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Total patents: more than 10k.
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Total patents: more than 10k.
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for invention","granted":true,"earliest_filing_date":"2010-10-29","grant_date":"2013-05-14","anticipated_term_date":"2031-03-03","has_disclaimer":false,"patent_status":"ACTIVE","publication_count":2,"has_spc":false,"has_grant_event":true,"has_entry_into_national_phase":false},"abstract":{"en":[{"text":"An apparatus for and method of performing multi-photon light sheet microscopy (MP-LISH), combining multi-photon excited fluorescence with the orthogonal illumination of light sheet microscopy are provided. With live imaging of whole Drosophila and zebrafish embryos, the high performance of MP-LISH compared to current state-of-the-art imaging techniques in maintaining good signal and high spatial resolution deep inside biological tissues (two times deeper than one-photon light sheet microscopy), in acquisition speed (more than one order of magnitude faster than conventional two-photon laser scanning microscopy), and in low phototoxicity are demonstrated. The inherent multi-modality of this new imaging technique is also demonstrated second harmonic generation light sheet microscopy to detect collagen in mouse tail tissue. Together, these properties create the potential for a wide range of applications for MP-LISH in 4D imaging of live biological systems.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"}]},"abstract_lang":["en"],"has_abstract":true,"claim":{"en":[{"text":"1. A multi-photon excitation light sheet microscope comprising: a sample holder; at least one excitation source, said excitation source being capable of producing an excitation beam having a power of adequate intensity to induce significant levels of multi-photon excitation in a sample, and being disposed such that the excitation beam is directed along at least one excitation beam path, wherein the at least one excitation beam path is provided with an excitation focusing optics for producing a substantially two-dimensional multi-photon excitation light sheet that defines a sample excitation region which extends along the excitation beam path and transversely thereto and intersects with at least a portion of the sample holder; an imaging detector defining at least one detection beam path and capable of detecting an excitation generated signal contrast from the sample excitation region, the detector being disposed such that the detection direction of the at least one detection beam path is substantially orthogonal to the direction of the excitation beam path; and wherein the multi-photon excitation light sheet is comprised of photons that each have an energy such that the sum of the energy of at least two of said photons is sufficient to cause multi-photon excitation and thereby excite a signal contrast in said sample excitation region, and wherein said signal contrast is proportional to I n , where I is the instantaneous intensity of the excitation energy at the sample excitation region and n is the number of said at least two photons that cause multi-photon excitation.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"2. The microscope as claimed in claim 1 , wherein the number of photons that cause multi-photon excitation light sheet is two.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"3. The microscope as claimed in claim 1 , wherein the excitation source is a pulsed near-infrared laser selected from the group consisting of lasers having pulse duration in the nanosecond, picosecond, and femtosecond range.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"4. The microscope as claimed in claim 1 , wherein the detected excitation generated signal contrast is selected from the group consisting of fluorescence, second harmonic generation, third harmonic generation, sum frequency generation, and stimulated Raman scattering.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"5. The microscope as claimed in claim 1 , wherein the excitation focusing optics comprises a spherical lens through which the excitation beam is focused, and wherein the excitation beam is laterally scanned along a desired axis of the excitation beam to form the light sheet, said light sheet having an effectively uniform excitation intensity across said excitation region.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"6. The microscope as claimed in claim 1 , wherein the excitation focusing optics comprises a cylindrical lens, and wherein the sample excitation region is created by statically focusing the excitation beam through said cylindrical lens.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"7. The microscope as claimed in claim 1 , wherein the at least one excitation source is capable of creating two coaxial and oppositely aligned excitation beams, such that said sample excitation region is formed from the non-coherent adjoining of the excitation regions created by each of the excitation beams.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"8. The microscope as claimed in claim 1 , wherein the numerical aperture of the excitation focusing optics is adjustable.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"9. The microscope as claimed in claim 8 , wherein the adjustable numerical aperture comprises a beam expander with an adjustable expanding ratio.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"10. The microscope as claimed in claim 1 , wherein focal volume engineering is applied to the excitation beam to optimize for light sheet imaging.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"11. The microscope as claimed in claim 10 , wherein focal volume engineering is implemented using one of the techniques selected from the group consisting of having the numerical aperture of the excitation focusing optics being anisotropic along at least two axes of said excitation beam, and having the excitation beam be a Bessel beam.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"12. The microscope as claimed in claim 10 , wherein the focal engineering is implemented by one or more optical elements selected from the group consisting of two orthogonally oriented sequential adjustable slit apertures, a plurality of independently expanding beam expanders, liquid crystal spatial light modulators, digital micromirror device spatial light modulators, and axiconic lens.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"13. The microscope as claimed in claim 1 , wherein the sample holder is moveable relative to the sample excitation region along or about at least one axis.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"14. The microscope as claimed in claim 1 , wherein the sample excitation region is moveable relative to the sample holder along or about at least one axis.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"15. The microscope as claimed in claim 1 , wherein the sample excitation region is one of either substantially planar-shaped or linearly-shaped.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"16. A method of imaging an object using a multi-photon excitation light sheet microscope comprising: producing a multi-photon excitation beam, and directing said excitation beam along an excitation beam path; focusing said excitation beam to produce a substantially two-dimensional multi-photon excitation light sheet that defines a sample excitation region, said sample excitation region being disposed to extend along the excitation beam path and transversely thereto; placing a sample within said sample excitation region to generate a signal contrast; detecting said signal contrast along a detection beam path that is substantially orthogonal to the excitation beam path; and wherein the multi-photon excitation light sheet has sufficient excitation intensity to produce significant levels of multi-photon excitation, the energy of said photons being selected such that cumulatively sufficient excitation energy is provided to excite a signal contrast in said sample excitation region, and wherein said signal contrast is proportional to I n , where I is the instantaneous intensity of the excitation energy at the sample excitation region and n is the number of photons involved in the multi-photon excitation.