo at the Gastrula stage.
  • the chorion, developing blastoderm, yolk, and YSL layer can be seen as the major compartments of the developing embryo.
  • the chorion is a non-cellular protective layer surrounding the entire embryo, and the developing blastoderm is an additional layer engulfing the yolk.
  • YSL yolk syncytial layer
  • This is a multinucleated layer, 10 ⁇ m thick, composed of a non-yolk cytoplasm that begins to develop at the Blastula stage.
  • the multinucleated layer 1 surrounds the yolk ahead of the developing blastoderm, with complete coverage at the end of the Gastrula stage.
  • the YSL replaces a thin 2 ⁇ m thick non-nucleated yolk cytoplasmic layer. It is important to mention that the YSL would normally not be visible in bright field images, but has been included in the below image for clarity purposes.
  • gating of the laser pulse train allows for selection of the number of pulses irradiating asample. Since each laser pulse has a designated pulse energy (i.e. energy/pulse) , the selection of the number of laser pulses incident on the sample provides control over how much energy the sample will absorb.
  • the goal for intracellular delivery of genetic material and/or cryoprotectants, for example, is the successful creation of a transient pore.
  • the ⁇ optical pore' is created by the absorption of laser energy by the sample, thereby leading to the formation of a pore.
  • the gating of the pulse train preferably ensures that the critical amount of energy is absorbed by the sample, whereby more absorption could lead to pore widening and eventual cell/embryo death. However, if the amount of absorbed energy is less then the critical amount, no pore is created.
  • the preferred amount of deposited energy depends on the cell type, composition etc.
  • gating of the laser pulses using either a galvanometer or a mechanical shutter is provided.
  • the mechanical shutter or galvanometer is set to a specific ⁇ on/off time (mechanical shutter: the duration the aperture is opened, galvanometer: the duration over which the rotating mirror switches from a deflection angle A to a deflection angle B)
  • the repetition rate is multiplied by the ⁇ on/off' time giving the number of pulses irradiating the sample.
  • the ⁇ on/off time can be anywhere from milliseconds to microseconds.
  • laser parameters are changed based on where the embryo is being ablated.
  • the yolk is more resistant to pore formation then the developing cells, for example.
  • the interface separating the cells and 'the yolk is less resistant.
  • ea ' ch specific ⁇ zone' will have a slightly different laser power, energy absorption, and laser gating time for transient pore formation.
  • Figure 43 is an alternate representation of Figure 42 where all of the images in Figure 43 have been false colored to emphasize the size of the ablated region.
  • the range of colors presented in the images provides important information on the composition of the embryo.
  • Figure 44a illustrates an embryo pre-laser treatment. Focusing the laser pulses on the yolk, and properly gating the laser pulses, a small pore was created, represented by the arrows in Figures 44b and 44c. Figure 44c is a false color image of Figure 44b.
  • Figures 45a - 45d illustrate still images at specific time intervals post-laser treatment and confirm that the embryo continues to develop normally even after laser treatment. interesting, at 8 hrs post-ablation, evidence of a pore is no longer present. The pore was found to completely seal after laser treatment without detrimental effects on the growth and development of the embryo. At 78 hrs post-ablation hatching has already occurred and further growth and development was observed at 123.5 hrs post- ablation ( Figure 46) . At approximately 148 hrs post- ablation the newly hatched larvae was food seeking and avoided being imaged.
  • biopreservation has been limited to the invasive techniques of microinjection and electroporation.
  • our novel process, setup, and design achieve this application with precision and control, thereby circumventing DNA denaturation, disruptions in biochemical pathways, and cell lysis, which is common in prior art methodologies.
  • the method provides for the transfection of carbohydrates with 100% efficiency without irreparable cell damage. Consequently, our technique of cell loading is also applied to subcellular loading. This is very important for embryos, for example, as different subcellular compartments can be targeted and preserved for carbohydrate loading.
