WO2006037236A1

WO2006037236A1 - Laser apparatus and method for manipulating cells

Info

Publication number: WO2006037236A1
Application number: PCT/CA2005/001556
Authority: WO
Inventors: Abdulhakem Elezzabi; Jason Acker; Vikram Kohli
Original assignee: Canadian Blood Services
Priority date: 2004-10-08
Filing date: 2005-10-11
Publication date: 2006-04-13

Abstract

An apparatus and system employing laser energy to manipulate cells and biological compositions or systems are provided. An apparatus and method of the present invention advantageously allows the manipulation of cellular structures and biological compositions, in a substantially non-invasive manner. An apparatus or method as embodied by the present invention employs laser energy, and preferably femtosecond laser pulses, to manipulate cells, cellular structures and/or biological compositions. According to the present invention, laser energy may be employed to manipulate physiological and/or chemical properties of such substrates, both in vivo and in vitro.

Description

LASER APPARATUS AND METHOD FOR MANIPULATING

CELLS

TECHNICAL FIELD

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.

BACKGROUND OF THE INVENTION

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 photόdynamic therapy of the latter.

Despite impressive advances in the fields mentioned above, the use of light energy to manipulate properties of ^• individual cells without causing permanent damage remains a difficult task.

When a short pulsed laser interacts with a material sample two key processes occur. The first being the formation of a plasma, and the second being the formation of a shock wave. These two effects arise due to the optical breakdown of the material. 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.

To maintain cell functionality, 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. This is also true for thermal stresses, as the physics of diffusion., indicates that a local temperature rise results in a temperature gradient directed outside the focal volume. As a result, adjacent cells rise in temperature, and therefore are exposed to thermal stress. It is desirable to minimize thermal propagation, since the rise in cellular temperature changes the histological features of the cell. Since picosecond and nanosecond laser light pulses induce large mechanical and thermal stresses, over a broad radius, their usage for investigating cell manipulation is not well suited. Using such pulses would cause considerable cell death, and consequently induce additional histological changes to in adjacent cells. The use of femtosecond pulses to remove portions of chloroplast in plant cells, to transfect DNA vector into mammalian cells, . to nano-dissect human metaphase chromosomes and to perform intracellular and intratissue nanosurgery have been reported. However, these systems and methods still resulted in significant cell damage and death.

Femtosecond pulses have also been used to vaporize micrometer-sized regions of living cells. This ^λλknockout" approach was also found to cause substantial cellular damage and cell death.

Thus, in view of the above, it is desirable to develop an improved laser system for manipulating cellular structure and biological compositions and systems.

SUMMARY OF THE INVENTION

There is therefore provided an apparatus and method for the manipulation of cellular structures and biological compositions, in a substantially non-invasive manner. An apparatus or method . as embodied by the present invention employs laser energy, and preferably femtosecond laser pulses, to manipulate individual cells, cellular structures and/or biological compositions.

Biological compositions are intended to include mammalian and non-mammalian cells, tissues and organs, for example.

According to the present invention, 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. - A -

According to an embodiment of the present invention, 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. Furthermore, laser energy may be delivered to a substrate of the present invention via single pulse or via multiple pulses.

In one embodiment there is provided 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. By non-thermally modified it is meant that the pulse, duration is sufficiently short to prevent thermally induced damage to adjacent cellular structures.

In another embodiment, the apparatus may comprise means, such as a multi-channel microfluidic device for spatially and temporally controlling an environment surrounding the cellular structure.

In another aspect of the invention there is provided 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. According to preferred embodiments of the present invention, 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.

Further features and advantages of the present invention will become apparent from the following detailed description.

It will be noted that throughout the appended drawings, like features are identified by like reference numerals.

DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates an apparatus and system according to an embodiment of the present invention.

Figure 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.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The ability to non-invasively study and manipulate mammalian cells has important consequences for cell-based therapeutics. With the recent advancements in tissue engineering, cell transplantation and preservation, and genetic technologies, living cells as a therapeutic tool for clinical care have received wide attention. These emerging technologies depend on the characteristics of living cells, and thus maintaining and preserving the biological function of cell-based therapeutics remains one of the most ^• important challenges < facing reparative medicine.

The goal of 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. According to an embodiment of the present invention 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. Preferably, 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. ' This effectively renders the cellular manipulation process non-thermal. Cell damage due to thermal heating is inconsequential, and the short pulses allow for smaller cell dissections to be achieved within the focal spot where the non-linear multi-photon interaction process occurs. Using appropriate optical techniques, a sub-diffraction limited focal spot can be obtained, producing a significant- decrease in dissection size.

