Abstract:
Methods and systems are described using a non-linear optical system comprising a laser and a light delivery system comprising a single mode fiber, a mode converter, and a high order mode fiber, wherein the light delivery system that receives light from the source and provides a structured free-space beam having an embedded Gaussian beam. The light delivery system functions to illuminate a region of a sample and generate a non-linear response in a spatial region smaller than that associated with a Gaussian beam having a width comparable to the width of the embedded Gaussian beam. In another aspect, the light delivery system illuminates a region of a sample and generates a non-linear emission of radiation, is depicted. A further aspect of this embodiment includes an imaging assembly for detecting the non-linear emission and using a signal derived from the detected emission to generate a microscopic image of the sample.

Description:
CROSS REFERENCE TO RELATED APPLICATION 
       [0001]    The present application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 61/264,538, filed on Nov. 25, 2009, and U.S. Provisional Patent Application Ser. No. 61/265,271, filed on Nov. 30, 2009. These applications are incorporated by reference herein in their entirety. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    The present invention relates generally to non-linear optical systems, and in particular to improved non-linear optical systems and techniques employing higher-order mode optical fibers. 
         [0004]    2. Background Art 
         [0005]    In a non-linear optical system; such as a non-linear microscopy system, a pulsed laser beam is tightly focused onto a sample, causing an optical output to be generated therefrom. A non-linear signal can be derived from the optical output, and this non-linear signal can be used to generate a microscopic image of the sample. A number of different higher-order light-matter interactions may be used in a non-linear optical system, including two-photon fluorescence, second-harmonic generation, third-harmonic generation, Raman scattering, and the like. In a multiphoton emission process, the relationship between incident light intensity and emitted radiation is nonlinear. For example, for two-photon excitation, the relationship is quadratic. As a result of this nonlinear relationship, only the central spatial portion of a conventional Gaussian beam substantially contributes to the intensity of emitted radiation. Therefore, much more multiphoton radiation is generated where the laser beam is tightly focused than where it is more diffuse. Effectively, excitation is restricted to the focal volume, resulting in a high degree of rejection of out-of-focus objects Similar effects occur in other types of light-matter interactions, including second-harmonic generation, third-harmonic generation, Raman scattering, optically-induced chemical reactions, material breakdown and the like. 
         [0006]      FIG. 1A  is a diagram illustrating the basic principles of operation of an exemplary non-linear microscopy system  20  according to the prior art. A femtosecond laser  22  provides incident light in the form of a pulsed laser output that is guided by a single-mode fiber (SMF)  24  having an end face  26  that provides the pulsed laser beam  28  as a free-space output. The laser beam  28  is directed by a scanning mirror assembly  30  to an objective lens  32  that focuses the beam onto a sample  34  for which a microscopic image is to be generated. The laser beam  28  has an intensity that is sufficient to cause multiphoton excitation of fluorophores in an excitation volume of the sample. Fluorescence  36  is emitted having an intensity level indicating the amount of multiphoton excitation for example two-photon fluorescence. 
         [0007]    The number of photons required for excitation depends upon the particular type of light-matter interaction used to create fluorescence. In the present discussion, microscopy system  20  is assumed to use two-photon excitation. However, it will be appreciated that the present discussion applies to non-linear microscopy employing other types of light-matter interactions, including second-harmonic generation, third-harmonic generation, Raman scattering, and the like. 
         [0008]    The emitted fluorescence is detected by a suitable detector  38 , such as a photodiode, photomultiplier, or like device. Scanning the focused laser beam  28  over a region of the sample allows point-by-point intensity data to be gathered. Alternatively, the position of the beam could be kept fixed and the sample scanned in position with respect to the beam. An image generator  40  then uses the intensity data to generate a microscopic image of the scanned sample region. 
         [0009]      FIG. 1B  shows a diagram of a scanning confocal non-linear microscopy system  50  according to the prior art. Like the microscopy system shown in  FIG. 1A , system  50  includes a femtosecond laser  52  and a single mode fiber  54  having an end face  56  that provides a pulsed laser output  58 . A scanning mirror assembly  60  directs the laser output  58  to an objective lens  62  that tightly focuses it onto a sample  64 , thereby resulting in multiphoton excitation of fluorophores in an excitation volume and the emission of fluorescence  66 . 
