Sweeped Confocal Planar Excitation (SCAPE) Microscopy

Abstract

A spacer for an immersion objective lens may be fabricated by pressing a set of side walls against a mirror to form a liquid-tight cavity, filling the liquid-tight cavity with a first amount of UV-curable polymer, and curing the first amount of UV-curable polymer into a first solid mass that is attached to the mirror. The top surface of the first solid mass is then positioned near the objective lens with a second amount of UV-curable polymer occupying the space between the first solid mass and the objective lens. The position of the first solid mass is then adjusted until it reaches a final position relative to the objective lens. This adjustment may be aided by checking the collimation of the light reflected through the mirror. The second amount of UV-curable polymer is then cured.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Applications No. 63 / 159758 (filed March 11, 2021) and No. 63 / 160297 (filed March 12, 2021), each of which is incorporated herein by reference in its entirety.

[0002] Description of research funded by the federal government. This invention was made with government support under grant numbers NS108213, NS094296, NS104649, and CA236554 granted by the National Institutes of Health, and grant numbers 1644869, 0801530, and 0954796 granted by the National Science Foundation. The U.S. Government has certain rights in this invention. [Background technology]

[0003] Patent documents 1, 2, 3, 4, and 5 are each incorporated herein by reference and describe various methods for performing swept confocal planar excitation (SCAPE) microscopy. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] U.S. Patent No. 10061111 [Patent Document 2] U.S. Patent No. 10831014 [Patent Document 3] U.S. Patent No. 10835111 [Patent Document 4] U.S. Patent No. 10852520 [Patent Document 5] U.S. Patent No. 10908088 [Non-patent literature]

[0005] [Non-Patent Document 1] https: / / www.mypolymers.com / products [Non-Patent Document 2] https: / / www.thorlabs.com / thorproduct.cfm?partnumber=G608N3 [Non-Patent Document 3] https: / / www.cargille.com / optical-gels / [Overview of the Initiative] [Means for solving the problem]

[0006] One aspect of this application relates to a first method for fabricating a spacer for an immersion objective lens. The first method includes pressing a set of sidewalls against a mirror so that a portion of the mirror positioned between the set of sidewalls serves as the bottom of a negative mold, and so that the set of sidewalls cooperates with the bottom of the negative mold to form a liquid-tight cavity. The liquid-tight cavity is filled with a first amount of UV-curable polymer, the first amount of UV-curable polymer is cured into a first solid mass. The first solid mass has a bottom surface that adheres to the mirror and a top surface. The set of sidewalls is removed from the mirror without disturbing the adhesion between the bottom surface of the first solid mass and the mirror. The first method also includes positioning the top surface of the first solid mass near the objective lens, with a second amount of UV-curable polymer occupying the space between the top surface of the first solid mass and the objective lens. Following the positioning step, the position of the first solid mass is adjusted until the underside of the first solid mass reaches its final position relative to the objective lens. The second amount of UV-curable polymer is cured after the first solid mass has reached its final position.

[0007] Some examples of the first method further include the steps of projecting parallel light through an objective lens toward a mirror, and detecting the collimation property of the light reflected by the mirror. In these examples, it is determined that the first solid mass has reached its final position when the light reflected by the mirror is precisely parallel. In some cases, curing of the second amount of UV-curable polymer may be carried out by projecting UV light into the second amount of UV-curable polymer through an objective lens. In some cases, following the projection of UV light into the second amount of UV-curable polymer through an objective lens, additional UV light is applied to further cure the second amount of UV-curable polymer.

[0008] In some cases of the first method, at least the mirror portion that serves as the bottom of the negative mold has a dielectric surface. In some cases of the first method, at least the mirror portion that serves as the bottom of the negative mold is flat within a range of 250 nm. In some cases of the first method, the set of sidewalls is made of a polymer. In some cases of the first method, the set of sidewalls is made of PDMS (polydimethylsiloxane). In some cases of the first method, the UV-curable polymer contains BIO-133.

[0009] Some examples of the first method further include the step of removing the mirror from the underside of the first solid mass.

[0010] Another aspect of this application relates to a first imaging device. The first imaging device comprises a first set of optical elements, a second set of optical elements, a scanning element, a light source, a TAG lens, a cylindrical lens, and a reflecting surface. The first set of optical elements has a proximal end, a distal end, and a first optical axis, and includes a first objective lens located at the distal end of the first set of optical elements. The second set of optical elements has a proximal end, a distal end, and a second optical axis, and includes a second objective lens located at the distal end of the second set of optical elements.

[0011] In the first imaging device, the scanning element is positioned proximal to the proximal end of the first set of optical elements and proximal to the proximal end of the second set of optical elements. The scanning element is positioned to deliver a sheet of excitation light such that the sheet passes through the first set of optical elements in a proximal-to-distal direction and incident on a sample positioned distal to the distal end of the first set of optical elements, where the sheet of excitation light is projected into the sample at an oblique angle and at a position that changes depending on the orientation of the scanning element. The first set of optical elements sends detection light back from the sample to the scanning element in a distal-to-proximal direction. The scanning element is also positioned to deliver detection light such that the detection light passes through the second set of optical elements in a proximal-to-distal direction and forms an intermediate image plane distal to the distal end of the second set of optical elements.

[0012] In the first imaging device, a light source generates excitation light. A TAG lens has an optical axis and is positioned to receive excitation light such that (a) the excitation light passes through the TAG lens parallel to the optical axis of the TAG lens, and (b) the excitation light passes through the TAG lens eccentrically with respect to the optical axis of the TAG lens. A cylindrical lens is positioned in series with the TAG lens such that the sheet of excitation light exits from the series combination of the TAG lens and the cylindrical lens. A reflective surface is positioned to direct the sheet of excitation light toward the scanning element.

[0013] Some embodiments of the first imaging device further include a third objective lens positioned to direct light arriving from the intermediate image plane toward the camera. In some of these embodiments, the third objective lens and the second objective lens are optically coupled via a fluid chamber.

[0014] Some embodiments of the first imaging device further include a high NA acceptance angle fused fiber bundle positioned to relay light from an intermediate image plane distal to the second objective lens toward the camera. In some cases, in these embodiments, the fiber bundle may have a front face that is aligned with the image of the oblique light sheet formed by the second objective lens. Alternatively, in these embodiments, the fiber bundle may have a bevel cut edge that is aligned with the image of the oblique light sheet formed by the second objective lens to collect light and effect image rotation.

[0015] In some embodiments of the first imaging device, the light source generates pulses of excitation light, and the pulses of excitation light are compressed by a prism compressor prior to their arrival at the TAG lens. In some embodiments of the first imaging device, the reflective surface comprises a dichroic beam splitter. In some embodiments of the first imaging device, the cylindrical lens is positioned within the optical path between the TAG lens and the reflective surface, and the cylindrical lens expands the excitation light emerging from the TAG lens into a sheet of excitation light.

[0016] Another aspect of the present application is directed to a second imaging device. The second imaging device includes a first set of optical elements, a second set of optical elements, a scanning element, and a folding mirror. The first set of optical elements has a proximal end, a distal end, and a first optical axis, and the first set of optical elements includes a first objective lens disposed at the distal end of the first set of optical elements. The second set of optical elements has a proximal end, a distal end, and a second optical axis, and the second set of optical elements includes a second objective lens disposed at the distal end of the second set of optical elements. The scanning element is disposed proximal to the proximal end of the first set of optical elements and proximal to the proximal end of the second set of optical elements, and the scanning element is mounted at an angle deviating from perpendicular by 20 to 25 degrees with respect to either the first optical axis or the second optical axis. The folding mirror is disposed between the scanning element and either the second set of optical elements or the first set of optical elements.

[0017] In the second imaging device, the scanning element is positioned to send a sheet of excitation light such that the sheet of excitation light passes through the set of first optical elements in a proximal-to-distal direction and impinges on a sample positioned distally beyond the distal end of the set of first optical elements, where the sheet of excitation light is projected onto the sample at an oblique angle and the sheet of excitation light is projected onto the sample at a position that varies depending on the orientation of the scanning element. The set of first optical elements sends the detection light from the sample back to the scanning element in a distal-to-proximal direction. The scanning element is also positioned to send the detection light such that the detection light passes through the set of second optical elements in a proximal-to-distal direction and forms an intermediate image plane at a distal position beyond the distal end of the set of second optical elements.

[0018] Some embodiments of the second imaging device further comprise a third objective lens positioned to send the light reaching from the intermediate image plane towards the camera.

[0019] In some embodiments of the second imaging device, the scanning element is mounted at an angle deviating 20 - 25° from perpendicular with respect to the first optical axis, and the folding mirror is disposed between the scanning element and the set of second optical elements. In some embodiments of the second imaging device, the scanning element is mounted at an angle deviating 20 - 25° from perpendicular with respect to the second optical axis, and the folding mirror is disposed between the scanning element and the set of first optical elements.

[0020] In some embodiments of the second imaging device, the sheet of excitation light reaches the scanning element via the set of second optical elements, and the sheet of excitation light is introduced into the set of second optical elements via a second mirror positioned proximally with respect to the second objective lens. Optionally, in these embodiments, the second mirror has a chamfered linear first edge and at least one second edge, and the second mirror is mounted such that the chamfered linear first edge is closer to the second optical axis than at least one second edge. Optionally, in these embodiments, the second mirror is mounted on a translation stage.

