Slanted plane microscopy system and method

Abstract

Provision of a slope microscopy system and method. [Solution] A system and method for slant microscopy are disclosed. The slant microscopy system is configured to image a sample having a refractive index n0, and the system includes a first microscope arranged to receive light emitted from the sample, and a first objective lens having at least one of a first immersion medium having a first numerical aperture NA1 ≥ n0 and a first refractive index n1 approximately equal to n0, and a second microscope including a second objective lens, the combination of the first and second microscopes having a magnification M RR The microscope is configured to generate an intermediate image of a sample accompanied by a second objective lens, and comprises a second microscope having a second numerical aperture NA2, and a third microscope having a third objective lens focused on the intermediate image and having a third numerical aperture.

Description

[Background technology]

[0001] Conventional optical microscopy provides high-resolution imaging for a wide range of applications. For example, in certain situations where biological samples are being imaged, it is usually desirable to obtain optically segmented images, corresponding to thin slices in the axial direction through the sample. The advantages of optically segmented imaging include improved image contrast through reduced contribution from the out-of-focus plane and the ability to produce three-dimensional (3D) images. So-called "light sheet" microscopy is an optically segmented imaging technique in which a thin "sheet" of light is used to illuminate the sample. This approach is beneficial in many applications, particularly for imaging living biological samples, because the illumination beam dose is lower than in various other conventional optically segmented imaging techniques, and therefore this approach is significantly less phototoxic to the sample. Despite its advantages, light sheet microscopy has not been widely adopted historically due to the cumbersome nature of most implementations, which use two orthogonally positioned lenses: one lens for delivering the light sheet (to illuminate the sample) and the other lens for acquiring the image. Due to the resulting geometric constraints, these implementations are typically incompatible with many biological sample preparation techniques, including glass slides, dishes, and multiwell plates.

[0002] A form of optical sheet microscopy called oblique plane microscopy has been developed, in which three objective lenses are used to tilt the image plane, thereby achieving focused imaging of oblique planes in a sample. Oblique plane microscopy allows oblique planes in a sample to be illuminated and imaged using the same objective lenses, and is therefore compatible with standard glass slides and other sample preparation techniques, while also retaining the advantages of minimal photobleaching and phototoxicity associated with optically segmented imaging. The concept of oblique plane microscopy and its implementation is described in "Optically sectioned imaging by oblique plane microscopy" by C. Dunsby on December 8, 2008 (OPTICS EXPRESS, Vol. 16, No. 25) (hereinafter referred to as "Dunsby"). The oblique plane microscopy system presented by Dunsby offers advantages over conventional optically segmented microscopy methods. However, the index of performance of the optical system, specifically the numerical aperture (NA), is too low for this technique to be useful in many applications. For example, while conventional high-resolution microscopy can achieve a high aperture value of 1.33 for aqueous samples, the Dunsby system achieves a theoretical aperture value of only 0.74 for water-immersion objective lenses.

[0003] A modification of the slanted-plane microscopy system described by Dunsby is presented by Yang et al., “High Numerical Aperture Epi-illumination Selective Plane Illumination Microscopy” (hereinafter referred to as “Yang”), published as a preprint paper on bioRxiv on February 28, 2018. Referring to Figure 1, the Yang system is a single-objective slanted-plane selective illumination microscope in which a water-immersion objective lens (O1) 110 is used for both sample illumination and fluorescence acquisition. According to Yang, the illumination light sheet has an incident angle of 60 degrees (°) with respect to the optical axis of O1, and the remote imaging module includes two objective lenses (O2 and O3) 120, 130, which are arranged at an angle of 30° so that the intermediate image produced by O2 is focused and re-imaged by O3. The light 140 is focused by an optical system 150 on a detector 160 for imaging. Yang has disclosed that the tilted arrangement of O2 and O3 conventionally causes a loss of numerical aperture (PNU) because it tilts a portion of the light cone generated by O2 out of the collectible range of O3 (as in the Dunsby system, for example). For high PNU systems where O3 is tilted relative to O2, it has been impractical in previous implementations for O3 to have an even larger collectible cone when the PNU of O2 is high enough to ensure sufficient coverage of the PNU of O1. Yang addresses this problem by selecting a mismatched pair of objective lenses for the remote imaging module, using an air objective lens (NA=0.9) for O2 and a water-immersion objective lens (NA=1.0) for O3, and separating the focal space between O2 and O3 with a cover glass located at the intermediate image plane 170, keeping water on one side and air on the other. According to Yang, the z' position of the cover glass can be adjusted to minimize spherical aberration, and the refractive index difference between the working medium of O2 (air and water) and O3 compresses the angle of the O2 light cone, thereby reducing numerical aperture loss. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] C. Dunsby, “Optically sectioned imaging by oblique plane microscopy”, OPTICS EXPRESS, Vol.16, No.25 [Non-Patent Document 2] Yang et al.,”High Numerical Aperture Epi-illumination Selective Plane Illumination Microscopy” [Overview of the project] [Means for solving the problem]