As mentioned above, the present invention has wide application for a variety of cellular manipulations. For example, 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. Accordingly, the present invention can be employed to cryopreserve oocytes, sperm, embryos and other reproductive material. Furthermore, due to continual increases in mammalian extinction rates, considerable efforts have been directed towards genetic resource banking of endangered species. The present invention provides an improved technique for the preservation of reproductive materials, such as sperm, oocytes and embryos such that genetic materials can be preserved well after the extinction of the animal, for example.

According to one embodiment of the present invention, we report on the feasibility of using high-intensity femtosecond laser pulses (10^~15 seconds) for performing membrane surgery and nanosurgical cell isolation on live mammalian cells. When sub-10 to 500 femtosecond laser pulses were focused to an intensity of 10¹⁰ - 10¹³ W/cm²/pulse onto cells, sub-micron to micron dissection cuts were made on the biological membrane without morphologically compromising the cell. We also demonstrate localized nanosurgical ablation of focal adhesions adjoining epithelial cells, and provide a novel process for cell isolation. In both studies membrane integrity was maintained, and cell collapse and disassociation were not observed .

In accordance with the present invention, 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. According to a preferred embodiment, the pulse duration of the laser is less than 20 femtoseconds. Advantageously, 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. Simply, there is no time for electrons to transfer energy to the bulk material or ions since these processes occur on longer time scales (picoseconds to nanoseconds) . Therefore, cell damage due to thermal heating is avoided, and the short pulses allow for smaller pores to be achieved. Using appropriate optical techniques, a diffraction limited focal spot can be obtained, yielding a femtolitre focal volume, thereby producing a significant decrease in pore size. In addition, 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. For example, as herein illustrated, 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. For example, an embodiment of the present invention is described below for manipulating cellular material. As disclosed, the pulse laser technique of the present invention is able to create tiny perforations in the biological plasma membrane without inducing irreparable damage. In addition, 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. At the focal spot, we generate intensities as large as 10¹⁰ - 10¹³W/cm², which can be controlled using an energy selecting optical element. 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. As the incident pulses traverse through glass, the pulse is broadened, and thus the pulse length increases. Longer pulse durations induce larger mechanical, and thermal damage than shorter pulses. For instance, 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. When dealing with material such as proteins, lipids, carbohydrates etc., which cells are composed of, it is imperative that we minimize any induced mechanical and thermal stress, as cellular matter is highly susceptible to these specific stresses. According to an alternative embodiment of the present invention, a reflective objective is employed. A reflective objective preferably contains no glass elements, and hence ^■ the pulse is not broadened. This type of objective is very useful when pulse broadening is to be minimized. As an example, an oscillator producing a 500 - sub-10 femtosecond-pulse train will remain nearly 500 - sub-10 femtoseconds at the focus of the sample. Evidently, a reflective objective is a preferred embodiment of, the apparatus and method of the present invention, as it is advantageously provides a reduction in cellular damage. Our 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. These embodiments are more beneficial than conventional 100 femtosecond laser systems since shorter pulses induce less thermal and mechanical damage when focused onto cellular matter. In addition, this is advantageous since cell viability depends strongly on induced stress. Thus, in our design we place considerable importance on minimizing irreparable damage, thereby using short pulse laser systems and specially designed microscope objectives. An interesting feature of our design is that the stage, housing the sample, 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. In fact, when interfaced, 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. In addition, 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. In addition to precise stage control, 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.

Housed within the optical microscope are a series of filters for UV fluorescence, epi-fluorescence, and laser beam imaging. The 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. Briefly, to accurately image both the cell and the focused laser beam, the focus of the two image sources is preferably decoupled. In our setup, .we back illuminate the sample using white light, and deliver the focused femtosecond beam in the forward direction. Thus, two images are produced, and they are overlapped. 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. Thus, the focused images provide a controlled means for live evaluation of cell-laser interaction. It should be mentioned that when we refer to 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.

It should be mentioned that in conjunction with UV, epi, white light, and laser beam imaging, the present invention may also be employed in connection with third harmonic generation (THG) microscopy and optical coherence tomography (OCT) imaging. 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. When the two imaging techniques are combined, the result is a high-resolution image of the sample that is otherwise impossible to achieve with THG and OCT imaging alone. It is also interesting to mention that OCT is capable of in vivo imaging. Examples of imaging protocols that may be employed in accordance with the present invention, such as THG and OCT imaging, for example 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.

A variety of illumination sources can be employ in connection with embodiments of the present invention. As mentioned above, control of the temperature of the cell can be advantageous. Using conventional light sources, i.e. standard white light sources coupled to optical microscopes, generate a large source of heat. In fact 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.

lastly, it should be mentioned that with the above, design we can adjust the stage focus such that we modify intracellular matter. According to a preferred embodiment, 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.