[0021] Another aspect of this application relates to a third imaging device. The third imaging device comprises a set of first optical elements, a set of second optical elements, a scanning element, a plurality of light sources, at least one optical beam combiner, at least one pair of alignment mirrors, and a set of third optical elements. The set of first optical elements has a proximal end, a distal end, and a first optical axis, and the set of first optical elements includes a first objective lens located at the distal end of the set of first optical elements. The set of second optical elements has a proximal end, a distal end, and a second optical axis, and the set of second optical elements includes a second objective lens located at the distal end of the set of second optical elements. The scanning element is located proximal to the proximal end of the set of first optical elements and proximal to the proximal end of the set of second optical elements.

[0022] In the third imaging device, the scanning element is positioned to deliver a sheet of excitation light such that it passes through the first set of optical elements in a proximal-to-distal direction and incident on a sample positioned distally beyond the distal end of the first set of optical elements, where the sheet of excitation light is projected onto the sample at an oblique angle and at a position that changes depending on the orientation of the scanning element. The first set of optical elements sends detection light from the sample back to the scanning element in a distal-to-proximal direction. The scanning element is also positioned to deliver detection light such that it passes through the second set of optical elements in a proximal-to-distal direction and forms an intermediate image plane distally beyond the distal end of the second set of optical elements.

[0023] In the third imaging device, each light source has its own output beam at its respective wavelength. At least one optical beam combiner is positioned relative to the multiple light sources to direct the output beams from the multiple light sources onto a common path of excitation light. Each pair of alignment mirrors is positioned relative to each light source to adjust the alignment of its respective output beam, and at least one pair of alignment mirrors is configured to facilitate the alignment of all output beams within the sample. The third set of optical elements is configured to expand the output beams into a sheet of excitation light.

[0024] Some embodiments of the third imaging device further include a third objective lens positioned to direct light arriving from the intermediate image plane toward the camera.

[0025] In some embodiments of the third imaging apparatus, a sheet of excitation light reaches the scanning element via a second set of optical elements. The sheet of excitation light is introduced into the second set of optical elements via a second mirror positioned proximal to the second objective lens. The second mirror is positioned to receive the sheet of excitation light from the third set of optical elements and to direct the sheet of excitation light via an alternative route toward the proximal end of the second set of optical elements.

[0026] In some embodiments described in the preceding paragraph, the second mirror has a chamfered linear first edge and at least one second edge, and the second mirror is mounted such that the chamfered linear first edge is closer to the second optical axis than the at least one second edge. In some embodiments, the second mirror is mounted on a translation stage. [Brief explanation of the drawing]

[0027] [Figure 1a] This is a schematic diagram of a SCAPE embodiment that provides higher resolution than conventional SCAPE systems. [Figure 1b] This is a detailed view of the mirror 80 shown in Figure 1a. [Figure 2a] This figure shows various methods for fabricating an imaging chamber. [Figure 2b] This figure shows a template used for locating the sample. [Figure 2c] This diagram shows the alignment targets incorporated into the registration template. [Figure 2d] This figure shows how FEP film can be used to restrain a sample being imaged. [Figure 2e]This is a diagram showing a C. elegans nematode confined within a chamber. [Figure 3a] This is a block diagram of a system for stimulating living tissue by generating localized heating. [Figure 3b] This figure shows the local temperature rise caused by IR illumination as shown in the embodiment of Figure 3a. [Figure 4a] This is a block diagram of the basic SCAPE system. [Figure 4b] This diagram shows the geometric relationship between two objective lenses used to analyze numerical aperture (NA). [Figure 4c] This diagram shows the geometric relationship between two objective lenses used to analyze numerical aperture (NA). [Figure 5a] This is a diagram showing the design of a water-immersion chamber. [Figure 5b] This figure shows the design of another immersion chamber. [Figure 5c] This figure shows the design of another immersion chamber. [Figure 5d] This diagram shows the design for holding the cover glass on the objective lens. [Figure 6a] This figure shows the geometric relationship between two objective lenses used to analyze the zero working distance approach. [Figure 6b] This figure shows the geometric relationship between two objective lenses used to analyze the zero working distance method. [Figure 6c] This figure shows the geometric relationship between two objective lenses used to analyze the zero working distance method. [Figure 6d] This figure shows the geometric relationship between two objective lenses used to analyze the zero working distance method. [Figure 6e] This figure shows the geometric relationship between two objective lenses used to analyze the zero working distance method. [Figure 6f]This figure shows the expected angle-dependent reflection loss at the glass-to-water interface for a ZWD lens. [Figure 6g] This figure shows the expected angle-dependent reflection loss at the glass-to-water interface for ZWD lenses. [Figure 7] This figure shows an adapter for holding water droplets in the third objective lens. [Figure 8a] This figure shows an additional spacer attached in front of the immersion lens used as the third objective lens in the SCAPE system. [Figure 8b] This figure shows a method for casting and positioning the add-on spacer shown in Figure 8a. [Figure 8c] This figure shows a method for casting and positioning the add-on spacer shown in Figure 8a. [Figure 8d] This figure shows a series of steps for manufacturing an add-on spacer. [Figure 8e] This figure shows another set of steps for manufacturing an add-on spacer. [Figure 8f] This figure shows a series of steps for attaching an add-on spacer to the third objective lens. [Figure 8g] This figure shows an add-on spacer attached to the third objective lens. [Figure 8h] This figure shows a series of different steps for fabricating an add-on spacer and attaching that spacer to a third objective lens. [Figure 8i] This figure shows a series of different steps for fabricating an add-on spacer and attaching the spacer to a third objective lens. [Figure 9a] This is a schematic diagram of a two-photon SCAPE embodiment using a TAG lens. [Figure 9b] Figure 9a shows a method for aligning the third objective lens O3 with the camera telescope in the embodiment shown. [Figure 9c] This figure compares the optical path through the TAG lens in the embodiment of Figure 9a with an alternative method. [Figure 9d]Figure 9c shows the curvature of the optical sheet for various methods shown. [Figure 9e] This figure shows the fluorescence measurements obtained when scanning an eccentrically aligned TAG lens with various amplitude modulations. [Figure 9f] Figure 9a shows the change in the illuminated field when the beam passing through the TAG lens is moved off-axis. [Figure 9g] Figure 9a shows the simulation results illustrating the expected asymmetric beam modulation when light is transmitted eccentrically through the TAG lens. [Figure 9h] Figure 9a shows the simulation results illustrating the expected asymmetric beam modulation when light is transmitted eccentrically through the TAG lens. [Figure 9i] This diagram shows the camera being rotated so that it reads rows as horizontal pixels. [Figure 9j] This figure shows how to incorporate asymmetrical magnification into an O3 telescope to compress the image in the Z region without rotating the camera. [Figure 10a] This diagram shows how various components are attached to the dual-camera SCAPE system. [Figure 10b] This diagram shows how various components are attached to the dual-camera SCAPE system. [Figure 10c] This diagram shows how various components are attached to the dual-camera SCAPE system. [Figure 10d] This diagram shows how various components are attached to the dual-camera SCAPE system. [Figure 11a] This is a schematic diagram of a death-scan type axially resolved two-photon embodiment of SCAPE. [Figure 11b] This figure shows an example of an image obtained using the embodiment of Figure 11a. [Figure 11c] This figure shows an example of an image obtained using the embodiment of Figure 11a. [Figure 11d] This figure compares various methods for acquiring high-speed 3-D2 photon images. [Figure 12a] These are energy level diagrams for stimulated Raman scattering and coherent anti-Stokes Raman scattering. [Figure 12b] This figure shows an example of localized pulse train generation. [Figure 12c] The figure shows an example of hierarchical pulse splitting: 8-path splitting. [Figure 12d] This figure shows hierarchical pulse splitting using optical fibers and fiber connectors. [Modes for carrying out the invention]

[0028] Various embodiments are described below in detail with reference to the attached drawings, where similar reference numerals represent similar elements.

[0029] This application describes several improvements to the SCAPE system and / or alternative methods for implementing the SCAPE system. In this specification, O1, O2, and O3 refer to the first objective lens, the second objective lens, and the third objective lens in the SCAPE system from sample to detector, respectively. The following acronyms are used herein: ZWD = zero working distance, FOV = field of view, NA = numerical aperture, NIR = near-infrared, GDD = group delay dispersion, PSF = point image distribution function, WD = working distance.

[0030] Section 1: High-Resolution Multispectral SCAPE Design with High Detection NA Figure 1a is a schematic diagram of a novel SCAPE configuration (referred to herein as "Y-SCAPE") primarily designed for multispectral imaging of C. elegans nematodes, but also applicable to other imaging applications such as cell imaging, in-situ sequencing, expansion sequencing, and histopathological diagnosis in fresh tissue. This design includes a novel set of lenses, as well as modifications to the layout and adjustment points. The major advantages of the design in Figure 1a are significantly higher resolution and higher processing capacity than some conventional SCAPE systems, while maintaining a relatively wide field of view with high detection NA using two air immersion lenses as O2 and O3.

[0031] The embodiment in Figure 1a includes a set of first optical elements 10-14 having a proximal end, a distal end, and a first optical axis. The set of first optical elements includes a first objective lens 10 located at the distal end of the set of first optical elements. The embodiment in Figure 1a also includes a set of second optical elements 20-24 having a proximal end, a distal end, and a second optical axis. The set of second optical elements includes a second objective lens 20 located at the distal end of the set of second optical elements. The embodiment in Figure 1a also includes a scanning element 50 located proximal to the proximal end of the set of first optical elements 10-14 and proximal to the proximal end of the set of second optical elements 20-24.