[0005] The Yang system achieves an aperture value of 1.06, which is much higher than that of the Dunsby system, but still less than NA=n0 (=1.33 for aqueous samples), which can be theoretically achieved using conventional high-resolution optical microscopy. The aspects and embodiments relate to a slant microscopy system that achieves an aperture value approaching the theoretical NA=n0 limit achievable with conventional high-resolution microscopy, while also providing advantages (e.g., reduced photobleaching and phototoxicity, improved image contrast, and the ability to generate 3D images) associated with optically segmented imaging and a convenient and practical implementation arrangement, which can be used in conjunction with standard bench microscopy and common sample preparation techniques, including glass coverslips and multiwell plates. Thus, the aspects and embodiments may provide a nearly uncompromising solution for single-objective-lens slant microscopy suitable for a wide range of applications.

[0006] According to one embodiment, the slanted microscope inspection system is configured to image a sample having a refractive index n0, and the system includes a first microscope arranged to receive light emitted from the sample, the first microscope having a first objective lens having at least one of a first immersion medium having a first numerical aperture NA1 ≥ n0 and a first refractive index n1 approximately equal to n0, and a second microscope having a second objective lens, the combination of the first and second microscopes having a magnification M RR The system is configured to generate an intermediate image of a sample accompanied by a second objective lens having a second numerical aperture NA2, and the intermediate image is formed in a second immersion medium having a second refractive index n2, with a magnification M RR The third microscope includes a second microscope having a refractive index ratio (n1 / n2) approximately equal to that of the first and second immersion media, and a third objective lens focused onto an intermediate image and having a third numerical aperture, wherein the third objective lens has an optical axis tilted with respect to the optical axis of the second objective lens by an angle of inclination such that the third microscope images a slope in the intermediate image corresponding to a slope in the sample, and is configured and arranged to collect substantially all of the light from the second microscope.

[0007] In one embodiment, the refractive index of the sample is in the range of 1.33 to 1.41. In one embodiment, the first objective lens is a silicone immersion lens. In another embodiment, the first numerical aperture is NA1 ≥ 1.35. In another embodiment, the first refractive index is n1 = 1.41. In another embodiment, the second immersion medium is air such that the second refractive index is n2 = 1.0. In one embodiment, the second numerical aperture is NA2 ≥ 0.95.

[0008] In another embodiment, the ratio of the second numerical aperture to the second refractive index (NA2 / n2) is greater than or equal to the ratio of the first numerical aperture to the first refractive index (NA1 / n1).

[0009] In one embodiment, the tilt angle is selected to optimize the acquisition efficiency of the microscopy system.

[0010] In another embodiment, the first objective lens is a glycerol-immersion type lens.

[0011] In another embodiment, the third numerical aperture is NA3≧n2.

[0012] In one embodiment, the third objective lens includes a solid glass frustum. In another embodiment, the third objective lens is configured with a working distance in the range of 0 to 20 μm in air.