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"17. The method as claimed in claim 16 , wherein producing the excitation beam includes using a pulsed near-infrared laser selected from the group consisting of lasers having pulse durations in the nanosecond, picoseconds, and femtosecond range.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"18. The method as claimed in claim 16 , wherein detecting the signal contrast includes using a detection technique selected from the group consisting of 2-photon-excited fluorescence, second harmonic generation, third harmonic generation, sum frequency generation, and stimulated Raman scattering.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"19. The method as claimed in claim 16 , wherein the signal contrast comes from molecules and structures endogenous to a biological sample.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"20. The method as claimed in claim 16 , wherein the signal contrast comes from exogenous labels that are introduced into a biological sample.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"21. The method as claimed in claim 16 , wherein the signal contrast is second harmonic generation coming from nano-sized probes which have been introduced into a biological sample.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"22. The method as claimed in claim 16 , further comprising spherically focusing and laterally scanning said excitation beam along a desired axis of the excitation beam path to form the light sheet such that said light sheet has an effectively uniform excitation intensity across said excitation region.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"23. The method as claimed in claim 16 , further comprising forming at least two coaxial and oppositely aligned excitation beams, such that said sample excitation region is formed from the non-coherent adjoining of the excitation regions created by each of the said excitation beams.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"24. The method as claimed in claim 16 , wherein the focusing of the excitation beam further includes adjusting the numerical aperture of a focusing optic.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"25. The method as claimed in claim 16 , wherein the focusing of the excitation beam further includes anisotropically adjusting the numerical aperture of a focusing optic such that the excitation beam is anisotropic along at least two axes.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"},{"text":"26. The method as claimed in claim 16 , wherein the imaging is performed in one of either a 3D or 4D mode.","lang":"en","source":"USPTO_FULLTEXT","data_format":"ORIGINAL"}]},"claim_lang":["en"],"has_claim":true,"description":{"en":{"text":"CROSS-REFERENCE TO RELATED APPLICATIONS The current application claims priority to U.S. Provisional Application Nos. 61/256,010, and 61/256,005 both cases filed Oct. 29, 2009, the disclosure of which is incorporated herein by reference. STATEMENT OF FEDERAL FUNDING The federal government has rights to current invention pursuant to a funding provided in accordance with grant numbers EY018241 and HG004071, issued by the National Institutes of Health, and grant number DBI0852883, issued by the National Science Foundation. FIELD OF THE INVENTION The present invention relates generally to light sheet illumination microscopes and microscopy, and more specifically to a light sheet illumination microscope and microscopy technique that uses multi-photon excitation. BACKGROUND OF THE INVENTION Advanced optical microscopy techniques offer unique opportunities to investigate biological processes in vivo. The ability to image tissues or organisms in three dimensions (3D) and/or over time (4D imaging) permits a wide range of applications in neuroscience, immunology, cancer research, and developmental biology. (See, e.g., Mertz, Curr. Opin. Neurobiol. 14, 610-616, (2004); Kerr, J. N. D. & Denk, W., Nature Reviews Neuroscience 9, 195-205, (2008); Friedl, P., Current Opinion in Immunology 16, 389-393, (2004); Bousso, P., Current Opinion in Immunology 16, 400-405, (2004); Provenzano, P. P., et al., Trends in Cell Biology 19, 638-648, (2009); Keller, P. J., et al., Science 322, 1065-1069 (2008); McMahon, A., et al., Science 322, 1546-1550 (2008); and Mavrakis, M., et al., Development 137, 373-387, (2010), the disclosures of each of which are incorporated herein by reference.) Fundamental light-matter interactions, such as light scattering, absorption, and photo-induced biological toxicity, set the limits on the performance parameters of various imaging technologies in terms of spatial resolution, acquisition speed, and depth penetration (how deep into a sample useful information can be collected). Often, maximizing performance in any one of these parameters necessarily means degrading performance in the others. (See, e.g., Ji, N., et al., Curr. Opin. Neurobiol. 18, 605-616, (2008) and Vermot, J., et al., HFSP Journal 2, 143-155 (2008), the disclosures of each of which are incorporated herein by reference.) Such tradeoffs in performance are seen in comparing two current well-known 4D fluorescence imaging techniques of two-photon laser scanning microscopy (2p-LSM) and one-photon light sheet (1p-LISH) microscopy: 2p-LSM excels in achieving high depth penetration in scattering tissues, while 1p-LISH allows higher acquisition speed and lower phototoxicity. In 2p-LSM, the images are generated by raster scanning the sample with tightly-focused point of near-infrared (NIR) light, inducing 2p-excited fluorescence signal only at the focus spot and thus generating 3D resolution. (See, e.g. Denk, W., et al., Science 248, 73-76 (1990) and Zipfel, W. R., et al., Nat. Biotechnol. 21, 1369-1377 (2003), the disclosures of each of which are incorporated herein by reference.) Signal and spatial resolution are maintained significantly deeper into scattering samples compared with modalities that use 1-photon excitation (such as confocal laser scanning microscopy (CLSM)), due to (i) the low scattering of NIR light, and (ii) the efficient non-imaging detection where both ballistic and scattered fluorescence photons contribute to the signal (as the 3D resolution is achieved through confinement of the excitation alone). The acquisition speed of 2p-LSM is, however, limited since the image is collected one voxel at a time. 1p-LISH microscopy is a century-old technology that has seen much development and refinement in recent years, under names ranging from Orthogonal. Plane Fluorescence Optical Sectioning (OPFOS), Thin Laser light Sheet Microscopy (TLSM), Selective Plane Illumination Microscopy (SPIM) ( FIG. 1A , high-speed imaging of live zebrafish heart), Objective Coupled Planar Illumination (OCPI) ( FIG. 1B , high-speed calcium imaging of neurons), ultramicroscopy ( FIG. 1C , blood vessel system of mouse embryo), and Digital. Scanned Laser Light Sheet Fluorescence Microscopy (DSLM) ( FIG. 1D , in toto imaging of developing zebrafish embryo), among others. (See, e.g., Siedentopf, H. & Zsigmondy, R., Ann. Phys .- Berlin 10, 1-39 (1902); Voie, A. H., et al., J. Microsc .- Oxf. 170, 229-236 (1993); Fuchs, E., et al., Opt. Express 10, 145-154 (2002); Huisken, J., et al., Science 305, 1007-1009 (2004); Holekamp, T. F., et al., Neuron 57, 661-672 (2008); Dodt, H. U. et al., Nat. Methods 4, 331-336 (2007); Huisken, J. & Stainier, D. Y. R., Development 136, 1963-1975 (2009); and Keller, P. J. & Stelzer, E. H. K., Curr. Opin. Neurobiol. 