[0032] The scanning element 50 is positioned to deliver the sheet of excitation light so that it passes through the first set of optical elements 10-14 in a proximal to distal direction, and incident on a sample positioned distal to the distal end of the first set of optical elements 10-14. The sheet of excitation light is projected into the sample at an oblique angle, and the sheet of excitation light is projected into the sample at a position that changes depending on the orientation of the scanning element.

[0033] The first set of optical elements 10-14 sends detection light from the sample back to the scanning element 50 in a distal-to-proximal direction. The scanning element 50 is also positioned to send the detection light so that it passes through the second set of optical elements 20-24 in a proximal-to-distal direction and forms an intermediate image plane at a distal position beyond the distal end of the second set of optical elements (i.e., to the left of the second objective lens 20 in Figure 1a).

[0034] In the embodiment shown in Figure 1a, the third objective lens 30 is positioned to direct light arriving from the intermediate image plane toward the camera 40. The camera may include a high-speed camera, an amplified camera, or a camera that is amplified in any other way to enable imaging at very high frame rates, and thus very fine sampling can be achieved during scanning over a wide field of view, resulting in both high resolution in three dimensions and very fast imaging speeds with a high signal-to-noise ratio and less photobleaching.

[0035] The embodiment shown in Figure 1a includes a plurality of light sources 60, each having its own output beam at its respective wavelength. The embodiment shown in Figure 1a also includes at least one optical beam combiner 64 positioned relative to the plurality of light sources 60 to direct the output beams from the plurality of light sources onto a common path of excitation light. At least one pair of alignment mirrors 62 are provided. Each pair of alignment mirrors is positioned relative to each light source 60 to adjust the alignment of its respective output beam (for example, by adjusting the angle and position of the beam). These pairs of alignment mirrors 62 are used to align all the output beams so that the output beams are aligned within the sample. In the shown embodiment, the optical path from the 405 nm light source 60 passes through lenses 66 and 67, and the optical paths from all the other light sources 60 pass through lenses 68 and 69.

[0036] Depending on the circumstances, the laser combiner may include one or more lens systems or other wavefront adjustment systems to adjust the beam size, divergence, or focus of the individual laser sources 60 before their outputs reach the common path, so that their outputs are aligned within the sample. This additional degree of freedom, in conjunction with the alignment mirror 62, may be required to pre-correct chromatic aberration and color effects within the lens system that could result in a misalignment of the illumination light sheet from each laser wavelength in the sample. This pre-correction requires free-space coupling of the combined laser wavelengths to the downstream optical system, which is not readily achievable when the laser wavelengths are combined and delivered through an optical fiber coupler, which is common in other multispectral microscopy systems. This technique enables very fast or simultaneous multispectral imaging by not requiring sequential adjustment of beam characteristics for each illumination wavelength.

[0037] A third set of optical elements 72-76 is configured to expand the output beam into a sheet of excitation light. The sheet of excitation light reaches the scanning element 50 via a second set of optical elements 20-24. More specifically, in the embodiment shown in Figure 1a, the sheet of excitation light is introduced to the second set of optical elements via a second mirror 80 positioned proximal to the second objective lens 20. This second mirror 80 is positioned to receive the sheet of excitation light from the third set of optical elements 72-76 and to direct the sheet of excitation light via an alternative route toward the proximal end of the second set of optical elements 20-24. In some preferred embodiments, the second mirror 80 has a chamfered linear first edge 81 and at least one second edge 82, as shown in Figure 1b. In these embodiments, the second mirror is mounted such that the chamfered linear first edge is closer to the second optical axis than at least one second edge. This second mirror 80 has the advantage of not using a dichroic beam splitter in the main optical path, thus enabling the use of a wider range of wavelengths.

[0038] In some cases, the second mirror 80 may be mounted on a translation stage that provides precise control of the position of the second mirror 80 in a direction perpendicular to the optical axis of the set of second optical elements 20-24, as indicated by the vertical arrow next to the second mirror 80 in Figure 1a.

[0039] The system can also include multiple laser lines and emission filter sets, which enable motionless switching between the fluorescent dye molecules being imaged, as well as hyperspectral imaging, allowing for spectral resolution of hundreds of possible color combinations.

[0040] In the embodiment shown in Figure 1a, the scanning element 50 is mounted at an angle deviating 22.5° from perpendicular to the first optical axis, and the folding mirror 55 is positioned between the scanning element and the set of second optical elements 20-24. However, in an alternative embodiment (not shown), the positions of the scanning element 50 and the folding mirror 55 may be swapped, in which case the scanning element 50 is mounted at an angle deviating 22.5° from perpendicular to the second optical axis, and the folding mirror 55 is positioned between the scanning element 50 and the set of first optical elements. The embodiments described in this paragraph advantageously increase the effective aperture compared to the conventional method in which the folding mirror 55 is omitted and the scanning element is mounted at an angle of 45° to both the second and first optical axes.

[0041] Depending on the circumstances, a cage system swivel mount (e.g., Thorlabs LC1A) may be provided for precise and easy alignment between the two telescopic arms (i.e., positioned between O2 and O3). Alignment may also be optimized using real-time camera-based visualization of O2 and O3 from above, superimposed on images showing ideal angles and positions derived from simulations. If O3 is implemented using the 40x 0.95NA lens shown in Figure 1a, it is preferable that the cover glass be carefully positioned in front of O3 to account for the cover glass compensation required for the 40x 0.95NA lens.

[0042] In some cases, intermediate lens telescopes 32, 36 positioned behind O3 may be provided to project the conjugate plane of the back focal plane of O3 into the image splitter 42. This provides a wider FOV along with better image uniformity. Furthermore, the generation of an intermediate image plane enables image cropping in both the Y and Z directions, which may be important for arranging dual-channel spectrally resolved images on the camera 40.

[0043] Table 1 lists the sets of elements that function well in the embodiment shown in Figure 1a, and Table 2 lists the distances between the centers of the various elements shown in Figure 1a.

[0044] [Table 1]

[0045] [Table 2]

[0046] Section 2: Sample Chamber Design This section describes how to fabricate sample holders for small specimens such as C. elegans nematodes, zebrafish, and microfluidics using lithography, PDMS, agarose, and related materials. For high-speed 3D imaging, it is preferable to carefully move the organism within the 3D field of view. Installation can be difficult if it involves fabricating a temporary chamber.

[0047] To obtain motion-blur-free images of freely moving animals, it is crucial to constrain the physical dimensions of the behavior arena. SU-8 soft lithography, followed by polydimethylsiloxane (PDMS), is a standardized and highly repeatable fabrication technique. However, the significant refractive index difference between PDMS (n=1.41) and water (n=1.33) induces severe aberrations in PDMS-based microchannels. Therefore, we developed a casting stamp technique using a PDMS mold as a secondary mold. The final behavior arena can be created by pressing PDMS onto agarose, thus maintaining a repeatable arena geometry. UV-curable polymers can also be used for casting from PDMS molds and are available in a range of refractive indices, including 1.33, which is more robust than agarose while precisely matching water for aberration reduction (Figure 2a).

[0048] The fabrication of an imaging chamber using lithography facilitates the positioning of the sample and its restraint to fit the microscope field of view. To accommodate the requirements of the objective lens and improve resolution, options such as glass coverslips, no coverslips, refractive index-matching polymers, and FEP coverslips may be used depending on the case. See Figures 2a to 2e. For capillary action to fill and protect the surface, the top may be covered with a material such as a coverslip, refractive index-matching polymer film, or FEP, as shown, for example, in Figure 2d.

[0049] The design (as shown, for example, in Figure 2b) may include a wide range of well sizes / regulatory positions to allow for the mounting and identification of multipurpose specimens. Final casting can be quick and inexpensive. Many designs can be made for experimental use. For example, if selecting nematodes of a specific size to fit into a microscope field of view, a multipurpose mold may have multiple different shapes and sizes available on a single slide. Sizes can be organized by a grid / referenceable symbol. The design may allow for the mounting of several specimens simultaneously. Polymer molds may be reusable.

[0050] The design can incorporate “alignment target” type structures on the slide for calibration, verification, and field of view. If made from polymer, the channels (e.g., as shown in Figure 2c) can accommodate tabs for 3D contrast to introduce and fill with fluorescein or similar. Lithography fabrication can also be used to include targets for system characterization, including 3D calibration and field of view uniformity assessment. These targets can be incorporated into each sample holder plate with minimal additional overhead costs.

[0051] Multilayer lithography can be used for 3D calibration and to make standardized targets that match the refractive index of the sample more permanent. FEP (fluorinated ethylene propylene) is a type of plastic with a refractive index close to that of water and can therefore be used instead of glass coverslips to constrain the sample without causing aberrations due to refractive index mismatch. Coupled with this high-resolution system and a "single objective lens" geometric arrangement, the inventors have found that incorporating FEP into their imaging chamber significantly improves residual aberrations in the system. See Figures 2d-2e.

