Two-term time lens

Abstract

A two-term time lens module, which may be integrated into a microscopy system, receives a first excitation beam and a second excitation beam from a laser source, temporally separates the first excitation beam from the second excitation beam, and provides the temporally separated first and second excitation beams to an imaging device, wherein an amount of temporal separation between the first and second excitation beams imparted by the two-term time lens module is based on at least one of a dispersion property of a material of the sample or a depth of the target location within the sample, such that the excitation beams temporally overlap within a predetermined distance of the depth of the target location and do not temporally overlap at other depths.

Description

TWO-TERM TIME LENSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 632,107, filed April 10, 2024, and titled “TWO-TERM TIME LENS”, the entire contents of which are herein incorporated by reference for all purposes.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.TECHNICAL FIELD

[0003] This disclosure relates to the field of microscopy. More particularly, this disclosure relates to optical systems and methods for two-photon microscopy with reduced out- of-focus fluorescence.SUMMARY

[0004] Multiphoton microscopy (MPM) uses multiple photons to excite a target molecule (e.g., a target fluorophore) and generates an image according to a response of the target molecule (e.g., a fluorescence signal). Compared to single-photon microscopy, MPM permits the use of longer wavelength pulsed excitation radiation allowing images to be generated of fluorophores located deeper in scattering samples. MPM also permits the use of excitation radiation having a lower energy, and thus permits imaging in situations where high- energy excitation radiation is difficult, impossible, or harmful. MPM includes two-photon (2P) microscopy (2PM), in which two photons are simultaneously or near-simultaneously absorbed to excite the target molecule, and three-photon (3P) microscopy (3PM), in which three photons are simultaneously or near-simultaneously absorbed to excite the target molecule.

[0005] According to one aspect of the present disclosure, a microscopy system is provided. The system comprises a laser source configured to output a first excitation beam and a second excitation beam; a two-term time lens module configured to temporally separate the first excitation beam from the second excitation beam; and a microscope configured to provide the temporally separated first and second excitation beams to a sample and obtain afluorescence image of a target within the sample, wherein an amount of temporal separation between the first excitation beam and the second excitation beam imparted by the two-term time lens module is based on at least one of a dispersion property of a material of the sample or a depth of the target location within the sample, such that the first excitation beam and the second excitation beam temporally overlap within a predetermined distance of the depth of the target location and do not temporally overlap at distances other than the predetermined distance from the depth of the target location.

[0006] According to another aspect of the present disclosure, an optical system is provided. The optical system comprises a two-term time lens module configured to receive a first excitation beam and a second excitation beam from a laser source, temporally separate the first excitation beam from the second excitation beam, and provide the temporally separated first and second excitation beams to an imaging device, wherein an amount of temporal separation between the first excitation beam and the second excitation beam imparted by the two-term time lens module is based on at least one of a dispersion property of a material of the sample or a depth of the target location within the sample, such that the first excitation beam and the second excitation beam temporally overlap within a predetermined distance of the depth of the target location and do not temporally overlap at distances other than the predetermined distance from the depth of the target location.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1 illustrates a comparison between 2PM and 3PM.

[0008] FIG. 2 illustrates a 3PM modality according to a comparative example.

[0009] FIG. 3 A illustrates a 2PM modality according to a comparative example.

[0010] FIG. 3B illustrates a 2PM modality according to a comparative example.

[0011] FIG. 3C illustrates a 2PM modality according to a comparative example.

[0012] FIG. 4A illustrates an imaging modality according to various aspects of the present disclosure.

[0013] FIG. 4B illustrates an imaging modality according to various aspects of the present disclosure.

[0014] FIG. 4C illustrates an imaging modality according to various aspects of the present disclosure.

[0015] FIG. 4D illustrates an imaging modality according to various aspects of the present disclosure.

[0016] FIG. 4E illustrates an imaging modality according to various aspects of the present disclosure.

[0017] FIG. 5 A illustrates a laser behavior according to a comparative example.

[0018] FIG. 5B illustrates a laser behavior according to a comparative example.

[0019] FIG. 6A illustrates a laser behavior according to various aspects of the present disclosure.

[0020] FIG. 6B illustrates a laser behavior according to various aspects of the present disclosure.

[0021] FIG. 7 illustrates an optical system according to various aspects of the present disclosure.

[0022] FIG. 8A illustrates a dispersion behavior according to various aspects of the present disclosure.

[0023] FIG. 8B illustrates a dispersion behavior according to various aspects of the present disclosure.