  • the present invention thus provides the ability to select specific subcellular compartments, for preservation applications.
  • the method can be applied to nerve welding. Specifically, using focused femotsecond pulses to surgical detach and attach severed nerve endings. The method provides the ability to fuse nerves with high post fusion survival rates.
  • the method may be beneficial for different field of activities such as surgery, and immunology.
  • nerve re-attachment has received very little progress due to the inherently tedious process.
  • the ability to fuse nerves together, without inducing irreparable damage, provides a new tedious free method for nerve welding.
  • a packaged technology accomplishing nerve welding has overwhelming consequences for surgical applications.
  • Single cell cutting and cellular welding may benefit applications such as: hybridoma, genetic engineering, and agricultural studies, as exemplified in Tian Y. Tsong, Biophys. J., vol. 60, pp. 297-306, (1991), which is herein incorporated by reference.
  • the beneficial application of hybridoma is exemplified by the teachings of Gordon H. Orians, H. Craig Heller, Life - The Science of Biology, Fourth Edition, Sinauer Associates, Inc., W. H. Freeman and Company, 1995, which is herein incorporated by reference.
  • biopreservation is to protect the integrity and functionality of cells, tissues and organs for cell 'based technologies.
  • the removal of cells from their native environment often results in cell death due to the inhibition and/or elimination of the cell's natural repair mechanisms.
  • the resulting absence of such protective physiological mechanisms leads to biological inactivity and eventual cell death (necrosis / apoptosis) .
  • a major challenge in drug delivery is the lack of carrier-mediated proteins which transfer the desired drugs into the intracellular matrix.
  • directly perforating the plasma membrane and surrounding the cell with the desired chemical provides a simple means for transfering the substance into the cytoplasm by diffusion.
  • subcellular organelles can be targeted, and drug delivery as well as gene therapy can be easily applied.
  • femtosecond laser based mediated transfection specific organelles such as mitochondria can be transfected for further investigation into the effect of drug delivery on mitochondrial disease.
  • direct gene therapy is applicable, as inhibitors or gene activators can be transfected for down-regulation (suppression) or over- expression (activation) of specific genes or proteins.
  • Stem Cell - Are undifferentiated cells that become specialized in place of those which have died.
  • the ability to locally manipulate the genetic information, and treat single gene defects, provides a direct means for curing common gene related diseases .
  • Cystic Fibrosis The most common genetic illness that prohibits the movement of salts from the body's tissue leading to chronic lung infection. Directly targeting the defective gene can correct for the genetic defect, and resume normal lung functionality. Gene transfer technology is expected to revolutionize the treatment of genetic diseases using DNA as a therapeutic device.
  • the goal of cellular physiology is to understand and classify the functionality of specific proteins, organelles and various other compositional constituents within cells.
  • molecular biologists can identify the specific functional roles the species plays, and classify the effect of direct inhibition or complete removal of the specific component.
  • Direct usage of our technique can remove specific subcellular species, thus providing a direct means for studying the effect of r ' its removal.
  • Microtubules provide mechanical stability, and serve as tracks that carry proteins from one part of the cell to another. More importantly, their function in cellular division is important, as they provide skeletal structure. However, the mechanisms by which the microtubules functions are still unknown.
  • Nerve Welding Current methods in repairing severed nerves requires the handheld precision of surgeons. The possibility of using- localized femtosecond pulses for nerve re-attachment will potentially impact the field of nerve surgery. Since femtosecond mediated nanosurgery produces precise, well defined, repetitive incisions, its application provides an easier approach to conventional , means.