[0052] Section 3 Additional Monitoring and Manipulation of Samples Figures 3a and 3b illustrate the incorporation of secondary sample imaging, intensive sample heating (e.g., laser heating), and active sample cooling for interacting with the sample, including tracking, noxious stimulation, and immobilization. These embodiments enable interaction with the specimen through real-time imaging of behavior, movement, and cellular signaling responses. Optionally, supplemental imaging by NIR and tracking may be performed. Optionally, one or more of the embodiments of sample imaging and manipulation shown in Figure 3a may be included.

[0053] Figure 3b shows a localized temperature rise due to IR illumination. More specifically, Figure 3b shows an image of the temperature-dependent change in FITC fluorescence emission (in agar). Using a visible beam allows for target alignment. The heating laser can be controlled by acquisition software so that its timing is carefully determined and / or its output is dynamically adjusted. This can be achieved, for example, by closed-loop analysis of images from a behavior camera.

[0054] The system was tested against targeted heating stimuli in the nematode C. elegans. The same or similar focusing or patterning techniques may be used for optogenetic stimulation.

[0055] Section 4 Maximizing Detected NA The detection NA is reduced in SCAPE due to the need to rotate the image between O2 and O3. Figures 4a–4c derive the conditions governing the detection NA in SCAPE based on the various lenses used for O1–O2–O3. They show, firstly, that high NA O3 in Y-SCAPE (i.e., the configuration shown in Figure 1a) provides a significantly improved detection efficiency compared to some previous designs. However, the field of view is limited in the current embodiment due to the commercial availability of suitable lenses, and in this case, a commercially available 40x 0.95NA lens as O3 and a 50x lens as O2 were selected to have sufficient space to position the lenses together.

[0056] This analysis supports the idea that a wider field of view can be obtained by using two immersion lenses as O2 and O3, and positioning them with water between them. The numerical aperture (NA) is not as good as Y-SCAPE, but it is better than some previous designs.

[0057] Referring next to Figures 4b and 4c, the inventors typically used an air objective lens with an angle of O2 = 48.59°, which corresponds to the approximate angle of the sheet in the sample when O1 is a water immersion lens with a 1.0 NA. A high NA O3 is desirable to collect as much light as possible while aligning the focal plane of O3 with the oblique image of the light sheet relayed from the sample. The choice of this lens is usually limited by the working distance available to physically position the lens, and the narrower field of view of lenses with higher NA (generally, higher magnification).

[0058] This is based on the formula NA = n sinα. The table below shows the angle α obtained when various combinations of NA and refractive index n are used with respect to the objective lens.

[0059] [Table 3]

[0060] Next, looking at Figure 4c, the inventors believe the following relationships exist: Seat angle = α1 - β; γ = α² / ² + α¹ - β - 4⁵; In the formula, 2γ is the angular range of light accepted by O3.

[0061] The previous configuration used a 1.0NA water objective lens for O1, a 0.75NA air objective lens for O2 (α1=48.59), and a 10x 0.45NA air lens for O3, which yielded an angle α2 of 26.74° (using NA=n sinα). Therefore, when β=0, γ=16.96°.

[0062] In the embodiment shown in Figure 1a, using O3 as a 0.95NA 40x air lens results in an angle α2 of 71.81°. Therefore, when β=0, γ=71.81 / 2-45+48.59=39.5°.

[0063] In another embodiment, when the inventors match O2 and O3 as a 1.0NA immersion lens (α1=α2=48.75°) with respect to the maximum sheet angle (β=0), γ=48.59 / 2-45+48.59=27.88°, which allows for a much wider field of view (>1mm) than the Y-SCAPE configuration in Figure 1a.

[0064] Section 5: Design of the Water Chamber We have previously explained that instead of using the two air objective lenses of Y-SCAPE (as in the embodiment in Figure 1a), three water immersion objective lenses can be used for O1, O2, and O3 to obtain not only a wider field of view but also better resolution and processing power compared to some previous SCAPE designs.

[0065] However, using water immersion lenses in O2 and O3 requires a water chamber. While many optical sheet systems use water chambers, they are generally used to hold the sample during imaging and have various requirements and constraints. Here, we describe a stable and safe design that reduces the possibility of leakage, evaporation, or contamination while maintaining degrees of freedom for alignment.

[0066] To accommodate the two immersion objective lenses in O2 and O3, the inventors designed various immersion chambers that support dynamic alignment (Figures 5a-5c).

[0067] Similar designs have also been made to hold a cover glass on the objective lens in a cover glass corrected system (Figure 5d). The inventors have found that cover glass correction can be an important factor for precision imaging. This includes including a cover glass between O2 and O3 when either O2 or O3 lens requires a cover glass. The inventors have manufactured various caps for use in holding those cover glasses. In some embodiments, precision alignment is preferred unless the cover glass can be permanently bonded to the objective lens. There may be some interrelationship between whether a cover glass is included or omitted in O2 and whether a cover glass is required in O1.

[0068] Section 6: Zero Working Distance Method (ZWD) The presence of two different immersion media between O2 and O3 allows for the acceptance of a much larger optical cone into O3. A novel system has been constructed that incorporates a zero working distance (ZWD) length lens as O3 into a standard SCAPE2.0 layout including a 20x 1.0NA water immersion main objective lens (O1). Adding this type of ZWD lens to a conventional SCAPE system provides a high-resolution version of SCAPE (even over a narrower field of view) for high-speed 3D intracellular imaging. Additional versions of this ZWD lens exist with wider fields of view and can be incorporated into this SCAPE design to provide a wider and more useful field of view by increasing resolution and light processing capacity (via an increased detection NA).

[0069] Figures 6a to 6e derive the basis for this ZWD method, showing that, in principle, 100% of the light coming from O1 is detectable regardless of the NA of O1 or O2 (as long as O2 has a sufficient NA to relay all of O1's NA and accidental losses are ignored).

[0070] The expected angle-dependent losses, polarization-dependent losses, and air-glass versus air-water refractive indices for n1-n2, along with the significant reflections expected for materials with higher refractive indices, are shown in Figures 6f-6g. This reflection reduces the amount of light entering O3, which depends on the refractive index (n2) of the material forming the ZWD interface, with the losses being greatest for higher refractive indices (e.g., glass) and for rays with large incident angles (e.g., rays labeled B).

[0071] Considering the NA and RI of O1, O2, and O3, referring to Figures 6a–6e, conventional SCAPE designs have typically used a 1.0 NA water immersion lens as O1. By using 0.75 NA air as O2, a full detection angle of 48.59° is maintained behind O2. Thus, a 1.0 NA water immersion lens as O3 (containing water up to the focal plane aligned with the image of the oblique light sheet) captures 100% of the light collected from the sample by O1. This is because 1.0 NA = 90° in air, and therefore any 1.0 NA objective lens as O3 paired with an air objective lens as O2 captures approximately 100% of the light from O2. This means that a standard 1.0 NA water immersion objective lens can be modified by adding a "water spacer" to meet this requirement.

[0072] The only possible benefit of switching to a non-dry immersion lens for O2 might be the ability to leverage the higher NA from O1. However, a 1.1 NA water lens in O1 produces 55.7° in O2, which (unless there are WD constraints) can be accepted by a 40 x 0.95 NA dry immersion lens in O2. Increasing the NA of O1 and O2 increases resolution and processing capacity, but increases oblique angles, reflection losses, and generally narrows the field of view.

[0073] Even when O1 and O2 have low NAs (e.g., 0.5 in air), the effect of ZWD will significantly improve the light detection efficiency. In some cases, when α1 is small and both O2 and O3 are air objective lenses, the amount of light detected by O3 may be zero (e.g., with respect to Figure 4c, 45 + β - α1 > α2 / 2). By using a ZWD lens at O3, the light collection efficiency can be greatly improved in this case, making it possible to use a low-magnification (and generally low-NA) lens as O1 for applications such as wide-field, long-working-distance, air-immersion distributed refractive index (GRIN) lenses.

[0074] Section 7: Multi-immersion adapter The adapter has the ability to enable the pairing of various lenses with different immersion while leveraging refractive index mismatch to capture more light at the optical interface between O2 and O3. These embodiments can also be used to hold a cover glass between O2 and O3 when a cover glass corrected objective lens is used. The inventors recognize the flexibility of this method to capture about 100% of the light from any system using various immersion lenses—with the only condition being that they have 1.0 NA.

[0075] Recognizing that a 1.0 NA immersion objective lens is sufficient for O3, the inventors recognize that the ability to add a spacer in front of a 1.0 NA immersion objective lens provides a versatile, low-cost, and wider-field-of-view option ZWD lens for obtaining a larger detection NA without the need for custom lenses such as lenses containing a glass frustum that provides a ZWD interface. Figure 7 shows various suitable 3D-printed adapters for holding water (or other immersion medium) droplets for these purposes, along with a front surface that is constrained by a coverslip (in the case of a coverslip-corrected immersion objective lens) or other refractive index matching material (such as FEP) to restrain the droplets of immersion medium.

[0076] Section 8: Zero Working Distance (ZWD) "Blob" Method Taking into account the difficulty of using truly liquid water for immersion, the inventors developed a technique for fabricating a spacer with a suitable refractive index that can be mounted in front of a 1.0 NA immersion lens to convert a 1.0 NA immersion lens used as a third objective lens (O3) into a ZWD lens to maximize the detected NA. This was achieved using a 1.0 NA, 2 mmWD, 20x water immersion objective lens and a UV-curable polymer with a refractive index of 1.33. This lens is not coverslip corrected, and therefore the spacer is formed as a single unit without a glass coverslip or other material at the focal plane. The material used also has low autofluorescence. Details in Figures 8a–8i show the fabrication, mounting, and alignment procedures for this technique (referred to herein as the “blob” technique), including the significant advantages of this technique over using a glass frustum-based ZWD lens as O3.