[0024] FIG. 8C illustrates a dispersion behavior according to various aspects of the present disclosure.

[0025] FIG. 9A illustrates a laser behavior according to various aspects of the present disclosure.

[0026] FIG. 9B illustrates a laser behavior according to various aspects of the present disclosure.

[0027] FIG. 10A illustrates a laser behavior according to various aspects of the present disclosure.

[0028] FIG. 10B illustrates a laser behavior according to various aspects of the present disclosure.

[0029] FIG. 10C illustrates a laser behavior according to various aspects of the present disclosure.

[0030] FIG. 10D illustrates a laser behavior according to various aspects of the present disclosure.DETAILED DESCRIPTION

[0031] The detailed description set forth below in connection with the appended drawings is intended as a description of various configurations and is not intended to represent the only configurations in which the subject matter described herein may be practiced. The detailed description includes specific details to provide a thorough understanding of various embodiments of the present disclosure. However, it will be apparent to those skilled in the art that the various features, concepts, and embodiments described herein may be implemented and practiced without these specific details.

[0032] Before any aspects of the invention are explained in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the following drawings. The invention is capable of other aspects and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Unless specified or limited otherwise, the terms “mounted,” “connected,” “supported,” and “coupled” and variations thereof are used broadly and encompass both direct and indirect mountings, connections, supports, and couplings. Further, “connected” and “coupled” are not restricted to physical or mechanical connections or couplings.

[0033] It is also to be understood that any reference to an element herein using a designation such as “first,” “second,” and so forth does not limit the quantity or order of those elements, unless such limitation is explicitly stated. Rather, these designations may be used herein as a convenient method of distinguishing between two or more elements or instances of an element. Thus, a reference to first and second elements does not mean that only two elements may be employed or that the first element must precede the second element in some manner.

[0034] Also as used herein, unless otherwise limited or defined, “or” indicates a nonexclusive list of components or operations that can be present in any variety of combinations, rather than an exclusive list of components that can be present only as alternatives to each other. For example, a list of “A, B, or C” indicates options of: A; B; C; A and B; A and C; B and C; and A, B, and C. Correspondingly, the term “or” as used herein is intended to indicate exclusive alternatives only when preceded by terms of exclusivity, such as, e.g., “either,” “one of,” “only one of,” or “exactly one of.” Further, a list preceded by “one or more” (and variationsthereon) and including “or” to separate listed elements indicates options of one or more of any or all of the listed elements. For example, the phrases “one or more of A, B, or C” and “at least one of A, B, or C” indicate options of: one or more A; one or more B; one or more C; one or more A and one or more B; one or more B and one or more C; one or more A and one or more C; and one or more of each of A, B, and C. Similarly, a list preceded by “a plurality of’ (and variations thereon) and including “or” to separate listed elements indicates options of multiple instances of any or all of the listed elements. For example, the phrases “a plurality of A, B, or C” and “two or more of A, B, or C” indicate options of: A and B; B and C; A and C; and A, B, and C. In general, the term “or” as used herein only indicates exclusive alternatives (e.g., “one or the other but not both”) when preceded by terms of exclusivity, such as, e.g., “either,” “one of,” “only one of,” or “exactly one of.”

[0035] The following discussion is presented to enable a person skilled in the art to make and use embodiments of the invention. Various modifications to the illustrated embodiments will be readily apparent to those skilled in the art, and the generic principles herein can be applied to other embodiments and applications without departing from embodiments of the invention. Thus, embodiments of the invention are not intended to be limited to embodiments shown but are to be accorded the widest scope consistent with the principles and features disclosed herein. The following detailed description is to be read with reference to the figures, in which like elements in different figures have like reference numerals. The figures, which are not necessarily to scale, depict selected embodiments and are not intended to limit the scope of embodiments of the invention. Skilled artisans will recognize the examples provided herein have many useful alternatives and fall within the scope of embodiments of the invention.