[0013] In one embodiment, the slant microscopy system further comprises a light source arranged to provide an incident beam of light and illuminate a slant in the sample, the illuminated slant corresponding to the imaged slant. In one embodiment, the incident beam of light is directed through a first objective lens. In another embodiment, a third microscope is rotatable to adjust the tilt angle. In one embodiment, the tilt angle is selected in part based on the divergence angle of the illumination light beam.

[0014] The first microscope may further include a first lens, and the first microscope has a first magnification M1, and the second microscope may further include f TL2 M RR =M1 * M2 -1 The system includes a second lens having a focal length selected to set the magnification M2 of the second microscope such that =(n1 / n2).

[0015] According to another embodiment, a slanted microscope inspection system configured to image a sample having a refractive index n0 includes a first microscope arranged to receive light emitted from a sample, the first microscope including a first objective lens having a first immersion medium with a first numerical aperture NA1 and a first refractive index n1, the first objective lens being configured based on a selected compromise between a first ideal condition of NA1 ≥ n0 and a second ideal condition of n1 being approximately equal to n0, and a second microscope including a second air-immersion objective lens, the combination of the first and second microscopes having a magnification M RRA second microscope configured to produce an intermediate image of a sample with =n1, wherein the second numerical aperture NA2 of a second air-immersion objective lens is selected to approach a third ideal condition for NA2 that is greater than or equal to the ratio of the first numerical aperture to the first refractive index (NA1 / n1); and a third microscope comprising a third objective lens focused on the intermediate image and having a third numerical aperture NA3≧1, wherein the third objective lens has an optical axis that is tilted with respect to the optical axis of the second objective lens by an angle of inclination such that the third microscope images a slope in the intermediate image corresponding to a slope in the sample.

[0016] In one embodiment, the first objective lens is a silicone immersion type objective lens.

[0017] In another embodiment, the tilt angle is selected to optimize the acquisition efficiency of the microscopy system.

[0018] In one embodiment, the third objective lens includes a solid glass frustum positioned to intersect the field of view of the second immersion objective lens. In one embodiment, the solid glass frustum has a chamfered edge. In another embodiment, the solid glass frustum has an end region sized to correspond to the field of view of the second immersion objective lens, which is shaped to be inserted into the field of view and to allow for the collection of available light from the second immersion objective lens. In yet another embodiment, the third microscope is rotatable to adjust the tilt angle.

[0019] Another embodiment relates to a method for configuring a microscopy system for imaging a slope in a sample, the microscopy system comprising a first microscope, a second microscope, and a third microscope. The method involves selecting a first objective lens for the first microscope based on a compromise selected between a first ideal condition and a second ideal condition for the first objective lens, the first ideal condition being that the first objective lens has a first numerical aperture NA1 ≥ n0, where n0 is an estimate of the refractive index of the sample, and the second ideal condition being that the first objective lens has a first refractive index [ka] The present invention includes having a first immersion medium having a first objective lens configured to direct an illumination light sheet onto a sample and collect light emitted from the sample; selecting a second objective lens for a second microscope, the second objective lens configured to collect substantially all light emitted from the first objective lens; configuring the second microscope to re-image a slope in the sample onto an intermediate image plane; selecting a third objective lens for a third microscope, the third objective lens positioned on the opposite side of the intermediate image plane from the second objective lens and configured to collect substantially all light emitted from the second objective lens while adapting to an inclination angle α; and positioning the third objective lens such that its primary optical axis is rotated by an inclination angle α with respect to the primary optical axis of the second objective lens.

[0020] In one embodiment, configuring a second microscope to re-image the slope in the sample is such that the magnification M of the image of the slope in the sample in the intermediate image plane is RR However, the conditions: [ka] The configuration of the second microscope's magnification M2 satisfies the following equation, where M1 is the magnification of the first microscope, n1 is the refractive index of the immersion medium of the first objective lens, and n2 is the refractive index of the immersion medium of the second objective lens. In another embodiment, the second microscope includes a lens, and configuring the magnification of the second microscope includes selecting the focal length of the lens. In one embodiment, selecting the second objective lens includes selecting an air-immersion type second objective lens. In another embodiment, selecting the first objective lens includes selecting a silicone-immersion type objective lens. In another embodiment, selecting the first objective lens includes selecting a glycerol-immersion type objective lens. In one embodiment, selecting the third objective lens includes selecting a third objective lens having a solid glass frustum. In another embodiment, positioning the third objective lens includes positioning the third objective lens such that the tip of the solid glass frustum intersects the field of view of the second objective lens. In another embodiment, selecting the second objective lens involves minimizing n2 and the second numerical aperture NA2 of the second objective lens being, under the condition: [ka] This includes selecting such a service as to satisfy the requirements.