A alternative setup, design, and process, according to another embodiment of the invention is provided whereby a femtosecond fiber laser is coupled to a multi-channel microfluidic device for achieving preferred applications, such as photodisruption procedures, for example. Figures 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. Thus the above described embodiment provide for the flexible adaptation of the system to study different cell lineages subjected to a variety of treatments. Furthermore, such a system can be easily transported while still maintaining the precision and accuracy that is desired.

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.

Attention should be directed towards the ability to change the repetition rate and wavelength of the laser, as provided in accordance with an embodiment of the present invention. 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.

In one embodiment the laser can be polarized. The polarization state of¹ 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. In recent studies, it has been shown that 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.

In another embodiment there is provided an- apparatus and method using a near field optics approach for manipulating cellular material. According to this embodiment 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.

In another aspect of the invention ' a near field optical technique using a fiber tip is provided. In this process, design, and setup, we use a femtosecond fiber laser coupled to a fiber optic tip for performing photodisruption experiments. When the femtosecond pulse is coupled to a fiber tip, tiny perforations in the biological membrane are achieved. This is accomplished by placing the fiber tip directly over the desired area. In conjunction with laser irradiation, imaging of the specific photodisruption¹ site is also achieved. Using irradiation with imaging, 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. 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. In this design, 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. With 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. Thus, our design is versatile, and anticipates functional changes .

) Examples of the components comprising the laser apparatus of the present invention are provided below (Figures 1 - 6) . As illustrated in Figure 1, 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. In accordance with one embodiment of the present invention, the microscope objective 8, is an air, water, or oil immersion type. Alternatively, according to preferred embodiment of the invention, the glass objective can be replaced with a reflective objective, producing smaller focal volumes, with uncompromised pulse lengths. Moreover, 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. Moreover, 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, θ, φ, 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. In addition, peizos, flextures, translation stages, and/or any other translation device are implemented based on the choice of precise movement and control. 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. These include: azimuthal, radial, linear, circular, and elliptically polarized Gaussian beams. The benefit of polarization selection determines the smallest obtainable focused spot achievable. The implementation of this element depends on the particular application being pursued. 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. The implementation of this element depends on the particular application being pursued. 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, θ, φ, 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. It should be noted that 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. In addition, 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 (ICT¹² 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 ^Λon-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. As illustrated in Figure 3, 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. 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. Alternatively, for studies involving smaller perforation sizes (i.e. smaller focal volumes), 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. It should be mentioned that as the apertured hole size decreases, the spot size decreases. This is important in perforation-based studies, as well as sub-wavelength imaging. In this delivery system, 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. Higher resolution images are obtained with smaller fabricated aperture holes. 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.

As exemplified in accordance with the illustrations discussed above, it is noted that 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.

Examples

Live Nanosurgery of Mammalian Cells

Part I As exemplified in Figures 13 - 22 nanoscale surgery of live Madin-Darby Canine Kidney (MDCK) cells, was performed and captured using a pulse laser apparatus of the present invention, as discussed above. Briefly, 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 10¹⁰ - 10¹³ W/cm² is used. Figure 14 exemplifies precise surgery using nanojoule pulses focused to 10¹³ W/cm² .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. As shown, 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. Thus the present invention provides a selective tool that allows for the manipulation of live cells through non-invasive means.

Live Nanosurgery of Mammalian Cells

Part II

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. In these images a IOOX 0.95 high numerical aperture (NA) microscope objective lens is used for performing live nanosurgery of Madin-Darby Canine Kidney (MDCK) cells. 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 μm is achievable with a IOOX 0.95 NA microscope objective lens.

Similar to the prior section, femtosecond pulses are coupled to a high NA microscope objective lens, and focused by the IOOX 0.95 NA microscope objective. In these images, 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 10¹⁰ - 10¹³ W/cm². What makes this section an extension of the previous is, 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. As illustrated in Figure 20 and 21 the cell's shape remains intact, and no changes in cell morphology are seen. Furthermore, Figure 21 depicts dimensional analysis, and for a 12 μm cell a cut size of 800 ran is achieved. Similarly, 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. When the laser beam traverses across the cell, 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.

Cellular welding has tremendous applications, especially in immunological studies. More specifically, our welding technique can be applied to nerve welding, specifically, the study of fusing nerve ending together.

Perforation based studies on live mammalian cells

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 10¹⁰ - 10¹³ W/cm². 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.

The ability to create perforations in live cells is important for transfection-based studies. Specifically, targeted transfection, drug delivery, gene therapy, and biopreservation all benefit from our novel approach. Since our technique is non-invasive, we readily achieve 100% transfection without inducing irreparable cell damage. Such damage includes disruption of the biochemical pathways, and DNA denaturation. These irreparable damages, including loss in cell viability, are common in microinjection and electroporation based studies.

Laser mediated transfection of live mammalian cells

In this study 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 10¹⁰ - 10¹³ W/cm². 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. 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.