[0077] Figure 8d shows the steps of a first method for fabricating what the inventors refer to in this section as a "blob" using BIO-133 (a UV-curable polymer with the same refractive index as water). The first step is to 3D print a negative mold. The next step is to mount a glass slide onto the 3D printed mold. The next step is to inject BIO-133 and then remove the gas using a vacuum. The next step is to add a second glass slide to the top to create a flat surface and then UV-cur the polymer. However, it should be noted that this first method has a drawback: the BIO-133 cannot be accessed from the side, which makes release difficult (as contact with the top and bottom surfaces should be avoided).

[0078] Figure 8e illustrates the steps of the second method for fabricating the “blob” discussed in this section. The first step is to 3D print a double negative mold. The next step is to inject PDMS, then cure the PDMS, and remove the PDMS from the first mold. At this point, the negative mold made from PDMS has a thin bottom. The next step is to remove the thin PDMS bottom to create a set of sidewalls surrounding the through-hole. The set of sidewalls may consist of multiple surfaces (e.g., four sidewalls in the case of a square) or only a single continuous surface (in the case of a cylinder). The next step is to press the PDMS sidewalls onto a high-flatness glass slide. The PDMS sidewalls adhere by reversible bonding. The next step is to inject BIO-133 and remove the gas using a vacuum. The next step is to add a second glass slide to the top to create a flat surface, followed by UV curing. The final step is to remove the PDMS sidewall and slide glass, leaving the cured BIO-133 polymer. Since oxygen weakens the UV curing of polymers and PDMS is oxygen permeable, the use of PDMS here creates a layer of uncured polymer between the PDMS-BIO133 interface, which facilitates removal.

[0079] Now, looking at Figure 8f, regardless of the method used to fabricate the "blob" (including, but not limited to, the two methods described above in relation to Figures 8d-8e), the blob and the third objective lens O3 are assembled to form an assembly using, for example, the steps shown in Figure 8f. More specifically, the first step in this example is to bring the polymer blob into contact with a clean, patterned glass slide (e.g., a resolution target). The next step is to gently bring a 3D printed support into contact with the polymer. This is intended to support the polymer later and prevent buckling. The support and polymer can be bonded to each other using UV adhesive. The next steps are to place the assembly under O3, fill the gap with water, and focus O3 at infinity using a tube lens until a focused image is present on the camera. The next step is to attach the 3D printed second device to the polymer assembly. The intention here is to fix the polymer blob in an optimal position by continuously observing the resolution target from the camera. However, it should be noted that when the procedure in Figure 8f is used, it can be difficult to ensure that the added blob is precisely aligned with the focal plane of the objective lens, with its (very smooth) front surface precisely aligned.

[0080] Figure 8h shows the steps of a third method for fabricating the blob discussed in this section, which yields very good performance and facilitates precise alignment of the blob's front surface with the focal plane of the third objective lens (O3). This method is similar in many ways to the method described above in relation to Figure 8e, except that a high-flatness mirror 95 is used instead of the lower slide glass, and a second slide glass is not added on top of the blob prior to UV curing. Thus, the steps of this third method are as follows: The first step is to 3D print a double negative mold. The next step is to inject PDMS, press the double negative mold into it, and then cure and remove the PDMS. At this point, we have a negative mold made from PDMS with a thin bottom. The next step is to remove the thin bottom of PDMS to create a set of side walls surrounding the through-hole. The set of sidewalls may consist of multiple surfaces (e.g., four sidewalls in the case of a square) or only a single continuous surface (in the case of a cylinder). The next step is to press the PDMS sidewalls against the mirror 95. The PDMS sidewalls adhere to the mirror 95 by reversible bonding. The next step is to inject BIO-133 and remove the gas using a vacuum. The next step is to UV cure it. The final step is to remove the PDMS sidewalls, leaving a cured BIO-133 polymer "blob" which is still attached to the mirror 95 at this point. In some embodiments, the cured blob has a thickness between 75 and 95% of the working distance of the objective lens to which the blob is ultimately mounted. For example, if the working distance of the objective lens is 2 mm, the cured blob may have a thickness of 1.8 mm.

[0081] In this third method, the front surface of the blob is formed on a very flat mirror 95, rather than on a coverslip or microscope slide. The dielectric front mirror is manufactured with an ultra-flat surface—with a precision within approximately a quarter wavelength range. Thus, the mirror is flat with a tolerance of less than 250 nm. This not only makes the mirror ideal for forming the ultra-flat coplanar surface of the blob, but the fact that the front surface of the blob contacts the mirror is used in alignment processes, as detailed below.

[0082] Referring next to Figure 8i, the entire rig is first aligned using a digital inclinometer. As shown, the initially molded blob 91, approximately 1.8 mm thick, which is still mounted on the molded mirror 95, is then positioned in front of the objective lens 30, with an amount of uncured BIO-133 polymer 92 between the clean glass front surface of the objective lens and the cured BIO-133 blob 91. The optical path marked with vertical stripes in Figure 8i represents parallel laser light that has been amplified and sent to the objective lens via a 50:50 beam splitter. This light is reflected by the mirror 95 (through the blob) and returns through the objective lens 30. The position of the mirror 95 (which is still mounted on the blob 91) is then adjusted in both 1-distance and 2D incline, while the parallel characteristics of the returning light are monitored, for example, using a sheer plate to check for precise parallelism. The reflected light becomes parallel only when the mirror (and therefore the front of the "blob") is precisely aligned with the focal plane of the objective lens 30. Once this state is reached, oxygen is purged from the environment around the polymer blob 91 (this ensures proper curing of the polymer), and UV light (represented by transverse stripes) is projected downward through the objective lens (via a dichroic beam splitter) to cure the liquid polymer 92 between the already cured "blob" 91 and the glass surface of the objective lens 30, providing a permanent bond. The mirror 95 is then peeled off from the front of the "blob" 91. Additional UV light may then be used to ensure complete curing of the polymer.

[0083] The most important step of the method shown in Figures 8h to 8i for fabricating a spacer for the immersion objective lens 30 is as follows: First, as seen in Figure 8h, the side walls of the negative mold are pressed against the mirror 95 so that the set of side walls cooperate with the bottom of the negative mold to form a liquid-tight cavity, with the portion of the mirror positioned between the set of side walls acting as the bottom of the negative mold. Next, the liquid-tight cavity is filled with a first amount of UV-curable polymer. The first amount of UV-curable polymer is then cured into a first solid mass 91. The bottom surface of the first solid mass 91 adheres to the mirror 95. Next, the set of side walls is removed from the mirror 95 without disturbing the adhesion between the bottom surface of the first solid mass 91 and the mirror. Next, the top surface of the first solid mass 91 is positioned near the objective lens with a second amount of UV-curable polymer 92 occupying the space between the top surface of the first solid mass 91 and the objective lens 30. Following positioning, the position of the first solid mass 91 is adjusted until the lower surface of the first solid mass (in direct contact with the upper surface of the mirror 95) reaches its final position relative to the objective lens 30. After the first solid mass 91 reaches its final position, a second amount of UV-curable polymer 92 is cured.

[0084] A good method for obtaining precise adjustment of the position of the first solid block 91 is to project parallel light through the objective lens 30 toward the mirror 95 while detecting the parallel characteristics of the light reflected by the mirror 95. The determination that the first solid block 91 has reached its final position is made when the light reflected by the mirror 95 becomes perfectly parallel. This can be achieved, for example, by using a shear plate.

[0085] A suitable method for curing the second amount of curable polymer 92 is to project UV light into the second amount of UV-curable polymer through the objective lens 30. If necessary, following the projection of UV light into the second amount of UV-curable polymer 92 through the objective lens, additional UV light is applied to further cure the second amount of UV-curable polymer.

[0086] After the second amount of UV-curable polymer has cured, the mirror 95 is removed from the underside of the first solid mass 91 so that the objective lens 30 can be used.

[0087] The zero working distance technique can be extended beyond the examples above. Any type of solid material (or constrained liquid) with a suitable refractive index can be used to modify existing immersion lenses. There are many UV-curable (or curable / activatable via, for example, time, heat, irradiation, or chemicals) compounds or adhesives with precise refractive indices that can be used to form permanent extensions to immersion lenses (not only water immersion lenses, but also lenses with a refractive index of 1.0 NA). See, for example, Non-Patent Document 1. RI (refractive index) matched gels with varying viscosities and minimal evaporation are also available. See, for example, Non-Patent Documents 2 and 3.

[0088] Another option is to use PDMS (refractive index approximately 1.43) in combination with lenses designed for tissue clearing imaging (clearance RI approximately 1.4), some of which have correction rings to allow for precise matching for the refractive index of the "blob" material. To capture more light in O3, precision-molded spacers may provide permanent modifications to those high NA, long WD lenses.

[0089] Cover glass corrected immersion lenses are also available with a glass front chamber, where the space is filled with a liquid or, for example, a curable polymer, as described herein. FEP-based front water chambers may be used for water immersion dipping lenses without cover glass correction. Certain other plastic or silicone materials may also be fabricated into solid blocks or chambers to match common silicone or oil immersion refractive indices. Multi-immersion and refractive index adjustable lenses may also be used due to the ease of material selection.