[0036] In comparative examples of 2PM, the depth of image is limited by the generation of out-of-focus (“background”) fluorescence. Some examples of transgenic mouse lines (i.e., genetically modified mice that have endogenously fluorescent neurons labeled with GCaMP) have dense labeling (i.e., a large amount of fluorophore per volume), which leads to a larger out-of-focus signal when attempting to image deep. In such examples, the viable imaging range may be limited to a depth of about 500-600 microns (pm) as shown in the image series on the far right of FIG 1. Comparative examples of microscopy have attempted to suppress the out-of-focus fluorescence with an excitation process that is effectively less likely to happen (e.g., three-photon absorption), thereby further localizing the excitation to the focal spot. In comparative examples of 3PM on densely labeled transgenic mice, the viable imagingrange has been extended to depths on the order of 1000 pm. FIG. 1 illustrates the effects of out- of-focus fluorescence on 2PM compared to 3PM. As can be seen from FIG. 1, out-of-focus fluorescence begins to dominate at depths of 500-600 pm, thus limiting the 2P imaging depth.

[0037] However, 3PM presents several problems that make it difficult or impossible to push the depth limit further. One such problem is that the longer wavelengths used for 3PM (e.g., 1300 nanometers (nm)) are more efficient at heating the brain than wavelengths used for 2PM (e.g., 940 nm). For example, although the 1300 nm wavelength is more power efficient (i.e., fewer photons are lost on their way to the focus), it is also more efficient at heating up the brain and therefore has lower thermal limits than wavelengths typically used for 2P excitations. Because the brain is mostly water, the thermal limits of average power incident on the brain’s surface are determined predominantly by water absorption. For the 3P excitation wavelength of 1300 nm, the in vivo thermal limit is about 100 milliwatt (mW). Comparatively, the thermal limits of 2PM at an excitation wavelength of 940 is double or triple this limit (e.g., about 250 mW).

[0038] Another problem is that the 3P absorption process itself is inefficient, due to its small action cross-section (as can be seen in FIG. 1). For example, laser pulse energies used in experiments for 3PM (using common fluorophores like GFP) are approximately 1-2 nanojoules (nJ). However, damage can occur at approximately 3-7 nJ in the brain when using high NA objectives. Thus, 3P excitation is always near the threshold for damage in vivo. FIG. 2 illustrates the penetration depth of 2 nJ pulses, subject to in vivo thermal limits. In particular, FIG. 2 shows pulse energy vs. depth for excitation pulses with a 0.5 megahertz (MHz) repetition rate at three different wavelengths and powers: 940 nm and 250 mW, 1300 nm and 100 mW, and 1700 nm and 50 mW. At these parameters, a 2 nJ laser pulse will penetrate 0.85 millimeters (mm) at 940 nm, 1.4 mm at 1300 nm, and 1.5 mm at 1700 nm, imposing a depth limit for processes requiring this energy, indicated in FIG 2 as the dash-dotted line.

[0039] Even though the low efficiency makes 3PM more effective at suppressing out- of-focus fluorescence to begin with, the low efficiency means that 3PM requires an energy per pulse of approximately fifteen times more compared to 2PM in order to achieve a similar signal. Therefore, if one were to use a 2P process, the required laser power would be fifteen times less than the 3P process. For example, for the same power budget, one would need only one- fifteenth the number of photons arriving at the focus to achieve the same brightness per laser pulse. Constrained by thermal in vivo limits, as well as the scattering and absorption properties of the cortex, this means that excitation pulses intended for 2P excitation can effectively reachmuch deeper for some wavelengths (> 2.2 mm) in cortex as seen in the FIG 2 (dashed line), and could be utilized in imaging assuming the out-of-focus fluorescence (also shown on the left side of FIG 1 for two different 2P excitation wavelengths in a scattering solution) can be minimized without reducing the efficiency of excitation at focus.

[0040] One comparative example of a methodology for utilizing 2P processes is referred to as non-degenerate two-photon absorption (ND-TPA or ND-2PA). This process uses two different wavelengths of excitation radiation to sum together at the focus and accomplish a 2P absorption event. By spatially separating the laser beams themselves, one can ensure that neither beam contains photons that sum to the requisite energy for an absorption event and that 2P absorption will only occur where the two beams overlap, thus reducing out-of-focus fluorescence. This is illustrated in FIG. 3A, which shows a top schematic view of the back- aperture of an objective in a spatially-separated ND-TPA setup, and FIG. 3B, which shows a side schematic view of the spatially-separated ND-TPA setup and highlights the two spatially separated laser beams at four different positions. As can be seen in FIG. 3B and 3C, at the focus region (i.e., the third image from the left in FIG. 3B) the two non-degenerate photons i and i combine to excite the target molecule as shown in the rightmost diagram of FIG. 3C; however, outside of the focus region (i.e., the leftmost two images and the rightmost image in FIG. 3B), the beams are spatially separated such that the absorption of two photons i or two photons i does not excite the target molecule as shown in the leftmost two diagrams of FIG. 3C. ND- TPA is more efficient than degenerate processes that use two photons of the same wavelength. However, comparative examples of ND-TPA that rely on spatially confining the beams are undesirably complicated and may require specialized equipment that is expensive and / or not readily available. Additionally, a limitation of such comparative examples is the necessity of an underutilized numerical aperture (NA) of the objective lens as shown in FIG. 3 A. Consequently, where the spatially separated beams cross, the focal region is comparatively large.