18, 624-632 (2008), the disclosures of each of which are incorporated herein by reference.) In 1p-LISH, ( FIG. 1E ) a planar sheet of light is used to illuminate the sample, generating fluorescence signal over a thin optical section of the sample, which is then imaged from the direction orthogonal to the light sheet, with a wide-field imaging camera. Axial sectioning results from the thinness of the light sheet, while lateral resolution is determined by the detection optics. The orthogonal geometry between the illumination and detection pathways of 1p-LISH, compared to the collinear geometry of conventional microscopes, not only enables higher imaging speed due to the parallel image collection (millions of voxels can be imaged simultaneously), but also reduces phototoxicity since only a single focal plane of the sample is illuminated at a time. The depth penetration of 1p-LISH into scattering biological tissue, however, is limited (only slightly better than CLSM), due to (i) the imaging requirement of the wide-field detection that requires ballistic fluorescence photons only and scattered photons would degrade the image quality, and (ii) the light sheet is spatially degraded beyond its original thinness due to scattering, as it is focused deep inside an optically heterogeneous sample. Accordingly, it would be advantageous to develop an optical microscopy technique that strikes a new balance between the imaging performance of 2p-LSM and 1p-LISH capable of providing new imaging capabilities heretofore unobtainable with conventional microscopy techniques. SUMMARY OF THE INVENTION The present invention is directed to a multiple-photon excitation light sheet microscope, where the optical signal contrast is generated by an excitation process that involves multiple (more than one) photons. In such an embodiment, if S equals such optical signal, and l the excitation light intensity, then S is proportional to l^n, where n is equal to or greater than 2, for a process that involves n photons. For example, for a two-photon process, n=2, for a three-photon process, n=3. In one embodiment, the microscope includes an excitation laser source that provides sufficiently high light intensity to induce significant levels of multiple-photon excitation. In another embodiment, the excitation source is a pulsed laser producing radiation in the near-infrared wavelength range (approximately 0.7-1.4 microns), having a pulse duration in the range of nanosecond, picosecond, or femotosecond. In yet another embodiment, the detected signal contrast can be two-photon-excited fluorescence, second harmonic generation, third harmonic generation, sum frequency generation, and stimulated Raman scattering. In still another embodiment, the microscope includes excitation focusing optics for producing a substantially two-dimensional sample excitation region which extends in the direction of an excitation axis of the excitation beam path and transversely thereto and intersects with at least a portion of the sample holder. In yet another embodiment, the microscope includes an imaging detector disposed such that the detection direction is substantially orthogonal to the sample excitation region. In such an embodiment, the imaging detector may be a charged-coupled device camera, which is a detector that registers the optical signal simultaneously with an array (one or two-dimensioned) of light sensitive elements, capturing an image of the sample. In still yet another embodiment, the excitation focusing optics of the microscope includes a spherical lens through which the excitation beam is focused, and the excitation beam is laterally scanned along a desired axis of the excitation beam to form an effectively uniform excitation intensity across said excitation region. In still yet another embodiment, the excitation focusing optics of the microscope includes a cylindrical lens, and the sample excitation region is created by statically focusing the excitation beam through said cylindrical lens. In still yet another embodiment, the excitation source is capable of creating two coaxial and oppositely aligned excitation beams, such that said sample excitation region is formed from the overlap of the two excitation beams. In still yet another embodiment, the numerical aperture of the excitation focusing optics is adjustable. In one such embodiment, the adjustable numerical aperture includes a beam expander with an adjustable expanding ratio. In still yet another embodiment, a so-called focal-volume-engineering process is applied to the excitation beam to optimize the focal region for light sheet imaging. Such focal volume engineering can be implemented by optical devices such as adjustable slit apertures, beam expanders, spatial light modulators, and so on, operated separately or in tandem. In still yet another embodiment, as an example of the focal volume engineering that can be applied, the numerical aperture of the excitation focusing optics is anisotropic along at least two axes of said excitation beam. This could be obtained by having two sequential adjustable slit apertures oriented so that the slits are orthogonal to each other, or by a spatial light modulator. In still yet another embodiment, the excitation beam could be transformed, via an axiconic lens or a spatial light modulator, to have the so-called Bessel beam profile (instead of the standard Gaussian beam profile). A focused Bessel beam could provide a larger field of view for LISH imaging, for the same thickness at the center of the light sheet. In still yet another embodiment, the microscope includes a sample holder that is moveable relative to the sample excitation region along or about at least one axis. In still yet another embodiment, the microscope includes a sample excitation region that is moveable relative to the sample holder along or about at least one axis. In still yet another embodiment, the excitation region is one of either substantially planar-shaped or linearly-shaped. The invention is also directed to a method of imaging an object using a multiphoton light sheet microscope. BRIEF DESCRIPTION OF THE DRAWINGS These and other features and advantages of the present invention will be better understood by reference to the following detailed description when considered in conjunction with the accompanying drawings and data, wherein: FIGS. 1A to 1D provide images and data from experiments taken using: (A) SPIM; (B) ultramicroscopy; (C) OCPI; and (D) DLSM; FIG. 1E provides a schematic of a conventional 1p-LISH microscope; FIG. 2A provides an optical schematic of a line illumination scheme, contrasting the spatial extent of the emitted signals from one-photon and multiphoton excitation; FIG. 2B provides schematics of the excitation region of both LSM and LISH microscopes; FIGS. 3A to 3C provides images and data of fly data before gastrulation at stage 5 slices at different planes: (A1-5) of an embryo imaged using 2p-LISH with the ventral side facing the detection objective, (B) and the light sheet along the xy-plane entering the embryos from lateral sides, and (C) with automated segmentation of nuclei using standard software both on the ventral nuclei (C, up) and the lateral side (C, down); FIG. 4 provides a schematic of a MP-LISH microscope in accordance with the current invention; FIGS. 5A to 5C provide schematics of and data from a MP-LISH microscope using a scanned light sheet where: (A) shows a top view schematic of setup around the sample area, where (B) the illumination light sheet (xy-plane) is generated by fast laser scanning in the y-direction, and (C) shows 3D renderings of a live Drosophila embryo at stage 8 (left) and stage 13 (right) with nuclear labeling (H2A-GFP) imaged with 2p-LISH, showing the ventral and lateral views, where the scale bar=50 μm. FIGS. 6A & 6B provide: (A) schematic of the main components of the two photon light sheet microscopy setup; and (B) imaging performance parameters of the setup including maximum intensity projections of representative xz-slices of sub-diffraction fluorescent beads