[0021] In one embodiment, the method further includes, in part, adjusting the tilt angle based on the divergence angle of the illumination light sheet.

[0022] According to another embodiment, a microscopy system for high numerical aperture low aberration imaging of an inclined image plane comprises a first objective lens having a first immersion medium with a first numerical aperture NA1 and a first refractive index n1, arranged to collect emitted light from a sample, and configured on a compromise selected between a first condition and a second condition, the first condition being [ka] And the second condition is, [ka] The first objective lens comprises a first objective lens, in which n0 is an estimate of the refractive index of the sample, and a second objective lens having a second numerical aperture NA2 and positioned and configured to collect substantially all emitted light from the first objective lens, wherein the combination of the first and second objective lenses comprises a second objective lens configured to produce an intermediate image of the sample, and a third objective lens focused onto the intermediate image, wherein the third objective lens has an optical axis that is tilted with respect to the optical axis of the second objective lens by an angle of inclination such that the third objective lens images an inclined plane in the intermediate image, and further has a solid glass frustum in which an end region having a size corresponding to the field of view of the second objective lens is inserted into the field of view and shaped to allow collection of substantially all emitted light from the second objective lens while adapting to the inclination angle.

[0023] In one embodiment, the first objective lens is further configured and arranged to deliver an illumination light beam to the sample.