[0090] To protect the "blob" element from dust or other environmental factors that may alter its optical properties, material shape, or optical integrity, it is advantageous to add a protective tube or housing around the finished modified lens, possibly with a removable cap. This can take the form of a capped chamber (without the addition of immersion fluid) as shown in Figures 5a–5c, which allows for adjustment of both lenses for alignment.

[0091] The advantages of this "blob" technique on objective lenses, such as NA 1.0 water objective lenses as ZWD O3, include: (1) The "blob" technique is a simple and inexpensive modification of a typical objective lens (e.g., a 1.0 NA water immersion lens) costing around US$6,000. (2) By using a deformable polymer material, damage during alignment is prevented and it can be molded to accommodate various O2 geometries. (3) The blob can be removed / replaced / regenerated as needed. (4) A wide field of view (e.g., >1 mm) can be obtained due to the well-characterized performance of the O3 lens used. (5) Reduced surface reflection to air compared to high NA glass: 1.33 interface. (6) A similar 100% receiving angle of light from O2 is achievable. Also, compared to air and glass, there is a better tolerance for out-of-focus in the image plane thanks to the smaller refractive index mismatch between air and water. The above is important because remote focusing mapping of oblique planes generally introduces small curvature of the oblique plane. However, it should be noted that this may be a limiting factor in the depth range available for the ZWD method.

[0092] Section 9 Two-photon SCAPE Figure 9a shows a schematic and experimental configuration of a two-photon ("2P") SCAPE system that can utilize the entire illumination sheet and yield 20 volumes per second of two-photon imaging in scattering tissues such as the brain of a living mouse. A 100W, 1MHz, 100μJ pulsed pump laser (Spectra-Physics) with pulse picking up to 500kHz was used to pump a non-collinear optical parametric amplifier (NOPA) to generate 940nm pulses.

[0093] The labels in Figure 9a are as follows: HWP: Half-wave plate used for beam attenuation, EOM: Electro-optic modulator for blocking the beam during galvanometer mirror flyback, TAG: Tunable acoustic gradient lens used for adjusting the axial position of the high-NA light sheet in the sample, CL: Cylindrical lens for generating the light sheet, L1 / 2: Achromatic lens for relaying, SL1 / 2: Scanning lens, TL1 / 2 / 3: Tube lens, O1 / 2 / 3: Objective lens, DM: Dichroic beam splitter (700nm short pass), EF: Emission filter. The camera was either a Zyla 4.2+ (Andor) or a HICAM Fluo (Lambert).

[0094] Imaging configurations were developed for experiments in awake and active mouse and zebrafish larvae receiving visual stimuli. The SCAPE imaging geometry is shown in the lower right corner of Figure 9a.

[0095] The embodiment in Figure 9a includes a set of first optical elements 10-14 having a proximal end, a distal end, and a first optical axis. The set of first optical elements includes a first objective lens 10 located at the distal end of the set of first optical elements. The embodiment in Figure 9a also includes a set of second optical elements 20-24 having a proximal end, a distal end, and a second optical axis. The set of second optical elements includes a second objective lens 20 located at the distal end of the set of second optical elements. The embodiment in Figure 9a also includes a scanning element 50 located proximal to the proximal end of the set of first optical elements 10-14 and proximal to the proximal end of the set of second optical elements 20-24.

[0096] The scanning element 50 is positioned to deliver the sheet of excitation light so that it passes through the first set of optical elements 10-14 in a proximal to distal direction, and incident on a sample positioned distal to the distal end of the first set of optical elements 10-14. The sheet of excitation light is projected into the sample at an oblique angle, and the position of the sheet of excitation light changes depending on the orientation of the scanning element.

[0097] The first set of optical elements 10-14 sends the detection light back from the sample to the scanning element 50 in a distal-to-proximal direction. The scanning element 50 is also positioned to send the detection light so that it passes through the second set of optical elements 20-24 in a proximal-to-distal direction and forms an intermediate image plane at a distal position beyond the distal end of the second set of optical elements (i.e., to the left of the second objective lens 20 in Figure 9a).

[0098] In the embodiment shown in Figure 9a, the third objective lens 30 is positioned to direct light arriving from the intermediate image plane toward the camera 40. Optionally, the third objective lens 30 and the second objective lens 20 are optically coupled via a fluid chamber 90.

[0099] However, in alternative embodiments, the high NA light-receiving angle-fused fiber bundle may be positioned to relay light from an intermediate image plane distal to the second objective lens 20 toward the camera. In these alternative embodiments, the fiber bundle may have a front surface that aligns with the image of the oblique light sheet formed by the second objective lens 20, or the fiber bundle may have a bevel-cut edge that aligns with the image of the oblique light sheet formed by the second objective lens 20 in order to collect light and enable image rotation.

[0100] Figure 9b shows a method for aligning the third objective lens O3 to the camera telescope in the embodiment of Figure 9a. This configuration allows for finer adjustment of the focal plane without significantly affecting the rest of the optical path of O3. As shown here, the mount of O3 is separated from the camera tube lens and camera mount. Previous embodiments had fine adjustment of the focus (parallel to the direction of the objective lens). However, we have found that lateral and vertical adjustment of this lens (and, where possible, rotation, tip, and tilt) is best performed by a micropositioner on the mount holding O3. While fine adjustment of its position has little bearing on the beam that leaves O3 and passes through the image splitter and tube lens in front of the camera, it can provide extremely important fine adjustment for focusing on the intermediate image plane.

[0101] In some cases, the system in the embodiment of Figure 9a may use a water chamber to couple the two water immersion objective lenses O2 and O3. Various designs have been developed for this water chamber to allow adjustment of the position of O3, etc. This water immersion configuration appears to be a viable option for imaging deeper depth ranges (e.g., about 400–450 micrometers) accessible using two-photon SCAPE.

[0102] Alternatively, improved light collection efficiency and resolution can be achieved using an air objective lens as O2 and a zero working distance (ZWD) lens as O3, along with a glass interface or material that matches the immersion medium of the immersion objective lens. If such a lens has an NA of 1.0, in principle, it should be possible to capture the entire angle of light that is captured by O1 and relayed through O2 (in this case, O2 would be replaced by an air lens with an NA of 0.75). Another ZWD configuration may use a high-NA light-receiving angle fused fiber bundle having a bevel-cut edge that is aligned at the focal plane by its front surface or aligned with the focal plane to collect light and achieve image rotation, in order to relay light from the mid-image plane behind O2. Here, the back side of the fused fiber bundle is imaged using a suitable O3.

[0103] However, while these ZWD options should enable efficient light collection, it is anticipated that their ability to capture the mid-oblique image plane along its entire Z range will be limited due to aberrations in the image between O1 and O2. Therefore, the water-water technique detailed above may be optimal for light collection along the entire (extended) depth range of the 2P-SCAPE.

[0104] An alternative approach to overcome this problem would be to use adaptive optics to optimize the illumination wavefront and generate an image as flat as possible behind O2 for efficient collection into O3.

[0105] Using a TAG lens in the embodiment shown in Figure 9a offers significant advantages. A TAG lens is a rapidly moduloable, tunable lens (e.g., Mitutoyo TAGLENS-T1, available from Mitutoyo America Corporation). Using a TAG lens at the position indicated in Figure 9a allows for the generation of a higher NA optical sheet with a narrower (but still stretched) waist to achieve more efficient nonlinear excitation in a thinner plane, and enables the very rapid sweeping of this waist up and down within the sample to generate an equivalent optical sheet to be imaged on the camera.

[0106] Figure 9c shows three promising methods for using TAG lenses. More specifically, panel 1 of Figure 9c (i.e., the upper panel) shows a first method that allows scanning of the focal plane in Z without changing the parallelism of the sheet. However, this method requires the system's laser to be focused within the lens, which was not possible with our selection of laser parameters (as the focused laser output could exceed the recommended damage threshold for the lens).

[0107] However, notably, the inventors have determined that safe generation of relatively uniform sheet excitation in the sample is possible by making two modifications to the layout shown in Panel 1. These two modifications are as follows: First, the TAG lens is combined with an external concave cylindrical lens (as shown in Panel 2, i.e., the central panel). Second, the TAG lens is mounted so that the beam is eccentric with respect to the optical axis of the TAG lens and crosses the TAG lens (as shown in Panel 3, i.e., the lower panel).

[0108] Returning to Figure 9a, the light source 160 generates excitation light. In the shown embodiment, the light source 160 generates pulses of excitation light, which are compressed in time by the prism compressor 166 before they reach the TAG lens 110.

[0109] The TAG lens 110 has an optical axis and is positioned to receive excitation light such that (a) the excitation light travels through the TAG lens parallel to the optical axis of the TAG lens, and (b) the excitation light travels through the TAG lens eccentrically with respect to the optical axis of the TAG lens. The cylindrical lens 115 is positioned in series with the TAG lens 110 such that the sheet of excitation light exits from the series combination of the TAG lens and the cylindrical lens. The reflective surface 58 (e.g., a dichroic beam splitter) is positioned to direct the sheet of excitation light toward the scanning element 50.

[0110] In the shown embodiment, the cylindrical lens 115 is positioned between the TAG lens 110 and the reflective surface 58, and the cylindrical lens magnifies the excitation light exiting the TAG lens into a sheet of excitation light.