Before transfection, fluorescence images depicted cells stained green from Syto 13 diffusion. Figure 24 is , a white light image of the cells before transfection and after transfection. It should be noted that after exposure the cells are still intact. Upon creating a perforation, ethidium bromide readily diffuses into the cell and stains the cell red/orange. To verify transfection, the cells were imaged under fluorescence to determine the diffusion of ethidium bromide. Verification was achieved with this methodology and all perforated cells were found to be successfully porated, as indicated by the diffusion of ethidium bromide.

Figure .24 illustrates verification of the transfection process. In addition, all cells that have been perforated have been done with 100% efficiency. The benefits of our study are evident. For drug delivery and gene delivery based studies, 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.

The non-invasive nature of our technique creates perforations that eventually seal; thereby cell viability is still maintained. Cell viability has been evaluated, and the results are presented below.

Selectively targeted transfection

In the previous section an illustration of laser mediated targeted transfection was presented. It was shown that live cells could be perforated with 100% efficiency. In this study we report on selectively targeted transfection by focused femtosecond pulses.

To achieve selectively targeted transfection 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.

Similar to ethidium bromide, 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) .

In this study only a few selective cells, within a collection of cells, are transfected. The selective nature illustrates the novelty of our process. Namely, the ability to selectively target any cell, and evaluate the effect of the transfected substance.

In one study of the present invention, cells are surrounded by Syto 13 and ethidium bromide. In the two transfected cells, a local perforation in the biological membrane was created, resulting in the diffusion of ethidium bromide. Since 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. Furthermore, 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.

In a second study according to an embodiment of the present invention 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.

i The ability to selectively target cells has tremendous benefits for drug delivery, gene therapy and biopreservation applications. Often it is desirable to transfect various cell types with different material, and using our novel technique we can accomplish this in a precise and well-controlled fashion. More specifically, emphasis should be directed on our methodology in tailoring this novel procedure.

Laser transfection of sugar for biopreservation applications

The study presented in this section is tailored to the biopreservation of live mammalian cells . In this study 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.

To achieve transfection 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. In this study the live cells were surrounded by 1.0 molar concentration of sucrose,, and upon creating the perforation, sucrose diffused into the cell.

To evaluate the transfection of sucrose, cellular increase is monitored. Initially, before transfection, when the cells are surrounded in a high molar sucrose concentration, the cells are initially in a shrunken state. The shrunken state is indicative of the cells being placed in a hypertonic solution resulting from a high molar concentration of sucrose outside the cell. Upon creating the perforation, sucrose diffuses into the cell causing the cell to swell in size. This is readily observed in Figures 27-32. From Figures 27 - 30, a time lapsed progression of the transfection process is provided. The first image in the sequence depicts the laser spot focused on the live mammalian cells. After a brief duration, 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.

As the cavitation subsides, an increase in cellular size is observed. This is seen in Figures 27 - 30. An increase in cellular size provides direct evidence of sucrose diffusion. Once the cavitation has subsided, and cellular increase is observed, the mammalian cells remain associated. In each image, we therefore illustrate 100% transfection. Figure 31 illustrates cellular swelling without reference to the position of the laser spot.

Sucrose concentration, pulse energy and pulse duration all play roles in cell survival. Figures 27 -31 clearly demonstrate cells which remained intact after the transfection process. However, Figure 32 depicts the case when the pulse duration is left on without significant pulse gating. An additional indication of cell damage other than cell morphology, cell collapse and disassociation is cellular blebbing. Figure 32 illustrates cellular blebbing without significant pulse gating. With our novel technique we accurately control pulse gating in a precise fashion thereby producing 100% transfection and maximized cell survival.

Furthermore, Figure 30 illustrates our non-invasive, highly localized approach to transfection. In the image 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.

At present several attempts have been made to preserve human oocytes and embryos. However, viability rates post cryopreservation, using microinjection or vitrification (in the presence of DMSO and other cryoprotectants) , has produced variable results. Specifically, cryopreservation with intracytoplasmic sperm injection (ICSI) has lead to an overall success rate of 1%, while 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)) . More recently, oocytes subjected to in-vitro fertilization (IVF) cryopreserved with trehalose using microinjection have been shown to have higher survival rates post thaw. However, variable survival rates were still observed, 13-66% (A. Eroglu et al. supra) . In addition, chromosome disorganization has been observed using conventional cryopreservation methods. Such disorganization can lead to cytogenetic anomalies, thereby compromising normal oocyte development. Therefore, it is evident that new alternative approaches for the cryopreservation of oocytes and embryos are needed. Consequently, the ability of our technique to transfect cells with 100% efficiency, without inducing irreparable cell damage, advantageously provides a new method for preserving cells that can benefit industries such as the aquaculture and reproductive industries .

Carbohydrates that can be used for biopreservation applications are known in the art. For example, 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. Alternatively, 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 characterization with respect to varying sucrose concentration

In this study we characterize cell viability after carbohydrate loading. From our studies we have found that 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.