         [0010]    The  FIG. 1B  microscope includes an output pinhole assembly  68 , which is provided by a using a detector lens  70  to focus the emitted fluorescence and provide it as an input into a second single-mode fiber (SMF)  72 , which in turn guides the focused fluorescence to a detector  74 , which provides fluorescence intensity data to image generator  76 . The use of pinhole assembly  68  adds depth (i.e., axial) resolution to the microscopy system  50 , as only signals generated in the focus of the incident light beam can efficiently couple into the second SMF  70  for measurement by the detector  72 . With this setup, a three-dimensional non-linear image of a sample can be obtained by scanning the sample transversely, as well as axially. 
         [0011]    In the microscopy systems shown in  FIGS. 1A and 1B , the pulsed laser beam used to provide incident light to the sample is guided from the laser to the objective lens using the fundamental LP 01  transverse mode, which has a near-Gaussian shape. In both systems  20 ,  50 , the single-mode fiber  24 ,  54  that guides the laser beam to the objective lens does not support propagation in higher-order modes, which have distinctly non-Gaussian shapes. 
         [0012]    Because of its near-Gaussian shape, an LP 01  mode laser beam can be focused to a narrower beam width than a higher-order mode laser beam. For this reason, current non-linear microscopy systems have used an LP 01  mode laser beam to provide incident excitation light. The localization of excitation in a non-linear optical system typically results in significantly higher spatial resolution than that achievable in a linear optical system. However, it would be desirable to improve the performance of microscopy systems even further. In particular, it would be desirable to find ways to enhance the signal resolution in multiple dimensions. 
       SUMMARY OF THE INVENTION 
       [0013]    Aspects of the invention are directed to improved non-linear optical systems and techniques. An embodiment of the present invention depicts a non-linear optical system comprising a source, such as a laser, and a light delivery system comprising a single mode fiber, a mode converter, and a high order mode fiber, wherein the light delivery system that receives light from the source and provides a structured free-space beam having an embedded Gaussian beam, wherein the embedded Gaussian beam has a width. The light delivery system functions to illuminate a region of a sample and generate a non-linear response in a spatial region that is smaller than a spatial region that would be obtained with a Gaussian beam having a width comparable to the width of the embedded Gaussian beam. Such non-linear responses may include, for example, second-harmonic generation or multi-photon material modification. Because of the structured nature of the beam, the spatial extent over which the non-linear effect is present is smaller than would be achieved with a Gaussian shaped beam. 
         [0014]    According to another embodiment of the present invention, a non-linear optical system comprising a source, such as a laser, and a light delivery system that receives light from the source and provides a structured free-space beam, wherein the light delivery system illuminates a region of a sample and generates a non-linear emission of radiation, is depicted. A further aspect of this embodiment includes an imaging assembly for detecting the non-linear emission and using a signal derived from the detected emission to generate a microscopic image of the sample. 
         [0015]    According to another aspect of the invention, a long-period grating is used as a mode converter. According to a further aspect of the invention, the higher-order mode of the laser light is created within an optical waveguide, while in another it is created outside of the waveguide using bulk-optic elements. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0016]      FIGS. 1A and 1B  are drawings of exemplary non-linear microscopy systems according to the prior art. 
           [0017]      FIG. 2A  is a graph comparing the respective intensity profiles of the LP 01  and LP 02  modes, and  FIG. 2B  is a graph comparing the squared intensity profiles of the LP 01  and LP 02  modes. 
           [0018]      FIGS. 3A and 3B  are diagrams of exemplary non-linear microscopy systems according to aspects of the present invention. 
           [0019]      FIGS. 4 and 5  are a pair of diagrams illustrating testing setups for comparing the resolution achievable by a non-linear microscopy system using the LP 01  and LP 02  modes. 
           [0020]      FIGS. 6A-6C  are a series of graphs showing the theoretical signals expected based on the computed intensity profiles for the LP 01  and LP 02  modes. 