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Abstract

L'invention concerne un appareil et un système utilisant l'énergie laser afin de manipuler des cellules et des compositions biologiques ou des systèmes. Un appareil et un procédé de cette invention permettent de manipuler, avantageusement, des structures cellulaires et des compositions biologiques, d'une manière pratiquement non effractive. Un appareil ou une méthode décrits dans ladite invention utilisent l'énergie laser et, de préférence, des impulsions laser femtoseconde, afin de manipuler des cellules, des structures cellulaires et/ou des compositions biologiques. Selon l'invention, l'énergie laser peut être employée pour manipuler des propriétés physiologiques et/ou chimiques de tels substrats, à la fois in vivo et in vitro.
PCT/CA2005/001556 2004-10-08 2005-10-11 Appareil laser et methode de manipulation de cellules WO2006037236A1 (fr)

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US20110060266A1 (en) * 2001-11-01 2011-03-10 Photothera, Inc. Enhanced stem cell therapy and stem cell production through the administration of low level light energy
WO2014132146A1 (fr) * 2013-02-26 2014-09-04 Koninklijke Philips N.V. Traitement non invasif de la peau au moyen de lumière laser
CN104956202A (zh) * 2012-10-30 2015-09-30 宾夕法尼亚州研究基金会 3d激光消融层析成像和光谱分析
US9437041B2 (en) 2012-10-30 2016-09-06 The Penn State Research Foundation 3D laser ablation tomography
US9835532B2 (en) 2012-10-30 2017-12-05 The Penn State Research Foundation 3D laser ablation tomography and spectrographic analysis
CN112835190A (zh) * 2021-01-04 2021-05-25 桂林电子科技大学 基于双芯光纤光操控和动态散斑照明显微成像方法和系统
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FR3124399A1 (fr) * 2021-06-28 2022-12-30 Universite De Bourgogne Neutralisation de parasites par irradiation laser.
WO2024019963A1 (fr) * 2022-07-17 2024-01-25 Fertility Basics, Inc. Système de communication modéré pour traitement de l'infertilité

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* Cited by examiner, † Cited by third party
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US10683494B2 (en) * 2001-11-01 2020-06-16 Pthera LLC Enhanced stem cell therapy and stem cell production through the administration of low level light energy
US20110060266A1 (en) * 2001-11-01 2011-03-10 Photothera, Inc. Enhanced stem cell therapy and stem cell production through the administration of low level light energy
US10913943B2 (en) 2001-11-01 2021-02-09 Pthera LLC Enhanced stem cell therapy and stem cell production through the administration of low level light energy
DE102006046925A1 (de) * 2006-09-28 2008-04-03 Jenlab Gmbh Verfahren und Anordnung zur Laser-Endoskopie für die Mikrobearbeitung
CN104956202A (zh) * 2012-10-30 2015-09-30 宾夕法尼亚州研究基金会 3d激光消融层析成像和光谱分析
US9437041B2 (en) 2012-10-30 2016-09-06 The Penn State Research Foundation 3D laser ablation tomography
US9835532B2 (en) 2012-10-30 2017-12-05 The Penn State Research Foundation 3D laser ablation tomography and spectrographic analysis
US9976939B2 (en) 2012-10-30 2018-05-22 The Penn State Research Foundation 3D laser ablation tomography
EP2914951A4 (fr) * 2012-10-30 2016-08-10 Penn State Res Found Tomographie d'ablation laser tridimensionnelle (3d) et analyse spectrographique
WO2014132146A1 (fr) * 2013-02-26 2014-09-04 Koninklijke Philips N.V. Traitement non invasif de la peau au moyen de lumière laser
US11058575B2 (en) 2017-10-25 2021-07-13 École Polytechnique Method and system for delivering exogenous biomolecules into the eye and method for forming pores into target cells of an eye
CN112835190A (zh) * 2021-01-04 2021-05-25 桂林电子科技大学 基于双芯光纤光操控和动态散斑照明显微成像方法和系统
CN112835190B (zh) * 2021-01-04 2022-08-09 桂林电子科技大学 基于双芯光纤光操控和动态散斑照明显微成像系统
FR3124399A1 (fr) * 2021-06-28 2022-12-30 Universite De Bourgogne Neutralisation de parasites par irradiation laser.
WO2024019963A1 (fr) * 2022-07-17 2024-01-25 Fertility Basics, Inc. Système de communication modéré pour traitement de l'infertilité

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