[0041] The present disclosure describes a 2PM modality that takes advantage of the large excitation efficiency of a 2P excitation and that rejects the out-of-focus fluorescence in another way: by temporally separating the two incident laser pulses. This modality utilizes a technology referred to herein as a “two-term time lens.” The two-term time lens system, module, and / or device involves changing the shape of and delay between two laser pulses such that they overlap at a later time and space governed by a target material’s dispersive properties. Unlike comparative examples that isolate the incident laser pulses in space, the two-term timelens isolates them in time such that they are not compressed, and do not temporally overlap at positions other than the focus. This involves a combination of two terms: the group delay (GD), and the group delay dispersion (GDD) or pulse spreading. Effectively, the dispersion of the target medium itself (e.g., the brain) compresses the pulses and “aligns” them in time as the pulse propagates.

[0042] The two-term time lens technique suppresses nonlinear phenomena involving the spectral overlap of the two laser pulses at all points until the laser pulses meet. The technique ensures that each pulse’s chirp and the group delay between the two pulses, as well as the NA of each beam, is appropriate such that the eventual overlap is maximized. Thus, the technique effectively suppresses out-of-focus fluorescence in the 2P modality, allowing deeper imaging than is possible with comparative techniques. While the following description provides examples of the two-term time lens technique in the context of MP imaging (e.g., MPM), the technique may be applied to any technology in which there are wavelengthdependent nonlinear effects and where it is desired to localize the effects in space.

[0043] FIGS. 4A-4E conceptually illustrate the beam geometries of a temporally- separated ND-TPA modality in accordance with the present disclosure, in which FIG. 4A shows a top schematic view and FIG. 4B illustrates a side schematic view at four different positions. As can be seen in FIGS. 4B-4E, at the depth of the focus region (i.e., the third image from the left in FIG. 4B) the two non-degenerate photons i and X2 temporally overlap and combine to excite the target molecule as shown in the rightmost diagram of FIG. 4D; however, at other depths (i.e., the leftmost two images and the rightmost image in FIG. 4B), the two beams are not simultaneously present (i.e., they are separated in time) and thus the absorption of two photons i or two photons X2 does not excite the target molecule as shown in the leftmost two diagrams of FIG. 4D. This achieves full NA using a 2P modality as shown in FIG. 4A. FIG. 4C illustrates the spatial and temporal separation of the two beams, wherein the graphs from top to bottom in FIG. 4C correspond to the positional diagrams from left to right in FIG. 4B.

[0044] Additionally, under conditions where the GDD experienced by i and X2 have opposite sign, a further degree of suppression of out of focus fluorescence can be achieved when exciting narrow band absorption as seen in FIG 4E. By adding a slight offset to the pulse compression and the geometric focus, the beams can be slightly chirped with opposite sign at the geometric focus such that a narrow band excitation can be achieved through the local spatiotemporal sum of complimentary instantaneous frequencies. In this example, the carrierwavelengths are 740 nm and 1700 nm, respectively, and can excite a target of interest with a 2P absorption peak at 1035 nm. This requires the local derivative of inverse group velocity at each carrier wavelength have an opposite sign as seen in FIG. 8C, discussed in more detail below.

[0045] In the top row of FIG. 4E, the electric fields of the laser pulses are shown as a function of time, where darker shading corresponds to a higher local instantaneous frequency, and lighter shading corresponds to a lower local instantaneous frequency. The pulse separation indicates a nonzero GD, and the pulse width indicates varying amounts of GDD or chirp, where longer pulses indicate the presence of more GDD. At the depth represented by the leftmost image of FIG. 4E, the beams are temporally long, out of spatial focus, and delayed in time; in other words, both GDD and GD are nonzero and comparatively large. At the depth represented by the second image from the left of FIG. 4E, the beams are also temporally long, out of spatial focus, and delayed in time. The GDD and GD are still nonzero, but are smaller than in the leftmost image. At the depth represented by the third image from the left of FIG 4E, the beams are temporally short, but out of spatial focus and delayed in time. At this point, the GDD is small but the GD remains nonzero. At the depth represented by the rightmost image of FIG. 4E (i.e., within a predetermined distance of a depth of a target), there exists a narrow band spatio-temporal sum of the beams in geometric focus. In this manner, the pulse widths of the two beams are spectral complements, thereby modifying the effective bandwidth of excitation. In other words, the GD at this location is minimal (i.e., is zero or nearly zero) and, while the GDD is nonzero, it is matched between the two beams.