[0024] Further aspects, embodiments, and advantages of these exemplary aspects and embodiments are discussed in detail below. Embodiments disclosed herein may be combined with other embodiments in any manner consistent with at least one of the principles disclosed herein, and references to “a certain embodiment,” “several embodiments,” “an alternative embodiment,” “various embodiments,” “one embodiment,” or equivalents are not necessarily mutually exclusive and are intended to indicate that a particular feature, structure, or characteristic described may be included in at least one embodiment. Not all expressions of such terms herein necessarily refer to the same embodiment. Various aspects and embodiments described herein may include means for carrying out any of the methods or functions described. The present invention provides, for example, the following: (Item 1) An inclined plane microscopy system configured to image a sample having a refractive index n0, a first microscope arranged to receive light emitted from the sample, the first microscope including a first objective lens having at least one of a first numerical aperture NA1 ≧ n0 and a first immersion medium having a first refractive index n1 substantially equal to n0, a second microscope including a second objective lens, wherein the combination of the first microscope and the second microscope is configured to generate an intermediate image of the sample having a magnification M RR and the second objective lens has a second numerical aperture NA2, and the intermediate image is formed in a second immersion medium having a second refractive index n2, and the magnification M RR is substantially equal to the ratio (n1 / n2) of the refractive index of the first immersion medium to the refractive index of the second immersion medium, a third microscope focused on the intermediate image, the third microscope including a third objective lens having a third numerical aperture, the third objective lens having an optical axis inclined with respect to the optical axis of the second objective lens by an inclination angle such that the third microscope images an inclined plane in the intermediate image corresponding to an inclined plane in the sample, and the third objective lens is configured and arranged to collect substantially all of the light from the second microscope, a system comprising. (Item 2) The inclined plane microscopy system according to item 1, wherein the refractive index of the sample is in the range of 1.33 to 1.41. (Item 3) The inclined plane microscopy system according to item 2, wherein the first objective lens is a silicone immersion lens. (Item 4) The inclined plane microscopy system according to item 3, wherein the first numerical aperture is NA1 ≧ 1.35. (Item 5) The inclined plane microscopy system according to item 4, wherein the first refractive index is n1 = 1.41. (Item 6) The inclined plane microscopy system according to item 3, wherein the second immersion medium is air such that the second refractive index is n² = 1.0. (Item 7) The oblique plane microscopy system described in item 6, wherein the second numerical aperture is NA2 ≥ 0.95. (Item 8) The slant microscope inspection system according to item 1, wherein the ratio of the second numerical aperture to the second refractive index (NA2 / n2) is greater than or equal to the ratio of the first numerical aperture to the first refractive index (NA1 / n1). (Item 9) The inclination angle is selected to optimize the collection efficiency of the microscopy system according to item 1. (Item 10) The first objective lens is a glycerol-immersed lens, as described in item 1, for the oblique plane microscope inspection system. (Item 11) The third numerical aperture is NA3 ≥ n2, as described in item 1, for the slant microscopy inspection system. (Item 12) The third objective lens is a frustum of solid glass, as described in item 1 of the inclined plane microscope inspection system. (Item 13) The oblique plane microscope inspection system described in item 12, wherein the third objective lens is configured to have a working distance in the range of 0 to 20 μm in air. (Item 14) The slope microscopy system according to item 1, further comprising light sources arranged to provide an incident beam of light and to illuminate a slope in the sample, wherein the illuminated slope corresponds to the imaged slope. (Item 15) The incident beam of light is directed through the first objective lens, as described in item 14 of the inclined plane microscopy system. (Item 16) The inclined plane microscopy system according to item 15, wherein the third microscope is rotatable to adjust the inclination angle. (Item 17) The inclination angle is selected in part based on the divergence angle of the illumination light beam, as described in item 16. (Item 18) The first microscope further includes a first lens, the first microscope has a first magnification M1, and the second microscope further includes M RR =M1 * M2 -1 The focal length f is selected to set the magnification M2 of the second microscope such that = (n1 / n2). TL2 The oblique plane microscope inspection system according to item 1, comprising a second lens having [a specific characteristic]. (Item 19) A slant microscopy system configured to image a sample having a refractive index n0, A first microscope arranged to receive light emitted from the sample, the first microscope includes a first objective lens having a first numerical aperture NA1 and a first immersion medium having a first refractive index n1, the first objective lens being configured based on a selected compromise between a first ideal condition NA1≧n0 and a second ideal condition n1 being approximately equal to n0, A second microscope including a second air-immersion objective lens, wherein the combination of the first microscope and the second microscope has a magnification of M RR A second microscope is configured to produce an intermediate image of the sample having =n1, and the second numerical aperture NA2 of the second air-immersion objective lens is selected to approach a third ideal condition in which NA2 is greater than or equal to the ratio of the first numerical aperture to the first refractive index (NA1 / n1), A third microscope focused onto the intermediate image, the third microscope includes a third objective lens having a third numerical aperture NA3≧1, the third objective lens having an optical axis tilted with respect to the optical axis of the second objective lens by an angle of inclination such that the third microscope images the slope in the intermediate image corresponding to the slope in the sample. A system equipped with