           [0021]      FIGS. 7A and 7B  are graphs showing experimentally measured curves for a linear detector and a second-order non-linear detector. 
           [0022]      FIGS. 8A and 8B  are a pair of diagrams of an experimental setup for comparing the confocal resolution of the LP 02  mode to that of the LP 01  mode. 
           [0023]      FIGS. 9A and 9B  are a pair of graphs showing the measured confocal signal for a linear detector and a second-order non-linear detector. 
           [0024]      FIG. 10  is a diagram illustrating a general example of a non-linear microscopy system according to aspects of the invention. 
           [0025]      FIG. 11  is a flowchart illustrating a general example of a non-linear microscopy technique according to aspects of the invention. 
       
    
    
     DETAILED DESCRIPTION 
       [0026]    Although the detailed description describes systems primarily surrounding non-linear microscopy, one of ordinary skill will appreciate the scope of the invention encompasses other non-linear optical systems and techniques employing higher-order mode optical fibers. 
         [0027]    In the case of a non-linear microscope, a higher-order mode excitation light can provide superior resolution compared with a fundamental LP 01  mode incident excitation light. The superior resolution arises from the respective shapes of the intensity profile of the higher-order mode or modes compared to the profile of the fundamental mode, and from the non-linear relationship between incident light intensity and fluorescence. 
         [0028]    Aspects of the present invention are described in the context of using the LP 01  and LP 02  modes used to provide excitation light in a two-photon non-linear microscope. However, it will be appreciated that the following discussion may be extended to other higher-order modes and other types of non-linear microscopes. In addition, it should be noted that creation and detection of fluorescence is but one example of an application that exploits the high spatial confinement of a high intensity region of the laser light. The confined laser light may be used to induce any number of non-linear effects, such as promotion of a multiphoton chemical reaction or material breakdown. As used herein, systems used to implement such applications are generally referred to as “non-linear optical systems.” 
         [0029]    The present discussion makes use of the M 2  parameter and the concept of an “embedded Gaussian” beam. The M 2  parameter is a measure of beam quality, i.e., the ability to be focused to a tight spot. The lowest possible value for M 2  is 1, which corresponds to a Gaussian beam. For a given light beam, the M 2  parameter is determined based upon the beam&#39;s divergence angle and width at its narrowest point, and quantifies how many times diffraction-limited the given beam is compared with a Gaussian beam. 
         [0030]    Even higher-order modes, such as the LP 02 , LP 03 , and LP 04  modes, have a non-Gaussian shape that includes a central lobe and one or more outer rings. In determining the M 2  values for these shapes, beam width is defined as the second moment of the transverse intensity distribution. 
         [0031]    The “embedded Gaussian” beam is a useful concept for understanding how non-Gaussian beams propagate through optical systems. If a Gaussian beam propagating through an optical system has a position-dependent beam width, ω(z), then it is possible to express the beam width of a non-Gaussian beam propagating through the same optical system, and having a known M 2  parameter, as M·ω(z), i.e., the product of the position-dependent beam width ω(z) and M, the square root of the M 2  value. 
         [0032]    Using this concept, it will be seen that for a non-Gaussian beam, comparing its performance to the embedded Gaussian beam performance gives a measure of the resolution enhancement available to non-Gaussian beams. 
         [0033]      FIG. 2A  shows a graph  100 , in which curve  101  shows the intensity profile for the LP 01  mode and curve  102  shows the intensity profile for the LP 02  mode. The two profiles have been scaled to have the same embedded Gaussian beam-width ω(z) to provide a direct visual comparison between them. The LP 01  mode intensity profile closely approximates a Gaussian shape, with a single central lobe  101   a . The LP 02  curve is characterized by a non-Gaussian shape having a central lobe  102   a  and an outer ring  102   b.    
         [0034]    The LP 01  mode has an M 2  value of just over 1, reflecting its near-Gaussian shape. The LP 02  mode has a non-Gaussian energy distribution. Using the second moment definition of beam width, the M 2  value for the LP 02  mode shown in  FIG. 2A  is approximately 3, and the M value is approximately 1.7. 