[0046] Comparative examples of ND-TPA utilize lasers having a relatively long pulse width. For example, FIGS. 5A and 5B illustrate the behavior of two excitation beams (e.g., lasers) each having a pulse width of 100 femtoseconds (fs), with one laser emitting at 850 nm and the other emitting at 1300 nm. The lines represent the trajectory of the carrier wavelength, and the shaded region surrounding each line represents the temporal extent of the laser pulse (full width half maximum (FWHM)) as it propagates through the brain to 2 mm (FIG. 5 A) and 4 mm (FIG. 5B), respectively. Where the shaded regions overlap, 2P excitation is temporally possible. Thus, FIGS. 5 A and 5B show that, even if one was to push well-bey ond depth limits imposed by in vivo thermal limits, in the comparative example, the temporal suppression of out-of-focus fluorescence is weak, and spatial separation of the pulses is required. This is due to pulse durations being long with respect to the difference in group delay incurred vs. thickness of cortex (see FIG 8B, described in more detail below).

[0047] Thus, the systems and methods in accordance with the present disclosure use short pulse durations. When the pulse durations become short (e.g., 25 fs), the temporal difference between excitation wavelengths can become large (see FIGS. 8 A and 8C, described in more detail below) and the two terms (GD and GDD) become meaningful. FIGS. 6 A and 6B illustrate this behavior for two lasers each having a pulse width of 25 fs, with one laser emitting at 740 nm and the other emitting at 1700 nm. It can be seen that there are large depth regions where there is no possibility for generation of out-of-focus fluorescence (i.e., regions where the shaded regions do not overlap). It can further be seen that, as the depth is increased, the region where the fluorescence is suppressed continues to grow. Thus, in contrast with the behavior seen in FIG. 5A and FIG. 5C, due to the choices of central wavelength and pulse duration, the techniques according to the present disclosure scale with depth.

[0048] In some examples, the two-term time lens technique may be used, for example, by microscopy labs that have paid the up-front costs of purchasing a laser that is intended for 3PM, because a microscope that can support such wavelengths is suited to take advantage of the two-term time lens technique and provide access to deeper imaging in the brain beyond the above-described limits of 3PM. Thus, the systems and methods described herein may be used with commercially available lasers that have a tunable output with short pulse durations and two wavelengths (e.g. signal beam and idler beam) that energetically sum to the second- harmonic generation (SHG) wavelength of the pump laser (e.g., 515-517 nm). This wavelength may be used to excite known fluorophores such as tdtomato and / or mScarlet, which have a strong 2P absorption peak near twice this wavelength, although other fluorophores may be used. Such lasers may be, for example, the Spirit-NOPA VISNIR (R), Monaco-OPERA-F (R), and Class5 White Dwarf (R) lasers. In the case of the Class5 White Dwarf (R), the two outputs can be specified at arbitrary wavelengths because they can generate two arbitrary wavelength signal beams, providing more freedom to choose the energy of the sum of the two photons. This enables the use of the two least attenuated wavelength bands for brain imaging near 1300 nm, and 1700 nm, and ND-TPA of indo-cyanine green (ICG). The emission from such a fluorophore would be weakly absorbed by the brain, allowing for efficient collection of photons. This could also be done by pumping two NOPA VISNIRs with a single Spirit laser, or pumping two OPERA-Fs with a single Monaco laser, ensuring that both excitation beams have exactly the same frequency.

[0049] In such examples, a two-term time lens device may be incorporated into the existing 3P optical table to condition the beams to have the appropriate group delay and groupdelay dispersion to overlap in space and time at the focus of the objective. In addition to temporal considerations, the beam sizes may be adjusted such that at the back aperture of the objective, a desired level of overlap of the point spread functions can be achieved. FIG. 7 illustrates one example of such a two-term time lens device.