imaged by 2p-LISH (B1), 1p-LISH (B2), and 2p-LSM (B3), were the scale bar=3 μm; FIGS. 7A to 7I provide images and data comparing image depth penetration in stage 13 H2A-GFP Drosophila embryos imaged with 2p-LISH (A-C), 1p-LISH (D-F), and 2p-LSM (G-I), where the scale bar=50 μm; FIGS. 8A to 8F provide images and data comparing the image axial resolution in stage 5 H2A-GFP Drosophila embryos imaged with 2p-LISH (A-B), 1p-LISH (C-D), and 2p-LSM (E-F), where the scale bar=50 μm; FIG. 9 provides a schematic comparing the excitation and detection optical paths in 2p-LISH, 1p-LISH and 2p-LSM; FIGS. 10A to 10C provide images and data comparing the image depth penetration in 45 hpf H2A-Cerulean zebrafish embryos imaged with 2p-LISH (A), 1p-LISH (B), and 2p-LSM (C), where the scale bar=100 μm; FIGS. 11A to 11D provide images and data of an analysis of a heart beating from high-speed 2p-LISH imaging of a 5.4 day old zebrafish embryo (130 hpf), the fluorescence signal is averaged in a small area of the heart (white square in A) and plotted depending on time (B), the variation of signal during 1 s (C) shows the fast acquisition rate at 70 fps allows capturing fast features during each beat cycle, and the Fourier analysis of the time variation of the fluorescence signal over the 14 s-time interval (D), where the scale bar in a is 20 μm; and FIGS. 12A and 12B provide images and data of two-photon fluorescence from DAPI-stained nuclei (A), second harmonic generation (SGH) signal from collagen (B) of a mouse tail collected using the same light sheet microscopy setup, and a combined view of the two signals (C), where the scale bar=50 μm. DETAILED DESCRIPTION OF THE INVENTION The current invention is directed to a novel multi-photon light sheet (MP-LISH) microscope and microscopy technique. The technique and device use a multi-photon excitation light to generate signal contrast in the light sheet, thus exploiting both the nonlinear excitation to achieve high depth penetration and the orthogonal geometry of light sheet to achieve high acquisition speed and low phototoxicity. Conventional LISH Microscopy A schematic of a conventional 1p-LISH microscope is provided in FIG. 1E . As shown, LISH is a microscopy technique where the illumination is done from the side of the sample, creating a diffraction-limited planar sheet of light going across the sample. (See, J. Huisken and D. Y. R. Stainier, Development 136, 1963-1975 (2009), the disclosure of which is incorporated herein by reference.) Detection of the emitted light is done at 90 degrees from the illumination direction, orthogonal to the light sheet. Z-sectioning is achieved since only one diffraction-limited plane is illuminated at a time. The sample may be scanned through the plane (or inversely the plane could be scanned through the sample) to allow coverage of the whole sample volume. Axial resolution of a LISH microscope is determined by the thinnest of the sheet and the detection optics. Lateral resolution of a LISH microscope is determined by the detection optics alone. The imaging field of view of a LISH microscope is determined by the so-called confocal parameter of the illumination, defined as the distance about the focus spot where the sheet thickness remains less than the squareroot of 2 times its smallest value. LISH microscopy differs from laser or raster-point-scanning (LPS or RPS) microscopy in the geometry of the illumination and detection optical pathways. RPS microscopy, which is a widely used imaging technology, uses a collinear (parallel or anti-parallel) geometry between the illumination and detection pathways. (See, e.g., Pawley, Handbook of Confocal Microscopy, 3 rd Edition, New York: Springer (2006), the disclosure of which is incorporated herein by reference.) This results in some inherent advantages for LISH microscopy. In particular, because of the orthogonal geometry between the illumination and detection directions, the lateral extent of the illumination focus (together with the detection optics) determines the axial resolution of the final image. Compare this with conventional LSM, where the final axial resolution is determined by the axial extent of the illumination focus. Since for a given focusing NA, the focus spot is always smaller laterally than axially, 2p-LISH needs to employ a substantially smaller focusing NA than conventional 2p-LSM to reach the same axial resolution (by a factor of approximately 10), which carries important implications, as will be discussed in detail later. In addition, particularly for imaging 3D biological samples, illumination in LISH is limited only to the plane that is being imaged, hence reducing photobleaching and phototoxicity; detection is done in parallel for the whole plane, usually with a CCD camera, hence time acquisition is fast, usually about 10-20 times faster than the LSM technique. Because of these inherent advantages, conventional 1p-LISH has been the subject of intensive study, and the literature discloses many recent implementations of the conventional LISH technique. (See Huisken and Stainier, referenced above.) Some of these techniques include Orthogonal Plane Fluorescence Optical. Sectioning (OPFOS), Selective Plane Illumination Microscopy (SPIM), Ultramicroscopy, Digital Scanned Laser Light Sheet Fluorescence Microscopy (DSLM), etc. Although these different implementations have different specialized features, they have one common critical feature: the illumination is done with a sheet of light orthogonal to the detection direction. In the DSLM technique, the light sheet is synthesized by scanning, via a movable device such as a galvanometer mirror, a low-NA focused beam of light. Seen from the side through the detection objective, the focused beam of light appears as a line of light. At any time instant the sample is illuminated by only a line of illumination, which when summed over the scanned space and over time, yields an illuminated light sheet. As the light sheet travels into the samples, scattering (and/or refraction) causes the light to diffuse out, degrading the diffraction-limited quality of the illumination light. As the light sheet degrades, its spatial extent increases and its sharpness decrease. Since the emitted signal is proportional to the instantaneous light intensity l at the sample, when l is spatially spread out, so is the signal Hence, spatial resolution and signal contrast are degraded for thick samples. This phenomenon is illustrated in FIG. 2 , which provides an optical schematic of line illumination scheme, showing the spatial extent of the emitted signals a LISH excitation. The lens represents the illumination objective, component. The figure shows the view as seen by the detector, looking along the −z direction towards the sample. Solid focusing lines depict the light propagation if there were no scattering/refraction in the sample. The light gray area depicts the spatial extent of the emitted signals from one-photon excitation, as the light sheet is degraded and diffused out due to scattering/refraction. This establishes for LISH microscopy an excitation depth penetration, i.e. the depth into a sample beyond which the light sheet has degraded so much that no useful information can be collected. Also, since the samples, particularly biological ones, are usually nonhomogeneous, the effects of scattering/refraction cause the light sheet intensity to be nonhomogeneous, and thus introducing artifacts into the resulting detected image. Inventive MP-LISH Microscopy The current invention describes a LISH microscope that uses multi-photon excitation to generate signal contrast, thus exploiting both the nonlinear excitation to achieve high depth penetration and the orthogonal geometry of light sheet to achieve high acquisition speed and low phototoxicity. The literature discloses a technique that reduces the detrimental effect of scattering/refraction to imaging: multiphoton (MP) excitation. In standard single photon (SP) excitation, one photon of the illumination light interacts with the sample and gives rise to an emitted photon (usually in the form of fluorescence). In MP excitation, the excitation step involves n number of photons, where n is equal to or greater than 2. The multiple number of photons interact with the sample essentially simultaneously, and then give rise to emitted radiation, which could be in the form of fluorescence, second harmonic generation, third harmonic generation, etc. (See, J. Pawley, Handbook of Confocal Microscopy, 3 rd Edition, New York: Springer (2006), the disclosure of which is incorporated herein by reference.) For MP excitation, the excitation probability, and hence the emitted signal, is proportional to l^n, where l is the instantaneous intensity of the laser light at the sample. This can be contrasted with the SP case, discussed above, where the signal is proportional to l. Because of the l^n dependence of the emitted signal in MP excitation, the signal is confined to a small region of space where l^n is above a certain threshold. Thus, as the laser light undergoes scattering/refraction and diffuse out in space in the sample, the signal intensity would decrease, but the signal would still only comes from a small volume, hence preserving the resolution. This phenomenon is shown schematically in FIG. 2A , where the spatial extent of the emitted signal from multiphoton excitation is restricted to the dark gray region, even though the light beam is degraded and spread out beyond it, due to the spatial confining effect of multiphoton excitation processes. In addition, MP excitation typical uses illumination light in the near infrared range, with wavelengths longer than visible light that is normally used for SP excitation, thus scattering effects are reduced since the scattering cross-sections of biological molecules are inversely proportional to the wavelength to the m power, where m is a number greater than zero. Finally, the longer wavelengths of MP excitation are also generally less phototoxic for biological samples, since there is less endogenous absorption from biomolecules at these wavelengths. Because of these advantages, MP excitation has been applied to LSM microscopy, and has been confirmed to have the above-mentioned advantages over SP-LSM microscopy. (See, J. Pawley, (2006), cited above.) However, despite these inherent advantages, no attempt has ever been made to apply MP excitation to LISH, and all implementations of LISH up to date have used excitation light in the visible range, ˜400-700 nm, and relying on SP processes to create the detected emitted light. (Examples of these one-photon processes include 1-photon absorption and fluorescence and Raleigh scattering.) The reason for this lack of interest is that developing a light sheet microscope based on multi-photon excitation has, in the past, raised concerns about whether sufficient fluorescence signal can be generated without reaching phototoxic levels of laser power when using low-NA-focused excitation light. However, it has been surprisingly discovered that with a spherically-focused beam, the average excitation probability within the focal volume for 2p excitation is proportional to NA 4 , and the total focal volume is proportional to NA −4 , hence the total excitation (given by the excitation probability times the focal volume) is independent of the focusing NA for a homogeneous distribution of fluorophores. (See, Zipfel, W. R., et al., Nat. Biotechnol 21, 1369-1377 (2003), the disclosure of which is incorporated herein by reference.) Thus, it has now been determined that 2p-LISH and conventional 2p-LSM should have the same average signal rate in imaging an extended 3D sample, under the same average laser excitation power, spatial sampling density (voxel size), and detection efficiency. This counter-intuitive result could be understood in another way by noting that even though for 2p-LSM the instantaneous signal rate at each voxel is higher due to the tighter focusing, these voxels are illuminated one at a time, while in 2p-LISH an entire row of voxels is being illuminated and imaged simultaneously. (This is illustrated schematically in FIG. 2B .) Hence longer voxel exposure time could be used in 2p-LISH to compensate for the lower instantaneous signal rate, making the final signal rate equal for the two modalities. As will be described in greater detail below, this result was experimentally verified by imaging a live Drosophila embryo with 2p-LISH, using imaging parameters that are typically used for 2p-LSM (acquisition time of 1 sec for frame of 400×900 pixels, and total average power of 30 mW at the sample). The acquired images ( FIG. 3A ) have approximately the same signal quality as that obtained through 2p-LSM with similar imaging parameters and samples: for instance, the signal-to-noise ratio (SNR) is high enough to permit automated nuclear segmentation with standard image processing software ( FIG. 3B ). (See, Supatto, W., et al., Nature Protocols 4, 1397-1412 (2009), the disclosure of which is incorporated herein by reference.) Another reason that further makes developing a light sheet microscope based on multi-photon excitation a non-obvious choice is that, even if it is realized that the signal rate is the same in 2p-LISH as in 2p-LSM, already a non-obvious realization as described in the previous paragraph, it can still be argued then that precisely because of the same signal rate between the two imaging modalites, 2p-LISH would have minimal benefit over the already established and well-commercialized technique of 2p-LSM. That is, the same inherent signal rate means that with the same excitaiton laser power, 2p-LISH is expected to achieve the same, not higher, image acquisition speed as 2p-LSM. Thus, while 2p-LISH is expected to have an advantage over 1p-LISH in depth penetration (see discussion above), it seemingly is not expected to have any advantage over the existing technique of 2p-LSM (which already employs the same two-photon excitation mechanism and hence would have about the same depth penetration), and hence, not worthy for development. The advantage of 2p-LISH over 2p-LSM comes from its lower phototoxicity quality, which comes from the usage of lower focusing NA resulting in lower peak excitation intensity. Because of the lower phototoxicity of 2p-LISH compared to 2p-LSM (the reasons for which will be demonstrated and discussed further below), more laser excitation can be used in 2p-LISH than in 2p-LSM in imaging a live biological sample before the onset of phototoxicity, thus yielding a higher signal rate and hence higher acquisition speed. Thus, one of the key advances in developing 2p-LISH lies critically in realizing the low phototoxicity quality of 2p-LISH compared to the conventional technique of 2p-LSM. The same signal rate of 2p-LISH and 2p-LSM is a result specific to the 2-photon excitation process. If the excitation process involves more than 2 photons, then the signal rate of the LISH modality will have less signal rate than the LSM counterpart. For example, 3-photon-excited fluorecence LISH microscopy will have lower signal rate than 3-photon-excited fluorescence LSM. Then the advantage of 3p-LISH over 3p-LSM would be less than that of 2p-LISH over 2p-LSM. However, the low phototoxicity quality, due to the low focusing NA, of 2p-LISH is expected to be still valid for MP-LISH involving more than 2 photons. Thus, MP-LISH is still expected to be able to tolerate higher laser excitation power before the onset of phototoxicity as compared to MP-LSM, thus possibly the higher laser power could make up for the decreased signal rate, and thus still renders MP-LISH more advantageous over MP-LSM. Turning to the MP-LISH microscope itself, the apparatus of the current invention is shown schematically in FIG. 4 . As shown, the basic MP-LISH setup includes an illumination objective ( 5 ) positioned along a first axis of a sample chamber ( 4 ). The sample chamber is attached to a sample holder and controller ( 3 ), preferably allowing control in x,y,z, and theta (rotational). A detection objective ( 2 ) and imaging device, such as, for example, a camera ( 1 ) are positioned in line of sight to the sample chamber along a second axis that is orthogonal to the first illumination axis. Black solid lines depict the optical axis of the illumination and detection beams, going through ( 5 ) and ( 2 ), respectively. Not shown are the mechanical supports of the system. (For a detailed description of a LISH microscope see, US Pat. Pub. No. 2009/0225413, the disclosure of which is incorporated herein by reference.) Comparing FIGS. 1E and 4 it becomes clear that the apparatus for MP-LISH ( FIG. 4 ) is facially similar to that used for 1p-excited fluorescence DSLM ( FIG. 1 ). (See, Keller, P. J., et al., Science 322, 1065-1069 (2008), the disclosure of which is incorporated herein by reference.) However, there are several critical changes: provisions must be made to send in the pulsed laser light along the illumination path, this includes ensuring that all of the optical components are compatible with the pulsed laser light (e.g. mirrors should reflect enough of that particular wavelength of the pulsed laser light so that illumination intensity is high enough to induce a multi-photon process at the sample, or that the optical components are not damaged by the high energy/intensity of the pulsed laser beam.) In turn, the detection pathway must be modified to include the addition of extra short-pass optical filter(s) to screen out the illumination radiation, which has longer wavelengths than the emitted signal radiation. Finally, because in MP-LISH microscopy, the signal depends very sensitively on the illumination NA, attention must be paid to achieve the optimal illumination NA, so that the highest signal level is achieved with an acceptable field of view. In practice, it turns out that for the same samples, one would need to use higher NA for MP than for SP excitation, and relying on additional methods to increase the field of view, as will be discussed in greater detail below). As will be described in greater detail in the exemplary embodiments to follow, the use of MP excitation in LISH microscopy as described herein improves the performance of the LISH microscope, particularly to mitigate the detrimental effects of scattering/refraction in samples, biological or non-biological The MP excitation is used to produce the emitted radiation signals, which could be, but are not restricted to, fluorescence, second harmonic generation, third harmonic generation, sum frequency generation, and stimulated Raman scattering. In carrying out the MP excitation, any suitable source of excitation energy may be utilized, however, in a preferred embodiment pulsed lasers are used in order to achieve the high instantaneous intensities required to produce significant levels of emitted signals (which are proportional to l^n for MP excitation processes). The puked lasers can be of any suitable type, such as, for example, nanosecond, picosecond, or femtosecond-duration pukes. The shorter the puke, the lower the total laser energy is needed to achieve the same level of instantaneous intensity. In light of this, for biological samples, in order to minimize thermal damage, femtosecond pukes (with duration of hundreds of femtosecond or shorter) are preferred. In turn, picosecond and nanosecond pulses might be more appropriate for non-biological samples, where thermal damage is less of a concern. Scanned Light Sheet In carrying out the MP excitation, the scanned sheet (via scanning of a line illumination) (as described in the DLSM technique) is preferred over the static sheet illumination. (See, J. Huisken & D. Y. R. Stainier, Development 136, 1963-1975 (2009), the disclosure of which is incorporated herein by reference.) The smaller spatial extent of the line, as compared to the sheet, helps to achieve high instantaneous intensity while minimizing total light energy irradiated onto the sample. Although a static light sheet, as shown schematically in FIG. 1E may be used with the MP-LISH apparatus of the current invention, in the preferred embodiment, shown schematically in FIG. 5A , the illumination light sheet is created by the fast lateral scanning along the y direction of the spherically-focused laser light (red in FIG. 5A-B ), generating a scanned sheet along the xy-plane, perpendicularly to the z detection axis (green in FIG. 5A-B ). (See, Keller, P. J., et al., (2008), cited above.) (It should be noted that although the illumination is bidirectional in this figure, that a scanned light sheet may be generated in with a unidirectional excitation source. A more detailed examination of the advantages of bidirectional excitation sources will be provided below.) A scanned light sheet can be generated by fast scanning of the beam, with a period of 1 ms to cover the full FOV. This kHz-speed is fast enough to produce an effectively uniform illumination intensity across the y-extent of the FOV, for imaging exposure times of tens of ms or more. For shorter exposure times, faster scanning hardware could be employed (e.g. resonant scanners or spinning polygon mirrors can scan in the range of 10-100 kHz). In this embodiment, the lateral extent of the illumination focus spot determines the thickness of the scanned light sheet, while the confocal parameter of the focal region (the distance over which the lateral extent remains less than two times its smallest value) determines the imaging field of view. As will be described in greater detail below, the scanned sheet feature provides dramatic and unexpected improvements in imaging capabilities for the inventive MP-LISH over the conventional static sheet, which is typically produced by focusing through a cylindrical lens. (See, Huisken, J., et al., Science 305, 1007-1009 (2004) and Palero, J., et al., Opt. Express 18, 8491-8498 (2010), the disclosures of each of which are incorporated herein by reference.) An example of the performance of this 2p-LISH system in live imaging of Drosophila and zebrafish embryos is shown in FIG. 5C . The data illustrates the striking results that could be obtained with this new imaging modality, by showing multi-view 3D renderings of a live Drosophila embryo at two time points in its embryonic development, exhibiting fine spatial resolution achieved even deep inside the embryo, all done at high enough imaging speed and negligible phototoxicity so that fast cellular dynamics could be followed throughout the entire embryonic development. In short a two-fold increase was found in depth penetration compared with 1p-LISH, and more than an order of magnitude increase in imaging speed compared to 2p-LSM, allowing 70 frame-per-second imaging of the beating heart inside a 5.4-day-old zebrafish. A scanned sheet implementation of LISH microscopy is advantageous over a static sheet by achieving higher excitation power throughput, better spatial uniformity along y-dimension of the FOV, and allowing convenient execution of non-coherent structured illumination to improve signal contrast. (See, Keller, P. J., (2008); and Keller, P. J. et al., Nat. Methods 7, 637-(2010), the disclosures of each of which are incorporated herein by reference.) In addition, it has been recently demonstrated that the scanned light sheet minimizes scattering artifacts compared to the static light sheet illumination used in SPIM. (See, Fahrbach, F. O., et al., cited below.) All of these benefits of scanned sheet versus static sheet exist in both 1p- and 2p-LISH. For the case of 2p-LISH, the spherically-focused scanned light sheet further yields an additional critical benefit over the cylindrically-focused static light sheet, in producing significantly more nonlinearly-excited fluorescence signal for the same average excitation power. In fact, for the same excitation power, the signal rate from