         [0035]    In a two-photon microscope, the relationship between incident light intensity and fluorophore excitation is quadratic. Thus, in comparing the respective resolutions achievable in a two-photon microscope using the LP 01  mode and the LP 02  mode, it is necessary to square their respective intensity profiles. The unsquared LP 01  and LP 02  mode intensity profiles can be viewed as indicating the probability that a single photon will be present at a given location at a given time. Thus, the square of the respective intensity profiles can be viewed as indicating the probability that two photons will be simultaneously present at a given location. 
         [0036]      FIG. 2B  shows a graph  110  in which curve  111  shows the squared intensity profile for the LP 02  mode and curve  112  shows the squared intensity profile for the LP 02  mode. After squaring, the LP 02  mode outer ring  112   b  has been substantially suppressed, and the central lobe  112   a  is now dominant. 
         [0037]    The dominant central lobe  112   a  of the squared LP 02  mode intensity profile  112  is significantly narrower that the central lobe  111   a  of the squared LP 01  mode intensity profile  111 . Thus, even though the beam width of the LP 02  mode is significantly greater that the LP 01  mode beam width, the squared mode intensity profiles indicates that, in a two-photon microscope, the LP 02  mode provides superior resolution. 
         [0038]      FIG. 3A  shows a schematic of an exemplary two-photon non-linear microscopy system  120  according to an aspect of the invention, in which a higher-order mode laser beam is used to provide incident excitation light. In the  FIG. 3A , microscopy system  120  comprises a laser light source  122 , such as a femtosecond laser, to provide a pulsed laser output that is initially guided by a single-mode fiber SMF  124 . Alternate light sources may use longer pulses or even generate continuous wave emission, though higher peak power is typically attained using shorter pulses. A long-period grating (LPG)  126  is connected to an output end of the SMF  124  and provides efficient excitation of a desired higher-order mode of the laser output. In the described practice of the invention, LPG  126  is preferably written directly into the HOM fiber  128 . It should be noted that, generally speaking, it would also be possible for LPG  126  to be written into a separate fiber, which is then spliced to the HOM fiber  128 . In the present example, the LPG  126  provides excitation of the LP 02  mode in a higher-order mode (HOM) fiber  128 . As discussed below, it would also be possible, to use other higher-order modes, including for example the LP 03  and LP 04  modes. Typically, even modes are desired due to their high localized peak intensity at the centerline. 
         [0039]    In addition, since light outside of the central region does not contribute to non-linear interactions, it is beneficial to use low-order higher-order modes since a larger fraction of the total power is present in the central lobe. In contrast, in so-called “Bessel beams,” optical power is distributed to many concentric rings, reducing the relative power carried in the center compared to the rings. In low-order modes, a greater fraction of the total power is carried in the central lobe, which is desirable for many nonlinear interactions. Thus, as used herein, a “higher-order mode of low order” refers to a fiber-guided higher-order mode having a central lobe, and which is commonly characterized as an LP 0n  mode, where n is less than 5. 
         [0040]    The HOM fiber  128  has an end face  130  from which the higher-order mode laser light is emitted as a structured free-space beam output  132  According to another aspect of the invention, the higher-order mode may be created after the laser light exits the waveguide using bulk-optic elements, such as axicons, phase plates, spatial light modulators, and the like. However, one advantage of a waveguide-based mode converter is the excitation of a specific mode or modes with little light coupled into unwanted modes compared to using bulk-optic elements. This allows for more efficient utilization of optical power. For the purposes of the present description, a beam exiting a waveguide in which the beam propagates as the LP 02  mode is referred to as an “LP 02 -structured beam.” LP 02 -structured beam output  132  is directed by a scanning mirror assembly  134  to an objective lens  136  that tightly focuses the laser light  132  onto a sample  138 . Fluorophores in the sample  138  that simultaneously absorb two photons enter into an excitation state, resulting in the emission of fluorescence  140  having an intensity indicating the amount of two-photon excitation occurring within the excitation volume. The focused laser light is scanned over a selected region of the sample  138 , thereby providing point-by-point emitted fluorescence intensity data over the scan region. The emitted fluorescence is detected by a suitable detector  142 , such as a photodiode or the like. An image generator  144  uses the detected signal to generate a microscopic image of the scanned sample region. 