[0111] The following explains why the combination of the two modifications identified above is beneficial: When the laser beam propagates through the center of the TAG lens (panel 2 in Figure 9c) (or, in an alternative embodiment, an electrically adjustable lens (ETL)), the x and y dimensions have the same wavefront modulation amplitude, which leads to curvature along the y dimension of the optical sheet, as seen in the left column of Figure 9d. However, when the laser beam propagates eccentrically within the TAG lens, as shown in panel 3 of Figure 9c, the wavefront modulation amplitude is mostly along the x dimension, as seen in the right column of Figure 9d, which results in a focus change (axial scanning) mainly along the x dimension, greatly improving the uniformity of the optical sheet along the y dimension.

[0112] Figure 9e shows fluorescence measurements when an eccentrically aligned TAG lens is scanned with various amplitude modulations. Figure 9f shows the change in the irradiation field when the beam is moved off-axis. Figures 9g to 9h show simulation results illustrating the expected asymmetric beam modulation when light is transmitted eccentrically through the TAG lens.

[0113] A 2P-SCAPE system has the ability to penetrate and image samples much deeper than a standard 1P-SCAPE. If more camera rows need to be read out to sample the required depth range, the image may need to be acquired more slowly overall, which can reduce either the x-direction field of view, the x-direction sampling density, or the overall volumetric imaging rate. While 200-300 camera rows may span the required depth range of a sample in a 1P-SCAPE, a larger depth number is beneficial in relation to a 2P-SCAPE. Each of the two options discussed immediately below increases the depth number.

[0114] The first option (see Figure 9i) is to rotate the camera orientation so that the camera reads rows as horizontal pixels (Y), enabling unlimited depth readout along the column. Rotating the camera in 2P-SCAPE so that Z' aligns with the column rather than the row allows for deeper imaging of the sample, along with a reduced horizontal (Y) field of view. This configuration is suitable for high-speed imaging of 400+ columns, achieving a depth of >400 microns at approximately 1 micron per pixel.

[0115] A second option (see Figure 9j) is to incorporate asymmetric (e.g., unilateral) magnification into the O3 telescope to compress the image in Z (without rotating the camera). For example, a cylindrical lens telescope could be inserted into the optical path between O2 and the camera to allow compression of oblique planar images along the z direction, so that fewer rows on the camera need to be read out to span a larger depth range. Most multi-depth 2P systems have large gaps between the Z planes, so reducing ours from 1 to (e.g.) 3 or 4 microns is not disadvantageous compared to competitors' systems, and therefore the above is a reasonable trade-off.

[0116] Section 10: Dual-camera SCAPE with a wide field of view Figures 10a–10d illustrate another single-photon SCAPE system using a D-mirror, three (expandable) excitation wavelengths, water / water objective lenses for O2 and O3, and two cameras for high-speed dual-color acquisition over a >1 mm FOV. These embodiments use a D-shaped mirror rather than a dichroic beam splitter to emit light. These embodiments also use two water immersion lenses as O2 and O3 (matched with O1) for higher NA detection compared to more conventional O2-O3 air-air SCAPE designs. In some embodiments, O2 and O3 are 20x, 1.0 NA, 2 mm WD water immersion lenses. These lenses are preferably aligned within a water chamber. The system incorporates two cameras for imaging to enable a wider field of view, higher resolution (higher sampling density), and imaging of two-color samples. An image splitter / image steering design accommodates both cameras. The computer architecture is designed to achieve full-speed readout of both cameras simultaneously. Figure 10c shows a suitable set of parameters for implementing a scanning telescope in these embodiments using finer rotation of the primary objective lens including a prism mirror, and Figure 10d shows a suitable set of parameters for implementing a scanning telescope in these embodiments using a Plössl lens that is coplanar with the back surface of the lens tube.

[0117] Section 11: Embodiment of DART-Descan Axial-Resolved Two-Photon Design Figure 11a shows a descan axially resolved two-photon embodiment referred to herein as “DART”. This embodiment uses an axial beam incident at an oblique angle rather than a full sheet. This beam is scanned in the x and y directions on the sample and descanned in the x and y directions so that the depth-resolved signals can be detected by a linear detector array or a series of individual detectors. This design preferably includes a telescope between the two axes of scanning galvanometer mirrors G1, G2 to optimize the method of scanning the beam to avoid positional displacement. The system may optionally use spatial light modulation (SLM) to shape the illumination beam.

[0118] These embodiments share the advantages of SCAPE's fast 3D imaging with a simpler implementation than competing fast 3D 2-photon systems that use complex means such as pulse delays to encode the origin of signals excited by line illumination. Compared to 2P-SCAPE, this embodiment provides higher resolution in x and y, limited only by the PSF of the incident beam, and scattering effects primarily affect z resolution. Thus, this technique can be conveniently used to penetrate deeper into scattering samples such as mammalian brains, with the potential for extension to imaging other forms of contrast, such as 3-photon fluorescence, second or third harmonic generation, coherent anti-Stokes Raman spectroscopy and stimulated Raman spectroscopy (CARS and SRS), fluorescence lifetime and phosphorescence lifetime.

[0119] These embodiments use linear detectors rather than cameras, but several methods exist for mapping laterally positioned portions of a descanned line image to multiple detectors. In one example, the position may be relayed to individual detectors using a series of polished optical fibers or tapered fiber bundles positioned in the image plane descanned after O2. Each channel of these detectors can be read at very high speeds, enabling parallel detection at a higher pixel rate than achievable with a camera. Each detector may also be more sensitive or have better noise equivalent power for high-bandwidth detection compared to camera pixels. This higher temporal bandwidth can be used to encode additional information such as fluorescence lifetime, spatial coding, or spectral coding.

[0120] Examples of images obtained with this prototype, which uses a linear photomultiplier detector array, are shown in Figures 11b and 11c. Although unoptimized, these images demonstrate high-resolution depth-resolved imaging results from various biological samples.

[0121] Since the beam must be raster-scanned across x and y to sample each volume, the laser repetition rate is one constraint in these embodiments. This constraint stems from the availability of a pulsed laser with sufficient power per peak, coupled with a suitable repetition rate (e.g., 4 MHz for 512x512x depth imaging at approximately 15 volumes per second) and a reduced achievable integration time per pixel compared to 2P-SCAPE. However, the inventors have recognized that two or more axial beams are usable, as long as the beams are separated by sufficient space to allow for isolated detection (Figure 11d). When projected onto a two-dimensional detector array, this compromise allows for an increase in the volumetric imaging rate of nx for additional axial beams of nx (and more detector channels of nx).

[0122] Notably, the volumetric imaging speed in these DART embodiments is currently limited by the 1–2 MHz repetition rate of the laser system (which provides far better excitation than an 80 MHz Ti:sapphire laser for equivalent average power). To improve the imaging speed, multiple axial beams can be generated and descanned onto a 2D detector array (e.g., an 8x8 element photomultiplier tube array). Each additional beam N increases the volumetric imaging speed by N times. Abundant power is available to generate these split beams, and the resolution is unaffected if the beams are sufficiently spaced apart (resulting in better xy resolution than 2P-SCAPE).

[0123] It should be noted that a significant improvement in detection efficiency can be achieved by using the zero working distance (ZWD) techniques described above in O3. These may include molten fiber bundles as described above, or lenses having glass or immersion medium matching extensions that allow collection of light entering O1 at all angles when paired with a suitable air objective lens in O2. These improvements may be beneficial to both single-beam and multi-beam embodiments of DART.

[0124] Section 12: DART for Raman Contrast Next, looking at Figure 12a, the inventors recognized that 2P-SCAPE and DART can be used to image Raman scattering contrast. These embodiments are referred to herein as “Raman DART”. Conventional Raman spectroscopy has very low signal levels, but stimulated Raman microscopy is a method that can significantly improve imaging speed and signal-to-noise ratio. Point scanning stimulated Raman microscopy (SRS) is performed by temporally modulating two combined beams—a pump beam and a Stokes beam. When the energy difference between these beams is equal to the Raman absorption band, the Stokes signal will increase and the pump signal will decrease. This action is nonlinear and therefore works best with pulsed lasers and has optical sectioning characteristics similar to two-photon excitation. Both signals are high, and the modulation of the signals is only a few percent of the signal (ΔI / I < 10⁻¹⁰ -4 However, single-channel lock-in detection can pick up the amplitude of luminance modulation, which enables imaging of Raman contrast.

[0125] The linear depth-resolved detection of DART and the use of a single-channel detector (preferably with high bandwidth) make this modulation and lock-in detection strategy feasible. Embodiments of Raman DART can enable rapid 3D imaging of Raman contrast by utilizing various alternative sources of contrast on the sample without requiring fluorescence emission.

[0126] In these embodiments of Raman DART, the improved volumetric imaging speed of Raman contrast can capture the unique in-vivo dynamics of objects such as neurotransmitters having specific Raman absorption bands. Contrast agents based on causing a shift in the Raman band can also be used, enabling the tagging of substances such as glucose for real-time mapping of the glucose uptake dynamics of cells. Disease contrast related to chemical changes and metabolic actions can be detected for clinical use. The 3D speed may enable in-situ clinical imaging without motion artifacts. In some cases, spectral multiplexing techniques - strategies such as spectral decomposition - that can map multiple different chemical substances interacting with each other simultaneously can be applied.