these features. (Item 20) The first objective lens is a silicone immersion type objective lens, as described in item 19 of the oblique plane microscope inspection system. (Item 21) The inclination angle is selected to optimize the collection efficiency of the microscopy system according to item 19. (Item 22) The oblique plane microscope inspection system according to item 19, wherein the third objective lens includes a solid glass frustum positioned to intersect the field of view of the second air-immersion objective lens. (Item 23) The aforementioned solid glass frustum has a beveled edge, as described in item 22, for the inclined plane microscopy system. (Item 24) The frustum of the solid glass is shaped such that an end region having a size corresponding to the field of view of the second immersion objective lens is inserted into the field of view, allowing for the collection of available light from the second immersion objective lens, as described in item 22. (Item 25) The inclined plane microscopy system according to item 22, wherein the third microscope is rotatable to adjust the inclination angle. (Item 26) A method for configuring a microscopic inspection system for imaging a slope in a sample, wherein the microscopic inspection system includes a first microscope, a second microscope, and a third microscope, and the method is The first objective lens for the first microscope is selected based on a compromise between a first ideal condition and a second ideal condition for the first objective lens, wherein the first ideal condition is that the first objective lens has a first numerical aperture NA1 ≥ n0, where n0 is an estimate of the refractive index of the sample, and the second ideal condition is that the first objective lens has a first refractive index [ka] The first immersion medium has a first objective lens configured to direct the illumination light sheet towards the sample and collect the light emitted from the sample. The selection of a second objective lens for the second microscope, wherein the second objective lens is configured to collect substantially all of the emitted light from the first objective lens, The second microscope is configured to re-image the inclined plane in the sample onto the intermediate image plane, The selection of a third objective lens for the third microscope, wherein the third objective lens is positioned on the opposite side of the intermediate image plane from the second objective lens and is configured to collect substantially all emitted light from the second objective lens while adapting to the inclination angle α, Position the third objective lens such that its primary optical axis is rotated by the inclination angle α relative to the primary optical axis of the second objective lens. Methods that include... (Item 27) Configuring the second microscope to re-image the slope in the sample means that the magnification M of the image of the slope in the sample in the intermediate image plane RR The condition is: [ka] The method according to item 26, comprising configuring the magnification M2 of the second microscope to satisfy the following, wherein M1 is the magnification of the first microscope, n1 is the refractive index of the immersion medium of the first objective lens, and n2 is the refractive index of the immersion medium of the second objective lens. (Item 28) The method according to item 27, wherein the second microscope includes a lens, and configuring the magnification of the second microscope includes selecting the focal length of the lens. (Item 29) The method according to item 26, wherein the selection of the second objective lens includes selecting an air-immersion type second objective lens. (Item 30) The method according to item 29, wherein the selection of the first objective lens includes selecting a silicone immersion type objective lens. (Item 31) The method according to item 29, wherein the selection of the first objective lens includes selecting a glycerol immersion type objective lens. (Item 32) The method according to item 27, wherein the selection of the third objective lens includes selecting a third objective lens having a solid glass frustum. (Item 33) The method according to item 32, wherein positioning the third objective lens includes positioning the third objective lens such that the tip of the solid glass frustum intersects the field of view of the second objective lens. (Item 34) The selection of the second objective lens is to minimize n2, and the second numerical aperture NA2 of the second objective lens is subject to the condition: [ka] The method described in item 27, which includes selecting such that the requirements are met. (Item 35) The method according to item 26, further comprising adjusting the tilt angle based in part on the divergence angle of the illumination light sheet. (Item 36) A microscopy system for high numerical aperture and low aberration imaging of an inclined image plane, A first objective lens arranged to collect light emitted from a sample, wherein the first objective lens has a first immersion medium having a first numerical aperture NA1 and a first refractive index n1, and the first objective lens is configured based on a compromise selected between a first condition and a second condition, the first condition being [ka] Therefore, the second condition is, [ka] n0 is an estimated value of the refractive index of the sample, and the first objective lens and A second objective lens having a second numerical aperture NA2, wherein the second objective lens is positioned and configured to collect substantially all emitted light from the first objective lens, and the combination of the first and second objective lenses is configured to produce an intermediate image of the sample. A third objective lens focused onto the intermediate image, wherein the third objective lens has an optical axis tilted with respect to the optical axis of the second objective lens by an angle of inclination such that the third objective lens images an inclined plane in the intermediate image, and the third objective lens further has a solid glass frustum, the end region having a size corresponding to the field of view of the second objective lens, which is inserted into the field of view and shaped to allow for the collection of substantially all emitted light from the second objective lens while adapting to the angle of inclination. A microscope inspection system equipped with the following features. (Item 37) The first objective lens is further configured and arranged to deliver an illumination light beam to the sample, as described in item 36. [Brief explanation of the drawing]