In this experiment we evaluate cell viability for 0.5, 0.4, 0.3, and 0.2 molar concentration of sucrose, using an input power of 270-275 mW (or 3.375 - 3.4375 nJ per pulse) . In each case Syto 13 and Ethidium Bromide were used to classify cell survival. The cells were stained approximately 30-45 min post transfection. Under fluorescence imaging, if the cells fluoresce green then the lipid membrane is intact and not compromised. Evidently, this indicates that the cells are alive and functional. On the other hand, if after transfection the mammalian cells fluoresce red/orange than the membrane has been compromised and cell death is imminent.

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.

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.

Keeping the gated shutter and input power approximately constant, 0.4 molar sucrose was investigated. A group of twelve cells were transfected in the first set, and only two cells have uncompromised membranes. Therefore, two cells remained alive while the remaining had died. In the second set, eight cells were investigated and six of the eight cells were transfected. From the six transfected cells three cells had intact membranes. The rest lost complete cell viability resulting in cell death. In the next data set, using a similar shutter time and input power, 0.3 molar sucrose was investigated. In the fist set, three cells were transfected, and in the second set four cells were transfected. From the two sets only two cells have lost complete cell viability. It should be mentioned that in^"the second set, the viability of one cell determined to be compromised based on the color contrast with respect to the adjacent cells.

Lastly, 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.

Thus in this particular example, 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. With 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. '

Applying this study to human embryos and oocytes cells, as well as other cell types for preservation, 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.

Live nanosurgery on fibroblast cells

In this study we demonstrate live nanosurgery on live fibroblast cells. 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 .

In Figure 33 two fibroblast cells are shown tethered together, where the width of the tethered region is ~1 μm. 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 10¹³ W/cm² 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.

As the focused femtosecond beam is traversed across the tethered region, vaporization of cellular material occurs. This can be seen in Figures 34 - 35 where the epithelial cell has begun to round up. Without wishing to be bound by theory, the rounding is caused by the focused femtosecond laser beam which detaches the cell from the adjacent cells as well as from the glass substrate. Once detached the fibroblast cell curls. The detachment and rounding is analogous to the enzymatic process of trypsinization. The basis of trypsinization involves the disassociation of cell-cell and cell-substrate bonds. In tissue culture preparation trypsin is often used to chemically detach mammalian cells before becoming confluent. A few minutes after trypsin exposure the cells disaggregate and round up into spherical form. However, prolonged exposure to trypsin has been shown to induce adverse effects on cells, and lipid vesicles. Similar spherical rounding is seen in images Figures34 and 35. In Figure 35, the ' laser pulse is turned off and further rounding is observed. Image 58 depicts an out-of-focus and an in-focus image of the rounded fibroblast cell.

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. Here we have shown a non-invasive nanosurgery procedure that allows live cells to be manipulated and controlled in a precise fashion with inducing irreparable cell damage.

' The ability to manipulate any cell type in a desired fashion has tremendous benefits for cellular biology. Specifically, cellular physiology can benefit the present invention. Moreover, specific proteins or groups of proteins, and^' DNA strands can be surgically cut for investigation into their functional role. Thus, we anticipate a plethora of new applications that will benefit from this non-invasive laser pulse technique, and foresee its integration into widely pursued cell-based technologies.

Presented below is brief outline of further exemplary applications that are being pursued. Only a few applications have been mentioned for illustration purposes. CeIl Nanosurgery Using Femtosecond Laser Pulses

METHODS

Culture Process

Chinese hamster fibroblasts (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 37⁰C 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 37⁰C, washed twice with supplemented medium, plated onto sterile untreated glass coverslips (12 mm² Fisher Brand) , and cultured at 37⁰C for 12 hours to allow the cells to attach.

Experimental Setup

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. To focus the femtosecond laser pulses, a 0.95 high numerical aperture microscope objective, was used, producing a spot size of -800 nm. Using a delivered average power of 410 mW, an intensity of 10¹³ W/cm²/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 4⁰C 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.

RESULTS

Cell Isolation Using High-Intensity Femtosecond Laser Pulses

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. In Figure 37a, two fibroblast cells are shown tethered together by focal adhesions, where the width of the tethered region is ~1 μm. To achieve cell isolation, the dissection interface, as shown in Figure 37a, 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. After ablation, the two-fibroblast cells are clearly isolated and detached, as shown in Figure 37c,d. After the laser pulse is turned off, 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.

Membrane Surgery Using High-intensity Femtosecond Laser Pulses

Figure 38 depicts membrane surgery on a live MDCK cell. When 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. In Figure 38b, the plasma membrane of the mammalian cell is dissected along the long axis of the 12 μm 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.

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. Here, under precise laser scanning, 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.