         [0041]      FIG. 3B  shows a scanning confocal two-photon microscopy system  150  according to a further aspect of the invention. Similar to the  FIG. 3A  microscopy system  120 , the  FIG. 3B  microscopy system  150  includes a femtosecond laser  152 , a single-mode fiber (SMF)  154 , a long-period grating  156 , and a higher-order mode (HOM) fiber  158  having an output end face  160  that provides an LP 02  mode pulsed laser beam  162  as an output. Laser beam  162  is guided by a scanning mirror assembly  164  and focused by an objective lens  166  onto a sample  168 , resulting in the emission of two-photon fluorescence  170 . 
         [0042]    The  FIG. 3B  microscopy system  150  further includes an output pinhole assembly  172 , comprising a detector lens  174 , a second single-mode fiber  176  having an input end face  178  and a second long-period grating  180  written into the second single-mode fiber  176 . The detector lens  174  focuses emitted fluorescence  170  onto end face  178 . This fluorescence is then guided by the second single-mode fiber  176  to the second long-period grating  180 . The focused emitted fluorescence is then guided to a detector  182 . An image generator  184  then uses the fluorescence data to generate a microscopic image of the scanned sample region. 
         [0043]    The output pinhole assembly  172  adds depth (i.e., axial) resolution to the microscopy system  150 , as only signals generated in the focus of the beam can efficiently couple into the second single-mode fiber  176  for measurement by the detector. With this setup, three-dimensional non-linear images of samples can be obtained by scanning the sample both transversely and axially. 
         [0044]    It should be noted that under non-linear excitation, two-photon fluorescence, or any emission induced by non-linear optical processes, will not look like a higher-order mode, but more like the fundamental mode, because the center peak is emphasized in the non-linear signal, as shown in  FIG. 2B . Therefore, for a non-linear confocal microscope based on an HOM fiber, the output pigtail should not be an NOM fiber, but rather a single-mode fiber. In a linear confocal microscope, the output fiber would still have to be an HOM fiber. 
         [0045]    The use of NOM fiber  128 ,  158 , in the  FIGS. 3A and 3B  microscopy systems improves both transverse resolution in a non-linear microscope as well as confocal resolution. HOM fiber is attractive in this application for a number of reasons, including its compatibility with an all-fiber setup, and its compatibility with femtosecond pulse delivery. 
         [0046]    The high dispersion of the HOM fiber enables better pulse delivery than SMF fiber. In particular, the dispersive properties of the HOM fiber can be engineered to compress or stretch the pulse during propagation, resulting in a more desirable pulse shape incident on the sample. Long-period grating technology enables efficient excitation of the desired higher-order mode and can also provide wavelength selectivity due to the resonant nature of the mode conversion. There are numerous examples of mode converters in the art, including those created using long-period gratings in waveguides. The spike-shaped central lobe of the squared LP 02  mode intensity profile, discussed above, enables increased transverse resolution in a non-linear microscope compared to the LP 01  mode. The high M 2  value of the LP 02  mode (approximately 3) enables increased confocal resolution compared to the LP 01  mode. 
         [0047]      FIGS. 4 and 5  are a pair of schematic diagrams illustrating testing setups  240 ,  270  that are designed to compare the resolution of a non-linear microscopy system using an LP 01  mode to one using an LP 02  mode. 
         [0048]    In the  FIG. 4  setup  240 , a pulsed laser beam from a 1550 nm femtosecond erbium laser  242  propagates in the LP 01  mode of a single-mode fiber  244 . In the  FIG. 5  setup  270 , a pulsed laser beam from a 1550 nm femtosecond erbium laser  272  is guided into a long-period grating  273  that excites the LP 02  mode of a higher-order mode fiber  274 . 
         [0049]    In both testing setups  240 ,  270 , the fiber laser output  246 ,  276  is collimated by a first lens  248 ,  278 . The collimated light  250 ,  280  is then focused by a second lens  252 ,  282 . The focus  254 ,  284  is then imaged by a third lens  256 ,  286  onto a suitable linear or non-linear detector  258 ,  288 , such as an indium gallium arsenide (InGaAs) photodiode, a silicon (Si) photodiode, or the like, that produces a two-photon photocurrent. 