[0050] In FIG. 7, the two-term time lens system comprises a laser device 70, a two- term time lens module 700, and a microscope 80. The laser device 70 may provide two excitation beam outputs, a signal / idler beam 72 and a signal / idler beam 74, that have the same repetition rate and different wavelengths. The two-term time lens module 700 (also referred to as a two-term time lens device) comprises two dispersion compensation devices which may include pulse compressors 712, 714 (e.g., prisms or other tunable technology), a group delay line 720 for one of the beams (i.e., the beam having the faster group velocity, such as, as illustrated, the signal / idler beam 72), a variable telescope 730 for one of the beams (as illustrated, the signal / idler beam 74, although in some implementations the variable telescope 730 may be disposed along the path of the signal / idler beam 72) such that the focal volumes of the beams are identical, and a beam combiner 740. The two-term time lens module 700 is configured to impart an amount of temporal separation between the excitation beams, wherein the amount is based on at least one of a dispersion property (including two or more dispersion properties) of a material of the sample or a depth of the target location within the sample, such that the first excitation beam and the second excitation beam are compressed to temporally overlap within a predetermined distance of the depth of the target location and do not temporally overlap at distances other than the predetermined distance from the depth of the target location.

[0051] The nonlinear pulse compressors 712, 714 may include a fiber, free space, a nonlinear crystal (e.g. sapphire / YAG), a varifocal lens, combinations thereof, and the like, and respectively reduce the Fourier transform limits (the shortest achievable pulse duration when compressed) in time of the excitation beams (e.g., to 25 fs or shorter, or to 10 fs or shorter). However, if the laser device 70 is capable of outputting sufficiently narrow pulses (e.g., <25 fs), the nonlinear elements of the pulse compressors 712, 714 may be omitted while retaining linear elements such as prisms. Where a linear compression element is present, it may be located at any point along the individual beamlines (e.g., between the signal / idler beam output 74 and the beam combiner 740). However, in practical implementations, the linear compression element may be co-located with the nonlinear pulse compressor 712 and / or the nonlinear pulse compressor 714. The delay line 720 imparts a group delay on the first excitation beam or thesecond excitation beam. The variable telescope 730 adjusts a beam characteristic (e.g., a beam size, a focal volume at the target, etc.) of the first excitation beam or the second excitation beam. The beam combiner 740 spatially combines the excitation beams. The microscope 80 provides the temporally separated (and spatially overlapping) excitation beams to a sample (not illustrated) and obtains a fluorescence image of a target within the sample. In some implementations, the microscope 80 may be replaced with a different imaging device.

[0052] The two-term time lens module 700 thereby increases the spatio-spectral spacetime simultaneity of the wavelengths, and suppresses the nonlinear effects elsewhere. While FIG. 7 illustrates a single delay line 720, in practical implementations each beam may have its own delay line with one delay line providing a longer delay. The two different wavelengths i and X2 may be selected based on the multiphoton absorption wavelength t of the target1 1 fluorophore according to the relationship= 2 / (— + — ). For example, excitation Z1 X2 wavelengths of 1300 and 1700 nm may be used to excite a fluorophore with a 2P absorption peak of 1473 nm; or excitation wavelengths of 740 and 1700 nm may be used to excite a fluorophore with a 2P absorption peak of 1031 nm. In some implementations, it may be possible to empirically measure characteristics of the target sample (e.g., scattering / absorption profile, absorption wavelength, etc.) and select the wavelengths based thereupon.

[0053] FIGS. 8A-8C illustrate the effects of a target material’s dispersion profile on wavelength selection. The group delay will be equal to the inverse group velocity (e.g., delay in fs caused by each pm of depth) multiplied by the approximate depth achievable (e.g., in pm) in in-vivo samples limited by scattering and absorption properties when utilizing the full power budget at the surface of the brain (see FIG. 2). In FIGS. 8A-8C, the group delay is plotted vs. excitation wavelength for seawater, which provides a good approximation of the brain; seawater provides a wavelength-dependent inverse group velocity that varies between approximately 4.49 fs / pm and 4.53 fs / pm from 0.680 - 1.8 pm. In FIG. 8A, the relative group delay at a depth of 2.2 mm is shown for excitation wavelengths of 1300 nm and 1700 nm (which serves as a conservative accessible 2P depth according to FIG. 2). In this case, the relative group delay is about 49 fs, enabling short pulses (< 25 fs) to be temporally distinct as they propagate through cortex. In FIG. 8B, the relative group delay at a depth of 1.1 mm (which serves as an approximate accessible 2P depth according to thermal limits shown in FIG. 2) is shown for excitation wavelengths of 850 nm and 1300 nm. In this case, the relative group delay is only about 6 fs. In FIG. 8C, the relative group delay at a depth of 1.1 mm is shown for excitation wavelengths of 740 nm and 1700 nm (which again serves as an approximateaccessible 2P depth according to thermal limits shown in FIG. 2). In this case, the relative group delay is increased to about 23 fs. Thus, the combination of wavelengths illustrated in FIG. 8B may not provide sufficient temporal separation for brain imaging, and one could use the combination of wavelengths illustrated in FIG. 8C instead), which allow for out-of-focus fluorescence suppression especially in cases of narrow band excitation in accordance with FIG. 4E since the sign of GDD (local slope of group delay) is reversed. Smaller relative group delays may also be compensated for by using narrower pulse widths in time.