DISCUSSION AND CONCLUSION

The application of high-intensity femtosecond lasers pulses for cell manipulation provides a novel approach for cell-based therapeutics and nanosurgery applications. In this study, we have shown that localized femtosecond laser pulses can precisely isolate individual cells as well as perform membrane surgery. When the laser pulses were focused by a high numerical aperture objective, ablation of cellular material occurred within the focal volume. This is evident in Figure 38c, where the mammalian cell is manipulated with multiple cuts, and the surgery is achieved without evidence of membrane re-orientation, cell collapse, and bleb formation. We suggest that the lack of membrane re-orientation and cell collapse is due to the coalescing of the upper and lower plasma membranes when the mammalian cell is ablated. Permanent incisions without resealing of the lipid bilayer would likely lead to bleb formation, cell collapse, and necrosis. Alternatively, phospholipids which have re-oriented in sealing the incision would provide no evidence of membrane disruption. Since neither of these cases is observed, we conclude that coalescence of the upper and lower membrane has likely occurred, thereby preventing the cell from disassociating.

Moreover, since the time scale for thermal heat diffusion is on the order of picoseconds to nanoseconds, the propagation of heat outside the ablation region is insignificant. Therefore, thermal shock to the biological sample is eliminated and precise nanosurgery is maintained. This is evident from Figures 37 and 38, where the ablation region is contained to the sub-micron focal spot, and the mammalian cell is observed to be morphologically intact without mechanical or thermal disruption outside the irradiation region.

Clearly, the use of 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. Alternatively, 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.

Embryonic manipulation using femtosecond laser pulse technique

In accordance with one embodiment, 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. Recently, 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.

In the present embodiment, we have investigated the applicability of using a high-intensity femtosecond laser pulse technique for selectively permeabilizing embryos for the introduction of cryoprotectants into the yolk. Due to the multicompartmental system of Brachydanio rerio embryos, and the low permeability of cryoprotectants, the creation of local transient pores fasciltates a non¬ invasive method for introducing cryoprotectants into the embryo. Efficient cryoprotectant diffusion, facilitated by the optical pore, prevents the formation of intraembryonic ice, thereby improving post-thaw viability rates. Since the yolk is composed of a high content of lipids, the membrane reseals without structural or biological damage. This has been verified on mammalian cell cultures where survival rates post-laser transfection of greater than 90% were found when cells were suspended in an optimal hypertonic solution. As discussed herein above, mammalian cells have been porated using focused femtosecond laser pulses and were successfully permeabilized in the presence of cryoprotectant sugar. From our analysis we determined that the cryoprotectant loading efficiency was > 98% with > 90% post-laser cell survival. The benefits of using a femtosecond laser pulse technique of the present invention for permeabilizing embryos, is the ability of this technique to overcome the permeability barrier, which has hindered successful cryopreservation in the past. Since the permeability of cryoprotectants. change as function of developmental stage, and insufficient permeation result in later embryo stages, 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. Underlying the blastoderm, which also surrounds the yolk, is the yolk syncytial layer (YSL) . This is a multinucleated layer, 10 μm thick, composed of a non-yolk cytoplasm that begins to develop at the Blastula stage. The multinucleated layer¹ surrounds the yolk ahead of the developing blastoderm, with complete coverage at the end of the Gastrula stage. The YSL replaces a thin 2 μm 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.

The ability for using focused femtosecond laser pulses of the present invention, in precision embryonic manipulation is evident with the precise ablation of zebrafish embryos as illustrated in Figure 40. In this example, laser pulses were focused onto the yolk for ablation without affecting the non-cellular chorion. In this example, the laser pulses were not gated to perform controlled ablation. From the sequence of images (a-d) it is evident that the laser pulse can be focused beyond the chorion. We also expect that with preferred pulse gating the degree of ablation can be properly controlled for the creation of transient pores. Gating of the laser pulses for reversible pores was performed in a different study, with assessment on embryo growth. The data presented illustrates, in accordance with an embodiment of the present invention that proper gating, as discussed further herein below, creates precise ablation. Post- ablation survival showed that the embryo grows and matures normally. This assessment was done visually, by comparing standard developmental images to the images obtained in accordance with a laser pulse technique of the present invention.

According to one embodiment of the present invention, 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.

According to a preferred embodiment of the present invention, gating of the laser pulses using either a galvanometer or a mechanical shutter is provided. According to this embodiment, if the mechanical shutter or galvanometer is set to a specific ^Λon/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 ^Λon/off' time giving the number of pulses irradiating the sample. The ^Λon/off time can be anywhere from milliseconds to microseconds. According to another embodiment of the present invention, laser parameters are changed based on where the embryo is being ablated. Generally, the yolk is more resistant to pore formation then the developing cells, for example. Similarly, the interface separating the cells and 'the yolk is less resistant. According to this embodiment, ea^'ch specific ^λzone' will have a slightly different laser power, energy absorption, and laser gating time for transient pore formation.