the scanned sheet is higher than from the static sheet by a factor equals to the ratio of field of view in y divided by sheet thickness, which is about 200 in our experimental implementation. This can be seen in the following analysis. Referring to the coordinate system definition in FIG. 6 and its inset described below, a comparison can be made between a scanned light sheet spherically focused to a certain characteristic width ω 0 in both the z- and y-directions, and a static light sheet cylindrically focused to the same width ω 0 in the z direction while being uniform in the y direction over a range of ω 0 , with N being a positive real number. ω 0 denotes the extent of the FOV in the y-direction (in the experimental implementation described below, yielding a field of view of about μm with ω 0 μm). The excitation intensity equals the average excitation power divided by the beam cross-sectional area, and thus is given by: where P is the same average excitation power used in both cases. The 2p-excited fluorescence signal rate, which is proportional to the squared of the intensity, averaged over the full extent of Nω 0 along the y-direction, is then: The reduction of the signal by the factor of (1/N) for the scanned sheet case in Eq. (3) above reflects the scanning that has to be done for the wide beam to cover the wide FOV along the y-direction, producing an effective spatial duty cycle of (1/N). It can be seen then from EQs. (3) and (4) that the average signal in the scanned sheet case is a factor of N larger than in the static sheet case, using the same excitation power. This comes directly from the quadratic dependence of the 2p-excited fluorescence signal on the excitation intensity. Since experimentally N is in the range of a few hundred, the difference between the two signals is significant. This substantially higher signal rate from the scanned sheet is critical in allowing optimized usage of the light power levels available from commercial laser sources in imaging of biological samples without inducing phototoxicity, as will be discussed further in the discussion section below. Alternative MP-LISH Embodiments Bidirectional Illumination: To increase the field of view of MP-LISH, the illumination may be done from opposite sides of the sample. A schematic of an MP-LISH apparatus including a bidirectional light source is provided in FIGS. 5A-B . As shown, in this embodiment, the illumination beams from the +x and −x directions are adjusted so that their fields of view slightly overlap at the center of the sample, effectively doubling the final field of view. In the literature it has been shown that bidirectional illumination would also improve SP-LISH, but in this case, because of the degraded light sheet, the illumination from opposite sides has to be done sequentially, then the two resulting images have to be merged computationally later to yield the final image (See, Huisken, J. & Stainier, D. Y. R., Opt. Lett. 32, 2608-2610 (2007), the disclosure of which is incorporated herein by reference.) In the case of MP-LISH, because the signal is spatially confined due to its l^n dependence, the illumination from opposite could be done concurrently, saving in time acquisition and complexity of data acquisition controls. The resulting image would then have about twice the field of view, with the same resolution and contrast as illuminated from one side at a time. Adjustable Illumination NA: The ability to adjust the NA of the illumination may be used to provide greater flexibility for the MP-LISH technique, since the signal depends quite sensitively to the NA, as described above. One way to achieve this is to have the illumination light go through a beam expander with adjustable expanding ratio, which then yields an adjustable illumination beam diameter, which in turns allow for fine-tuning the illumination NA. Focal Volume Engineering: Taking into consideration that in LISH microscopy (MP or SP), the lateral resolution of the captured image is determined by the detection optics, independent of the illumination NA; and for MP, the signal is proportional to l^n, it would be possible to engineer the spatial extent of the focal volume of the illumination light so that it is optimized for a particular sample. For example, an anisotropic NA could be used for the illumination to obtain more uniform signal profile in a scattering sample, effectively increasing the depth penetration. Referring to FIG. 2A , for a particular sample, a particular NA_z is used along the z-axis for the excitation, to meet whatever specification for axial resolution that is needed. If sheet illumination is used the NA along the x-direction would be NA_x˜0, and if standard line illumination is used NA_x=NA_z. Because of the scattering in the sample, and assuming that the center of the focal volume is significantly inside the sample, the light intensity has decreased significantly at the focal center, decreasing the signal contrast and thus also the excitation depth penetration. This scenario can be mitigated by using NA_x>NA_z. The stronger lateral focusing takes light energy away from the right side part of the sample, where the illumination first penetrates the sample, and put it more to the left towards the focal center, increasing the signal contrast in this deeper region, hence improving the signal uniformity over the entire sample and increasing the depth penetration. The larger NA_x illuminates more of the sample laterally away from the focal center, but does not degrade the detected lateral resolution, since that is solely determined by the detection optics. And, by increasing only NA_x, leaving NA_z unchanged, in trying to get more signal at the larger depth, the optimal axial (z) resolution may be maintained. Anisotropic NA could also be done with NA_xa sample holder;\n
at least one excitation source, said excitation source being capable of producing an excitation beam having a power of adequate intensity to induce significant levels of multi-photon excitation in a sample, and being disposed such that the excitation beam is directed along at least one excitation beam path, wherein the at least one excitation beam path is provided with an excitation focusing optics for producing a substantially two-dimensional multi-photon excitation light sheet that defines a sample excitation region which extends along the excitation beam path and transversely thereto and intersects with at least a portion of the sample holder;\n
an imaging detector defining at least one detection beam path and capable of detecting an excitation generated signal contrast from the sample excitation region, the detector being disposed such that the detection direction of the at least one detection beam path is substantially orthogonal to the direction of the excitation beam path; and\n