[0054] In cases where the laser pulses have a very short pulse width, the GDD term becomes very impactful and the two-term time lens device may have additional benefits. FIGS. 9A and 9B illustrate the behavior of two lasers each having a pulse width of 10 fs, with one laser emitting at 740 nm and the other emitting at 1700 nm. As above, the lines represent the trajectory of the carrier wavelength, and the shaded region surrounding each line represents the temporal extent of the laser pulse (FWHM) as it propagates through the brain to 2 mm (FIG. 9A) and 4 mm (FIG. 9B), respectively. Where the shaded regions overlap, 2P excitation is possible. Thus, in comparison with FIGS. 6A and 6B, FIGS. 9A and 9B show that, by reducing the pulse duration in this way, the pulses are pre-stretched more significantly at the surface of the brain and only short at the focus. This may be used in the case where there is a likelihood that, at the surface, one of the wavelengths has a small but nonzero absorption cross-section. In order to achieve the 10 fs pulse duration, the lasers may be compressed by approximately three times, which can be done in free-space or in hollow core capillaries through self-phase modulation. This spectral broadening device may be incorporated into the two-term time lens module (e.g., module 700 of FIG. 7) as well, which would allow for the bandwidth to be tuned.

[0055] FIGS. 10A-10D illustrate a comparison between the pulse behavior at four different pulse widths, with one laser emitting at 1300 nm and the other emitting at 1700 nm. FIG. 10A illustrates a pulse width of 50 fs, FIG. 10B illustrates a pulse width of 25 fs, FIG. 10C illustrates a pulse width of 15 fs, and FIG. 10D illustrates a pulse width of 10 fs. As shown in FIGS. 10B-10D, the temporal independence of the laser pulses and the GDD effects suppress out-of-focus fluorescence especially well for pulse widths of 25 fs or narrower for these wavelengths.

[0056] Other examples and uses of the disclosed technology will be apparent to those having ordinary skill in the art upon consideration of the specification and practice of the invention disclosed herein. The specification and examples given should be consideredexemplary only, and it is contemplated that the appended claims will cover any other such embodiments or modifications as fall within the true scope of the invention.

[0057] The Abstract accompanying this specification is provided to enable the United States Patent and Trademark Office and the public generally to determine quickly from a cursory inspection the nature and gist of the technical disclosure and in no way intended for defining, determining, or limiting the present invention or any of its embodiments.

Claims

CLAIMSWhat is claimed is:

1. A microscopy system, comprising: a laser source configured to output a first excitation beam and a second excitation beam; a two-term time lens device configured to temporally separate the first excitation beam from the second excitation beam; and a microscope configured to provide the temporally separated first and second excitation beams to a sample and obtain a fluorescence image of a target within the sample, wherein an amount of temporal separation between the first excitation beam and the second excitation beam imparted by the two-term time lens module is based on at least one of a dispersion property of a material of the sample or a depth of the target location within the sample, such that the first excitation beam and the second excitation beam temporally overlap within a predetermined distance of the depth of the target location and do not temporally overlap at distances other than the predetermined distance from the depth of the target location.

2. The system of claim 1, wherein the two-term time lens device includes a delay line configured to impart a group delay on the first excitation beam or the second excitation beam.

3. The system of claim 1, wherein the two-term time lens device includes a first delay line configured to impart a first group delay on the first excitation beam and a second delay line configured to impart a second group delay on the second excitation beam, wherein the second group delay is different from the first group delay.