[0025] Various aspects of at least one embodiment are discussed below with reference to the accompanying figures, which are not intended to be drawn to exact scale. The figures are included to illustrate and further understand the various aspects and embodiments, and are incorporated into and form part of this specification, but are not intended to define the limitations of this disclosure. In the figures, each identical or substantially identical component illustrated in the various figures is represented by a similar number. For the sake of clarity, not all components are labeled in all figures. [Figure 1]Figure 1 is a schematic diagram of one embodiment of a slant microscopy inspection system. [Figure 2A] Figure 2A is a functional block diagram showing the light collection path for one embodiment of the inclined light sheet microscopy system according to a side view of the present invention. [Figure 2B] Figure 2B is a functional block diagram of one embodiment of a slanted light sheet microscopy system, showing an example of the illumination light path. [Figure 3] Figure 3 is a schematic diagram illustrating, in aspect of the present invention, the relationship between the theoretical acquisition angle of various microscopes in a slanted light sheet microscopy system and the resulting acquisition / emission path numerical aperture. [Figure 4] Figure 4 is a process flow diagram illustrating an embodiment of the design methodology for a slanted light sheet microscopy inspection system according to aspects of the present invention. [Figure 5] Figure 5 is a schematic diagram illustrating the parameters of an embodiment of the illumination light sheet. [Figure 6A] Figures 6A-C are schematic diagrams of an embodiment of a third objective lens that may be used in the embodiment of the microscopy system of Figure 2A, according to aspects of the present invention. [Figure 6B] Figures 6A-C are schematic diagrams of an embodiment of a third objective lens that may be used in the embodiment of the microscopy system of Figure 2A, according to aspects of the present invention. [Figure 6C] Figures 6A-C are schematic diagrams of an embodiment of a third objective lens that may be used in the embodiment of the microscopy system of Figure 2A, according to aspects of the present invention. [Figure 7] Figure 7 is a flowchart of one embodiment of a method for configuring a microscopic inspection system according to aspects of the present invention. [Figure 8] Figure 8 is a schematic diagram showing the discharge path of one embodiment of the system shown in Figure 2A, from an aspect of the present invention. [Figure 9] Figures 9A and 9B are schematic diagrams showing the angular ray transmission paths of the theoretical model of an embodiment of the system in Figure 8. [Figure 10] Figure 10 is a schematic diagram showing the discharge path of an embodiment of the system in Figure 8, from an aspect of the present invention. [Figure 11] Figure 11 is a schematic diagram showing an embodiment of a third objective lens that may be used in the systems of Figures 2A, 8, and 10, in aspect of the present invention. [Figure 12A] Figures 12A-C are schematic diagrams showing an embodiment of the third objective lens of Figure 11, according to a side view of the present invention. [Figure 12B] Figures 12A-C are schematic diagrams showing an embodiment of the third objective lens of Figure 11, according to a side view of the present invention. [Figure 12C] Figures 12A-C are schematic diagrams showing an embodiment of the third objective lens of Figure 11, according to a side view of the present invention. [Modes for carrying out the invention]