wherein the multi-photon excitation light sheet is comprised of photons that each have an energy such that the sum of the energy of at least two of said photons is sufficient to cause multi-photon excitation and thereby excite a signal contrast in said sample excitation region, and wherein said signal contrast is proportional to In, where I is the instantaneous intensity of the excitation energy at the sample excitation region and n is the number of said at least two photons that cause multi-photon excitation."],"number":1,"annotation":false,"title":false,"claim":true},{"lines":["The microscope as claimed in claim 1, wherein the number of photons that cause multi-photon excitation light sheet is two."],"number":2,"annotation":false,"title":false,"claim":true},{"lines":["The microscope as claimed in claim 1, wherein the excitation source is a pulsed near-infrared laser selected from the group consisting of lasers having pulse duration in the nanosecond, picosecond, and femtosecond range."],"number":3,"annotation":false,"title":false,"claim":true},{"lines":["The microscope as claimed in claim 1, wherein the detected excitation generated signal contrast is selected from the group consisting of fluorescence, second harmonic generation, third harmonic generation, sum frequency generation, and stimulated Raman scattering."],"number":4,"annotation":false,"title":false,"claim":true},{"lines":["The microscope as claimed in claim 1, wherein the excitation focusing optics comprises a spherical lens through which the excitation beam is focused, and wherein the excitation beam is laterally scanned along a desired axis of the excitation beam to form the light sheet, said light sheet having an effectively uniform excitation intensity across said excitation region."],"number":5,"annotation":false,"title":false,"claim":true},{"lines":["The microscope as claimed in claim 1, wherein the excitation focusing optics comprises a cylindrical lens, and wherein the sample excitation region is created by statically focusing the excitation beam through said cylindrical lens."],"number":6,"annotation":false,"title":false,"claim":true},{"lines":["The microscope as claimed in claim 1, wherein the at least one excitation source is capable of creating two coaxial and oppositely aligned excitation beams, such that said sample excitation region is formed from the non-coherent adjoining of the excitation regions created by each of the excitation beams."],"number":7,"annotation":false,"title":false,"claim":true},{"lines":["The microscope as claimed in claim 1, wherein the numerical aperture of the excitation focusing optics is adjustable."],"number":8,"annotation":false,"title":false,"claim":true},{"lines":["The microscope as claimed in claim 8, wherein the adjustable numerical aperture comprises a beam expander with an adjustable expanding ratio."],"number":9,"annotation":false,"title":false,"claim":true},{"lines":["The microscope as claimed in claim 1, wherein focal volume engineering is applied to the excitation beam to optimize for light sheet imaging."],"number":10,"annotation":false,"title":false,"claim":true},{"lines":["The microscope as claimed in claim 10, wherein focal volume engineering is implemented using one of the techniques selected from the group consisting of having the numerical aperture of the excitation focusing optics being anisotropic along at least two axes of said excitation beam, and having the excitation beam be a Bessel beam."],"number":11,"annotation":false,"title":false,"claim":true},{"lines":["The microscope as claimed in claim 10, wherein the focal engineering is implemented by one or more optical elements selected from the group consisting of two orthogonally oriented sequential adjustable slit apertures, a plurality of independently expanding beam expanders, liquid crystal spatial light modulators, digital micromirror device spatial light modulators, and axiconic lens."],"number":12,"annotation":false,"title":false,"claim":true},{"lines":["The microscope as claimed in claim 1, wherein the sample holder is moveable relative to the sample excitation region along or about at least one axis."],"number":13,"annotation":false,"title":false,"claim":true},{"lines":["The microscope as claimed in claim 1, wherein the sample excitation region is moveable relative to the sample holder along or about at least one axis."],"number":14,"annotation":false,"title":false,"claim":true},{"lines":["The microscope as claimed in claim 1, wherein the sample excitation region is one of either substantially planar-shaped or linearly-shaped."],"number":15,"annotation":false,"title":false,"claim":true},{"lines":["A method of imaging an object using a multi-photon excitation light sheet microscope comprising:\n
producing a multi-photon excitation beam, and directing said excitation beam along an excitation beam path;\n
focusing said excitation beam to produce a substantially two-dimensional multi-photon excitation light sheet that defines a sample excitation region, said sample excitation region being disposed to extend along the excitation beam path and transversely thereto;\n
placing a sample within said sample excitation region to generate a signal contrast;\n
detecting said signal contrast along a detection beam path that is substantially orthogonal to the excitation beam path; and\n
wherein the multi-photon excitation light sheet has sufficient excitation intensity to produce significant levels of multi-photon excitation, the energy of said photons being selected such that cumulatively sufficient excitation energy is provided to excite a signal contrast in said sample excitation region, and wherein said signal contrast is proportional to In, where I is the instantaneous intensity of the excitation energy at the sample excitation region and n is the number of photons involved in the multi-photon excitation."],"number":16,"annotation":false,"title":false,"claim":true},{"lines":["The method as claimed in claim 16, wherein producing the excitation beam includes using a pulsed near-infrared laser selected from the group consisting of lasers having pulse durations in the nanosecond, picoseconds, and femtosecond range."],"number":17,"annotation":false,"title":false,"claim":true},{"lines":["The method as claimed in claim 16, wherein detecting the signal contrast includes using a detection technique selected from the group consisting of 2-photon-excited fluorescence, second harmonic generation, third harmonic generation, sum frequency generation, and stimulated Raman scattering."],"number":18,"annotation":false,"title":false,"claim":true},{"lines":["The method as claimed in claim 16, wherein the signal contrast comes from molecules and structures endogenous to a biological sample."],"number":19,"annotation":false,"title":false,"claim":true},{"lines":["The method as claimed in claim 16, wherein the signal contrast comes from exogenous labels that are introduced into a biological sample."],"number":20,"annotation":false,"title":false,"claim":true},{"lines":["The method as claimed in claim 16, wherein the signal contrast is second harmonic generation coming from nano-sized probes which have been introduced into a biological sample."],"number":21,"annotation":false,"title":false,"claim":true},{"lines":["The method as claimed in claim 16, further comprising spherically focusing and laterally scanning said excitation beam along a desired axis of the excitation beam path to form the light sheet such that said light sheet has an effectively uniform excitation intensity across said excitation region."],"number":22,"annotation":false,"title":false,"claim":true},{"lines":["The method as claimed in claim 16, further comprising forming at least two coaxial and oppositely aligned excitation beams, such that said sample excitation region is formed from the non-coherent adjoining of the excitation regions created by each of the said excitation beams."],"number":23,"annotation":false,"title":false,"claim":true},{"lines":["The method as claimed in claim 16, wherein the focusing of the excitation beam further includes adjusting the numerical aperture of a focusing optic."],"number":24,"annotation":false,"title":false,"claim":true},{"lines":["The method as claimed in claim 16, wherein the focusing of the excitation beam further includes anisotropically adjusting the numerical aperture of a focusing optic such that the excitation beam is anisotropic along at least two axes."],"number":25,"annotation":false,"title":false,"claim":true},{"lines":["The method as claimed in claim 16, wherein the imaging is performed in one of either a 3D or 4D mode."],"number":26,"annotation":false,"title":false,"claim":true}]}},"filters":{"npl":[],"notNpl":[],"applicant":[],"notApplicant":[],"inventor":[],"notInventor":[],"owner":[],"notOwner":[],"tags":[],"dates":[],"types":[],"notTypes":[],"j":[],"notJ":[],"fj":[],"notFj":[],"classIpcr":[],"notClassIpcr":[],"classNat":[],"notClassNat":[],"classCpc":[],"notClassCpc":[],"so":[],"notSo":[],"sat":[]},"sequenceFilters":{"s":"SEQIDNO","d":"ASCENDING","p":0,"n":10,"sp":[],"si":[],"len":[],"t":[],"loc":[]}}