         [0050]    A knife edge  260 ,  290  is translated through the focus  254 ,  284  of the beam and the amount of photocurrent on the detector  258 ,  288  is measured as a function of the knife edge position. The steepness of this curve versus position is a measure of the resolution of the microscope. 
         [0051]    The lenses are chosen such that both beams have the same embedded Gaussian width in the collimated region  250 ,  280  just before the focusing lens  252 ,  282 . This ensures that at the beam focus.  284  the LP 02  beam is approximately M times larger in width than the LP 01 , where the M 2  of the LP 02  beam is approximately 3. 
         [0052]      FIGS. 6A-6C  are a series of graphs  300 ,  310 ,  320  showing the theoretical signals expected based on the computed intensity profiles for the LP 01  and LP 01  modes. 
         [0053]    The  FIG. 6A  graph  300  shows the signal vs. knife edge position for the LP 01  mode (curve  301 ) and the LP 02  mode (curve  302 ) if the detector responds linearly to the intensity of the beam. 
         [0054]    The  FIG. 6B  graph  310  shows the signals for the LP 01  mode (curve  311 ) and LP 02  mode (curve  312 ) for a second-order non-linear detector that responds to the intensity squared. In the case of the linear detector, the LP 02  curve is dominated on the edges by the outer ring, and thus the slope of the curve is smaller than for the LP 01  beam. However, for the non-linear detector the outer ring of the LP 02  beam is suppressed, and the slope of the LP 02  curve is sharper than the LP 01 , showing the potential for increased resolution for the LP 02  beam in an apparatus which exploits non-linear or multi-photon interaction with matter, such as a microscope. 
         [0055]    The  FIG. 6C  graph ( 320 ) shows the signals for the LP 01  mode (curve  321 ) and LP 02  mode (curve  322 ) for a third-order non-linear detector. An even greater expected improvement is shown, with further suppression of the wings and a resolution for the LP 02  which is almost twice that of the LP 01 . 
         [0056]      FIG. 7A  is a graph  330  showing experimentally measured curves for the LP 01  mode (curve  331 ) and the LP 02  mode (curve  332 ) for a linear detector  258 ,  288 .  FIG. 7B  is a graph  340  showing experimentally measured curves for the LP 01  mode (curve  341 ) and the LP 02  mode (curve  342 ) for a non-linear detector  258 ,  288 . Third-order measurements were not made in these experiments. 
         [0057]    Note the correspondence between the experimental curves  331 ,  332 ,  341 , and  342  in  FIGS. 7A and 7B  and the theoretical curves  301 ,  302 ,  311 , and  312  in  FIGS. 6A and 6B . In the case of linear detection (curves  301 ,  302 ,  331 , and  332 ), the LP 02  provides relatively poor resolution due to the outer ring, but for non-linear detection (curves  311 ,  312 ,  341 , and  342 ) the outer ring is suppressed, the central spike dominates, and the slope of the LP 02  measurement is larger than the slope for the LP 01 . The benefit of higher-order modes would be even greater for higher-order non-linearities such as third-harmonic generation, which would provide better suppression of the LP 02  outer ring. 
         [0058]    These measurements show that the transverse resolution of the LP 02  can be better than the transverse resolution of the LP 01  in a non-linear microscope. HOMs also add increased benefit in the confocal setup due to the faster diffraction of beams with large M 2  values. 
         [0059]      FIGS. 8A and 8B  show experimental setups  360 ,  380  for comparing the confocal resolution of the LP 02  mode to the LP 01  mode. In the  FIG. 8A  setup  360 , a pulsed laser beam from a femtosecond laser  362  propagates in the LP 01  mode of a single-mode fiber  364 . In the  FIG. 8B  setup  380 , a pulsed laser beam from a femtosecond laser  382  is guided by a single-mode fiber  383  into a long-period grating  384  that excites the LP o2  mode of a higher-order mode fiber  385 . 