[0127] Selecting the wavelength in these embodiments was done as follows. Targeting the CH2 vibration band including the Raman shift of 2845 cm -1 , the signal and idler wavelengths to be used (based on our OPA with a pump wavelength of 515 nm) can be solved based on (1 / λ signal )+(1 / λ idler )=1 / (515 nm), (1 / λ signal )-(1 / λ idler )=2845 cm -1 . This equation results in λ signal = 898.4 nm, and λ idler = 1207 nm.

[0128] Another option is to use the 1030 nm output of a common pump laser (e.g., Spirit 100 by Spectra Physics) as the Stokes beam, and set the signal beam of the OPA to an appropriate wavelength (797 nm for the CH2 band) as the pump beam. Both of these configurations utilize the fact that the two beams are pulsed and temporally synchronized with each other.

[0129] Several considerations exist when determining the modulation frequency in these embodiments. The modulation frequency should be much higher than the xy pixel rate, not only to allow for a sufficient period of modulation and demodulation, but also to avoid spurious modulation signals (even in the absence of a Stokes beam) resulting from scanning through a turbid biological sample. The modulation frequency should also be much higher than the laser noise, which is limited to <100 kHz. Typical point scanning microscopes attempt to scan the sample as quickly as possible to achieve pixel residence times in the microsecond range. In some embodiments, the modulation is typically set to at least 10 MHz. For a typical 80 MHz Ti:sapphire laser source, this corresponds to true source modulation (e.g., 4 pulses on, 4 pulses off). However, for DART-based line excitation, the advantages of simultaneous multilayer detection allow for a slight slowdown. However, to enable high-speed volumetric imaging, modulation rates in the MHz range may also be targeted. Since lasers with lower repetition rates (<1 MHz) may be used to increase peak output, it may be considered to use the laser pulse itself as the modulation. Given modulation f m For the beam that is always on, at least 2f m The pulse, and the f for the ON-OFF modulated beam. m Please note that the pulse should be present.

[0130] When using an OPA that generates pulses at 400 kHz, one suitable method would be to divide each pulse into, for example, 16 copies to obtain a uniformly spaced pulse train of 6.4 MHz. However, the inter-pulse delay in this case becomes 1 / 6.4 μs, which translates to 46.875 meters in free space, which is impractical (especially considering that the delay between the first copy and the intermediate copies is 8 × 46.875 > 350 meters). Therefore, other techniques for pulse division are preferable. For example, each pulse may be divided into multiple copies, each with a practical inter-pulse delay of 10 ns, corresponding to, for example, about 300 cm in free space, and the inter-pulse delay of the Stokes (or idler) wavelength is set to be equal to (or vice versa) twice the inter-pulse delay of the pump (or signal) beam. In this way, a local pulse train can be obtained from the signal pulse or idler pulse of each OPA, and two local pulse trains can be matched to achieve high-frequency modulation for SRS detection.

[0131] Figure 12b shows an example of local pulse train generation. This example illustrates the concepts of global and local (sub)pulse trains. The global pulse group train (400 kHz) is shown on the left, with each group consisting of eight subpulses with interpulse delays of 10 ns or 20 ns.

[0132] Figure 12c shows an example of 8-path splitting as a conceptual diagram of hierarchical pulse splitting. The input pulse enters the system from the lower left corner, and the output sub-pulse train exits at the upper right corner. In this figure, BS1-BS3 are 50 / 50 beam splitters, M is a mirror, and C is a pulse combiner (which can be implemented using beam splitters). In the 8-path splitting example in Figure 12c, the beam passes through the 50 / 50 beam splitters three times, with appropriate delay setups between consecutive passes. The principle of this hierarchical pulse splitting is shown in Figure 12c. Essentially, each 50 / 50 beam splitter doubles the number of sub-pulses and introduces an additional relative delay between two sub-pulses. The relative delay is doubled (or halved) step by step. In this way, the additional delay experienced by the pulse leaving the last combiner (indicated by C) can be mathematically explained as 4b1 + 2b2 + b3Δ, where bi is either 0 or 1 depending on the path the pulse takes following each beam splitter.

[0133] Figure 12d shows hierarchical pulse splitting using optical fibers and fiber couplers. The input pulse enters the system from one of the input ports of the first coupler (FC1), and the last coupler here (FC4) is used as a combiner from which an output subpulse train can be obtained from either output port (O1 or O2).

[0134] To obtain a local inter-pulse delay Δ=10ns (corresponding to a modulation frequency of 50MHz), the additional delays at each stage are 12m, 6m, and 3m, respectively. This is difficult to achieve with free-space optics. One way to facilitate this is to use a fiber-based approach that employs a 2x2 coupler as a beam splitter and optical fibers of appropriate length for generating the delay.

[0135] Considering a group refractive index of approximately 1.5 in silica-based optical fibers for both wavelengths, a 2-meter optical fiber will produce a delay of 10 ns, a 4-meter optical fiber will produce a delay of >20 ns, and an 8-meter optical fiber will produce a delay of >40 ns. Longer fibers are required to obtain even longer resolutions and lower modulation frequencies for stimulated Raman scattering imaging. Such longer fibers will introduce a considerable amount of dispersion into the final subpulse, and the delayed subpulse will also experience proportionally more group delay dispersion (GDD), thus resulting in a longer pulse duration. In addition, nonlinear effects (such as self-phase modulation) will begin to act, altering the spectrum of the subpulse. One solution is to introduce an appropriate amount of negative chirping at each delay stage to offset the GDD introduced by the extra inter-fiber propagation. Such negative chirping can be achieved using free-space setups based on fiber-based devices such as prism pairs, diffraction grating pairs, or grism pairs, or custom-fabricated fiber Bragg diffraction gratings.

[0136] Although the present invention has been disclosed in relation to specific embodiments, numerous modifications, alterations, and changes to the described embodiments are possible without departing from the scope and scope of the invention as defined in the appended claims. Therefore, the present invention is not limited to the described embodiments and has the entire scope and equivalents defined by the following claims. [Explanation of Symbols]

[0137] 10 First optical element, first objective lens 12 First optical element 14. First optical element 20. Second optical element, second objective lens 22 Second optical element 24 Second optical element 30. Third objective lens, immersion objective lens 32 Relay Lens Telescope 36. Relay Lens Telescope 40 Cameras 42 Image Splitter 50 scanning elements 55 Folding Mirror 58 Reflective surface 60 Light source, laser source 62 Alignment Mirror 64 Optical Beam Combiner 66 lenses 67 Lens 68 lenses 69 lenses 72 Third Optical Element 74. The third optical element 76. The third optical element 80 The Second Mirror 81 First edge 82 Second edge 90 Fluid Chamber 91 Blob, first solid mass 92 liquid polymer, second amount of UV-curable polymer 95 High Flatness Mirror 110 TAG lenses 115 Cylindrical lens 160 light source 166 Prism Compressor BS1 50 / 50 Beam Splitter BS2 50 / 50 Beam Splitter BS3 50 / 50 Beam Splitter C Pulse Combiner FC1 First Coupler FC4 final coupler G1 Scanning Galvanometer Mirror G2 Scanning Galvanometer Mirror M Mirror

Claims

1. A method for manufacturing a spacer for an immersion objective lens, The steps of pressing the set of side walls against the mirror such that the portion of the mirror positioned between the set of side walls functions as the bottom of the negative mold, and the set of side walls cooperates with the bottom of the negative mold to form a liquid-tight cavity, The steps include filling the liquid-tight cavity with a first amount of UV-curable polymer, A step of curing a first amount of ultraviolet-curable polymer into a first solid mass, wherein the first solid mass has a lower surface and an upper surface that adhere to the mirror, The steps include removing the set of side walls from the mirror without disturbing the adhesion between the lower surface of the first solid mass and the mirror, The steps include positioning the upper surface of the first solid mass near the objective lens, with a second amount of UV-curable polymer occupying the space between the upper surface of the first solid mass and the objective lens, Following the positioning step, the step of adjusting the position of the first solid mass until the lower surface of the first solid mass reaches the final position relative to the objective lens, A method comprising the step of curing a second amount of ultraviolet-curable polymer after the first solid mass has reached the final position.

2. The steps include projecting parallel light through the objective lens toward the mirror, The method further includes the step of detecting the parallel characteristics of the light reflected by the mirror, The method according to claim 1, wherein the determination that the first solid mass has reached the final position is made when the light reflected by the mirror becomes precisely parallel.

3. The method according to claim 2, wherein the step of curing the second amount of UV-curable polymer is carried out by projecting ultraviolet light into the second amount of UV-curable polymer through the objective lens.

4. The method according to claim 3, wherein additional ultraviolet light is applied to further cure the second amount of ultraviolet-curable polymer, following the projection of ultraviolet light into the second amount of ultraviolet-curable polymer through the objective lens.

5. The method according to claim 1, wherein at least the portion of the mirror that functions as the bottom of the negative mold has an inductive surface.

6. The method according to claim 1, wherein at least the portion of the mirror that functions as the bottom of the negative mold is flat within a range of 250 nm.

7. The method according to claim 1, further comprising the step of removing the mirror from the lower surface of the first solid mass.

8. The method according to claim 1, wherein the set of side walls is made of polymer.

9. The method according to claim 1, wherein the set of side walls is made of polydimethylsiloxane (PDMS).

10. The method according to claim 1, wherein the UV-curable polymer comprises BIO-133.

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