4. The system of claim 1, wherein the two-term time lens device includes: a first pulse compressor configured to reduce an achievable pulse width in time of the first excitation beam, a second pulse compressor configured to reduce an achievable pulse width in time of the second excitation beam, and a linear pulse compressor configured to cause the reduced achievable pulse width in time of the first excitation beam and the reduced achievable pulse width in time of the secondexcitation beam to be located within a predetermined distance of the depth of the target location.

5. The system of claim 1, wherein two-term time lens device includes a first pulse compressor configured to reduce a first pulse width in time of the first excitation beam, and a second pulse compressor configured to reduce a second pulse width in time of the second excitation beam, such that the first pulse width in time is matched to the second pulse width in time within a predetermined distance of the depth of the target location.

6. The system of claim 5, wherein the first pulse compressor is configured to reduce the first pulse width in time to 25 femtoseconds or shorter, and the second pulse compressor is configured to reduce the second pulse width in time to 25 femtoseconds or shorter.

7. The system of claim 5, wherein the first pulse compressor is configured to reduce the first pulse width in time to 10 femtoseconds or shorter, and the second pulse compressor is configured to reduce the second pulse width in time to 10 femtoseconds or shorter.

8. The system of claim 1, further comprising a beam combiner configured to spatially combine the first excitation beam and the second excitation beam.

9. The system of claim 1, wherein a first wavelength of the first excitation beam and a second wavelength of the second excitation beam are based on at least one of the dispersion property of the material of the sample, the depth of the target location within the sample, or an absorption wavelength of a fluorophore at the target location.

10. The system of claim 1, wherein the two-term time lens device includes a variable telescope configured to adjust a focus of the first excitation beam or the second excitation beam.

11. An optical system, comprising: a two-term time lens device configured to: receive a first excitation beam and a second excitation beam from a laser source,temporally separate the first excitation beam from the second excitation beam, and provide the temporally separated first and second excitation beams to an imaging device, wherein an amount of temporal separation between the first excitation beam and the second excitation beam imparted by the two-term time lens module is based on at least one of a dispersion property of a material of a sample or a depth of the target location within the sample, such that the first excitation beam and the second excitation beam temporally overlap within a predetermined distance of the depth of the target location and do not temporally overlap at distances other than the predetermined distance from the depth of the target location.

12. The system of claim 11, wherein the two-term time lens device includes a delay line configured to impart a group delay on the first excitation beam or the second excitation beam.

13. The system of claim 11, wherein the two-term time lens device includes a first delay line configured to impart a first group delay on the first excitation beam and a second delay line configured to impart a second group delay on the second excitation beam, wherein the second group delay is different from the first group delay.

14. The system of claim 11, wherein the two-term time lens device includes: a first pulse compressor configured to reduce an achievable pulse width in time of the first excitation beam, a second pulse compressor configured to reduce an achievable pulse width in time of the second excitation beam, and a linear pulse compressor configured to cause the reduced achievable pulse width in time of the first excitation beam and the reduced achievable pulse width in time of the second excitation beam to be located within a predetermined distance of the depth of the target location.

15. The system of claim 11, wherein two-term time lens device includes a first pulse compressor configured to reduce a first pulse width in time of the first excitation beam, and a second pulse compressor configured to reduce a second pulse width in time of the secondexcitation beam, such that the first pulse width in time is matched to the second pulse width in time within a predetermined distance of the depth of the target location..

16. The system of claim 15, wherein the first pulse compressor is configured to reduce the first pulse width in time to 25 femtoseconds or shorter, and the second pulse compressor is configured to reduce the second pulse width in time to 25 femtoseconds or shorter.

17. The system of claim 15, wherein the first pulse compressor is configured to reduce the first pulse width in time to 10 femtoseconds or shorter, and the second pulse compressor is configured to reduce the second pulse width in time to 10 femtoseconds or shorter.

18. The system of claim 11, further comprising a beam combiner configured to spatially combine the first excitation beam and the second excitation beam.

19. The system of claim 11, wherein a first wavelength of the first excitation beam and a second wavelength of the second excitation beam are based on at least one of the dispersion property of the material of the sample, the depth of the target location within the sample, or an absorption wavelength of a fluorophore at the target location.

20. The system of claim 11, wherein: the first excitation beam has a positive group delay dispersion in the target material and a first group delay, the second excitation beam has a negative group delay dispersion in the target material and a second group delay different from the first group delay, such that a pulse width in time of the first excitation beam is a spectral complement to a pulse width in time of the second excitation beam within the predetermined distance from the depth of the target location, thereby to modify an effective bandwidth of excitation.

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