When the laser is left on, and focused on the yolk, the result is an increase in ablation followed by rupturing and collapse of the yolk. Figures 41 b and c depict this progressive collapse, where the yolk's content has begun to leak outside into the space between the chorion and yolk. It is apparent from the images that focused femtosecond laser pulses can be tailored for targeted ablation. However, to realize the prospect of a reversible pore, the laser beam dwell time, and average laser power must be accurately controlled to ensure closure of the pore, and more importantly, without inducing detrimental effects as shown in Figure 41.

To properly control both the beam dwell time and energy absorption by the sample, the focused laser pulses must be properly gated. In this experiment we employed a mechanical shutter with a gating speed of < 10 ms, and investigated if .the degree of ablation decreased. Figure 42a illustrates the entire embryo before ablation, while Figures 42 b-d depicts the embryo post-ablation. The arrows point to the location where ablation has occurred. From this experiment we found that proper control over the laser parameters provided localized ablation, with precise control over the size of the ablated region, and without evidence of rupture or collapse of the yolk region.

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.

Using similar laser parameters as employed above in accordance with the results illustrated in Figures 42 and 43, we assessed the survival rate of zebrafish embryos post-laser treatment. Still images were taken at specific time intervals to monitor the development of the embryo, and to confirm the non-invasiveness of using focused femtosecond laser pulses. 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.

Cell loading and subcellular loading - selective loading and targeting of embryos and oocytes

In this application we are investigating the transfection of carbohydrate-based compounds into live mammalian cells. The transfection process is based on creating tiny perforations, mediated by a focused femtosecond pulse on the biological plasma membrane. The method used for disrupting the membrane is accomplished through our novel process, setup, and design.

The ability to transfect carbohydrate-based compounds into live cells have overwhelming implications for biopreservation applications. At present, biopreservation has been limited to the invasive techniques of microinjection and electroporation. We provide a new non- invasive tool for preserving embryos, oocytes, and any other cell type with the capability of achieving 100% survival post cryopreservation. Moreover, 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.

Single cell cutting, nerve welding & cellular welding

In this application we are investigating the interaction of focused femtosecond pulses as applied to single cell cutting, nerve welding and cellular welding. Here, we apply the laser as a scalpel tool and as a welding tool. More specifically, we use the pulse laser technique setup, and design to selectively cut cells, nerves, and weld together cells. In cell cutting the laser is used to selectively cut an aggregate of cells and/or single cells from hard or soft tissue. The surgical procedure is performed with nanometer precision without inducing irreparable damage to either the adjacent cells or the removed cell. Once removed, the liberated cell can be further investigated for such studies as cellular physiology. Without wishing to be bound by theory, in cellular fusion/cellular welding, focused femtosecond pulses are used to locally photodisrupt the cellular membranes of adjacent cells (of similar or of different cell line) thereby inducing cell fusion.

For example, 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. For surgery applications, nerve re-attachment has received very little progress due to the inherently tedious process. According to another embodiment of the invention, we use the laser to selectively cut nerves, and re-attach them to various other nerve endings.. This is beneficial for accident trauma patients who often lose complete usage of arms, legs, and etc. due to severed nerves. The ability to fuse nerves together, without inducing irreparable damage, provides a new tedious free method for nerve welding. In addition, 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 ability to fuse cells together, using our process, setup, and design, advances the immunology sector. For instance, the welding of cells may provide a more efficient means for the production of hybridomes. 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.

It will be appreciated that many other cellular- applications can be envisioned and are considered to be included in the present description.

We propose a technique involving the use of femtosecond laser pulses focused onto cellular matter. With our technique, selectively targeted transfection of drugs can be delivered for drug delivery applications, biopreservation of fish embryos and human reproductive cells for infertility treatments, local nano-manipulation of genes, and nanosurgery of cellular and subcellular organelles are realizable.

Provided below is a synopsis of exemplary applications being pursued, which are achievable with our novel approach.

Biopreservation

It is widely known that issues surrounding the biopreservation of cell-based organisms have hindered current advancements in cell-based therapeutics. Long-term storage of living cells or tissues is critical^' for clinical applications. At present, the lack of understanding of the complex processes involved in biopreservation is the main factor hindering future developments.

The goal of 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) . Thus, we realize the importance of cellular preservation and the ability to sustain biological materials over long storage times without inducing cell death.

Provided below is a brief summary of applications benefiting from our novel transfection approach:

[2] Drug Delivery, Transfection & Gene Therapy

At present, a major challenge in drug delivery is the lack of carrier-mediated proteins which transfer the desired drugs into the intracellular matrix. However, 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. Furthermore, subcellular organelles can be targeted, and drug delivery as well as gene therapy can be easily applied. Using 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. Thus, 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.