         [0060]    In both testing setups  360 ,  380 , the fiber laser output  366 ,  386  is collimated by a first lens  368 ,  388 . The collimated light  370 ,  390  is then refocused by a second lens  372 ,  392  onto a mirror  374 ,  394  that is scanned axially. The reflected power from the beam propagates back through the optical system and is coupled back into the fiber  364 ,  385 . The backward propagating power is detected through the 5% tap of a 95/5 splitter  376 ,  396 . A circulator may also be used beneficially in the place of the tap. The reflected optical power is plotted as a function of mirror position. 
         [0061]      FIG. 9A  is a graph  400  shows the measured confocal signal for a linear detector. Even for the linear detection, the LP 02  (curve  402 ) has a much sharper response curve compared to the LP 01  (curve  401 ) due to the large M 2  value of the LP 01 . 
         [0062]    In  FIG. 9B  is a graph  410 , the response of a non-linear detector is simulated by squaring the signal from the linear detector. As can be seen in  FIG. 9B , non-linear confocal microscopy with the LP 02  mode (curve  412 ) offers much sharper confocal response as compared to the LP 01  mode (curve  411 ). 
         [0063]    According to a further aspect of the invention, the higher-order mode fibers described herein are optimized for particular applications of a non-linear microscope. Generally speaking, it is possible to optimize the mode shape to obtain better resolution in a higher-order mode non-linear microscope compared to a non-linear microscope employing LP 01  incident light. 
         [0064]    Through fiber design, the mode shape could be adjusted in the following ways: 
         [0065]    1. The M 2  value of the fiber can be adjusted. A lower M 2  value will potentially allow tighter focusing at the focal plane. 
         [0066]    2. The fraction of power in the center lobe and the outer ring of the LP o2  mode can be adjusted. Less power in the outer ring would decrease its influence in second order non-linear measurements. 
         [0067]    3. The amplitude and width of the outer rings could be traded off. By decreasing the amplitude of the outer ring, while simultaneously increasing the width, the power in the outer ring could be kept constant, decreasing the impact of the outer ring on non-linear measurements. 
         [0068]    4. The center lobe could potentially be made narrower, increasing the resolution. 
         [0069]    5. Different order modes, such as LP 03  or LP 04 , or combinations thereof could be used. In cases where multiple modes are combined, care must be taken with the relative phase relationship between modes to ensure that the beams combine in the desired fashion, e.g., constructively, on the sample. 
         [0070]    It is important to note that all of these design adjustments are interrelated. For exampling, adjusting the fraction of power in the outer ring, or its width and amplitude, will also change the M 2  of the beam. 
         [0071]      FIG. 10  a diagram of a general non-linear optical system  500  according to aspects of the present invention. System  500  includes an NOM light source  502  that provides as an output  504  a structured free-space beam. An illumination assembly  506  uses output  504  to illuminate a sample  508 , thereby generating an optical output  510  therefrom. An imaging assembly  512  then uses a non-linear signal  514  derived from the optical output  510  to generate a microscopic image  514  of the sample  508 . In the examples of the invention described above, the non-linear signal comprises a multi-photon fluorescence signal. Alternatively, the non-linear signal may comprise, for example, a second-harmonic signal, a third-harmonic signal, a Raman signal, or the like. 
         [0072]      FIG. 11  is a flowchart of a general non-linear microscopy technique  600  according to aspects of the present invention. The method comprises the following steps: 
         [0073]    Step  601 : Excite a higher-order mode of low order of a fiber-guided laser light. 
         [0074]    Step  602 : Provide as an output a structured free-space beam. 
         [0075]    Step  603 : Use the structured free-space beam to provide illumination to a region of the sample, thereby generating an optical output therefrom. 
         [0076]    Step  604 : Use a non-linear signal derived from the optical output to generate a microscopic image of the sample region. 
         [0077]    While the foregoing description includes details which will enable those skilled in the art to practice the invention, it should be recognized that the description is illustrative in nature and that many modifications and variations thereof will be apparent to those skilled in the art having the benefit of these teachings. It is accordingly intended that the invention herein be defined solely by the claims appended hereto and that the claims be interpreted as broadly as permitted by the prior art.