Provided below is a brief review of applications involving drug delivery, transfection, and gene therapy for which the present invention provides benefit:

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.

[3] Cellular physiology

The goal of cellular physiology is to understand and classify the functionality of specific proteins, organelles and various other compositional constituents within cells. By selectively suppressing specific components, 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.

Provided below is a brief synopsis of applications involving cellular mechanics using our novel technique:

Embryo development - Proper understanding of embryo development requires a complete understanding of cellular division and stabilization. A key process in cell division is the functionality of microtubules. 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.

[4] Other applicable applications

The precise nature of nano manipulation and nanosurgery provides further insight into a plethora of new applications using femtosecond-mediated manipulation. Such an application is presented below.

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.

The embodiment (s) of the invention described above is (are) intended to be exemplary only. The scope of the invention is therefore intended to be limited solely by the scope of the appended claims.

Claims

I /WE CLAIM :

1. An apparatus for modifying cellular properties of a biological sample, said apparatus comprising: 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 said one or more laser pulses with a predetermined amount of energy on said cellular structure; means for positioning said cellular structure relative to said focused laser beam; and wherein said properties, are non-thermally modified.

2. The apparatus as claimed in claim 1 wherein said means for focusing leaves said predetermined duration substantially unchanged.

3. The apparatus according to claim 1 or 2 wherein said femtosecond laser source is selected from a cavity dumped laser, diode pumped laser, amplified femtosecond laser, optical parametric amplifier, optical parametric oscillator and tunable femtosecond laser.

4. The apparatus according to any one of claim 1-3 wherein said focusing means is selected from air objectives, water objectives, oil immer-sion objectives and reflective objectives.

5. The apparatus according to claim 4 wherein said objective have a numerical aperture of between about 0.8 and about 0.95.

6. The apparatus of any one of claim 1-3 wherein said means for focusing is a fiber.

7. The apparatus as claimed in claim 6 further comprising means for delivering material to said cellular structure.

8. The apparatus as claimed in claim 7 wherein said means is for focusing is the fiber according to claim 6.

9. The apparatus of any one of claim 1-8 wherein said means for positioning is a^moving stage.

10. The apparatus according to any one of claim 1-9 further comprising means for spatially and temporally controlling an environment surrounding said biological sample.

11. The apparatus according to claim 10 wherein said means for spatially and temporally controlling is a microfluidic device.

12. The apparatus according to claim 10 wherein said microfluidic device is a multi-channel microfluidic device.

13. The apparatus according to any one of claim 1-12 further comprising means to control said pulses frequency and polarization.

14. The apparatus according to claim 13 further comprising a pulse shape controller.

15. The apparatus according to any one of claim 1-14 wherein said means for focusing comprise a means to produce a near field focused laser beam.

16. The apparatus as claimed in claim 15 wherein said means to produce a near field focused laser beam is selected from a cantilever and a tapered waveguide.

17. A method for modifying cellular properties of a biological sample, said method comprising: providing one or more laser pulses having a predetermined amount of energy such that said energy produces non-thermal photodisruption in said biological sample; and focusing said one or more pulses on said cellular structure for a time sufficient to produce said non-thermal photodisruption.

18. The method as claimed in claim 17 wherein said one or more laser pulses are femtosecond pulses.

19. The method as claimed in claim 18 wherein said femtosecond pulses have a duration that remains substantially unchanged by said focusing.

20. The method as claimed in any one of claim 17-19 wherein said focused pulses are spatially controlled to produce a beam shape having a cross section of a desired surface area.

21. A method for treating a cell, said method comprising: generating one or more focused laser pulses; applying said focused laser beam on a cellular structure such that an amount of energy is deposited in said cellular structure that produces a substantially non-thermal photodisruption of said cellular structure and

I wherein said application is for a time and encompassing a surface sufficient to effect treatment of said cell.

22. The method as claimed in claim 21 wherein said focused laser pulses are femtosecond laser pulses.

23. The method as claimed in claim 21 or 22 wherein said treatment comprises altering cell membrane permeability.

24. The method as claimed in claim 23 wherein said permeability allows carbohydrate-based molecule to cross said cell membrane.

25. The method as claimed in claim 24 wherein said treatment is biopreservation.

26. The method as claimed in claim 24 wherein said permeability allows nucleic acids to cross said cell membrane.

27. The method as claimed in claim 26 wherein said treatment is DNA transfection.

28. The method as claimed in claim 17 wherein said treatment is cellular welding.

29. The method as claimed in claim 28 wherein said cellular welding is nerve welding.

30. The apparatus of claim 1 wherein said cellular properties are physical and/or chemical properties.

31. The method of claim 17 wherein said biological sample is one or more germ cells or an embryo.