[0026] The aspects and embodiments provide solutions for imaging inclined planes at high numerical apertures with minimal aberrations and losses in numerical aperture or optical efficiency, which may be applied to a variety of imaging applications. One aspect and embodiment relates to a slanted plane microscopy system that uses a single objective lens to illuminate a sample and collect light from the sample, along with an imaging module that includes two additional objective lenses to produce a high-resolution image of the sample. As used herein, the term “objective” refers to an optical module that includes one or more optical elements such as lenses or mirrors, and is used herein synonymously with the term “objective lens.” As will be further discussed below, embodiments of the microscopy systems disclosed herein can achieve numerical aperture (NA) values ​​in the range of 1.2 to 1.3, approaching the limit of NA=n0 (=1.33 in aqueous samples) set by conventional non-optically segmented (but high-resolution) microscopes, and can provide a significant improvement in resolution over the systems disclosed by Dunsby (theoretical NA of only 0.74) and Yang (realized NA of 1.06).

[0027] Referring to Figure 2A, a functional block diagram of one embodiment of a microscopy system is illustrated. As shown, the microscopy system 200 includes a microscope module 202 and an imaging module 204 configured to image a sample 206. The microscope module 202 includes a first objective lens 210 (Obj1) and a first lens 212 (TL1), which are collectively referred to herein as the “first microscope”. In the embodiment shown in Figure 2A, the sample 206 is prepared on a coverslip 208 placed at the front focal plane or object image plane (IP0) of the first objective lens 210. However, in other embodiments, other sample preparation techniques such as dishes and multiwell plates may also be used. In some embodiments, the microscope module 202 may be a standard microscope that is converted into a slanted light sheet microscopy system 200 through the inclusion and arrangement of the imaging module 204, as discussed below. The first objective lens 210 is used both to direct the illumination beam 216 onto the sample 206 and to collect the emitted light 220 from the sample 206. Figure 2A shows only the light collection or imaging path of the microscopy system 200. For completeness, Figure 2B shows an example of an array of microscope modules 202, including the illumination path.

[0028] Referring to Figure 2B, for example, a light source 214, such as a laser, generates an illumination beam 216. The illumination beam 216 is coupled into the optical path of a first objective lens 210 using a beam coupling device 218 and focused onto the sample 206 by the first objective lens 210. In one embodiment, the beam coupling device 218 may be a beam splitter that reflects the illumination beam 216 toward the first objective lens 210, allowing the emitted light 220 from the sample 206 to pass through. However, in other embodiments, other arrays or beam coupling devices 218 may be used, as will be understood by those skilled in the art, assuming the advantages of the present disclosure. Similarly, in the embodiment shown in Figure 2B, the illumination path includes a lens 222 and a folding mirror 224, configured and arranged to direct the illumination beam 216 from the light source 214 toward the beam coupling device 218. However, in other embodiments, as will be understood by those skilled in the art, assuming the advantages of the present disclosure, any number and arrangement of optical elements (lenses or mirrors) may be used to direct the illumination beam toward the first objective lens 210. In some embodiments, the light source 214 and optical elements (e.g., 218, 222, 224) that direct the illumination beam toward the first objective lens 210 may be part of the microscope module 202. In other embodiments, one or more of these components may be separate from the microscope module 202, contained within the imaging module 204, or part of a separate module that can be coupled to the imaging module 204 and / or the microscope module 202.

[0029] Referring again to Figure 2A, in one embodiment, the imaging module 204 includes a second microscope, which includes a combination of lens 228 (TL2) and a second objective lens 230 (Obj2), and a third microscope, which includes a combination of a third objective lens 232 (Obj3) and a third lens 234 (TL3). The first, second, and third lenses 212, 228, and 234 are represented as single lenses in Figure 2A, but it should be understood that in practice, each may be implemented using one or more optical elements (lenses or mirrors). The imaging module 204 further includes a detector 226. The combination of the first lens 212 and the second lens 228 directs the light 220 (also referred to as emitted light or emitted ray) emitted from the sample 206 from the back focal plane (BFP1) of the first objective lens 210 to the back focal plane (BFP2) of the second objective lens 230. In the embodiment shown in Figure 2A, the first and second lenses 212 and 228 are in a relay configuration, with the microscope module 202 forming a first intermediate image plane (IP1) between the first and second lenses 212 and 228, and the second lens 228 relays the light 220 from its image plane to the back focal plane (BFP2) of the second objective lens 230. The third lens 234 focuses the light 220 from the back focal plane (BFP3) of the third objective lens 232 onto the final image plane (IP4), where it can be received by the detector 226. As shown in Figure 2A, the second and third objective lenses 230 and 232 are arranged to be inclined relative to each other such that the second intermediate image plane (IP2) formed at the focal plane of the second objective lens 230 is inclined by an angle α with respect to the third intermediate image plane (IP3) formed at the focal plane of the third objective lens 232. The angle α is referred herein to as the inclination angle of the third objective lens 232.

[0030] The imaging resolution of an aberration-free microscopy system is proportional to the numerical aperture (NA). Therefore, the higher the overall numerical aperture achieved, the better the imaging resolution of the system. The aspects and embodiments show a very high overall numerical aperture NA of the emission path, considering the various properties and parameters of the sample 206 being imaged and the light sheet (illumination beam) 216 used to illuminate the sample 206, as well as certain optical principles and mechanical constraints relating to the practical implementation of the microscopy system 200. em This document covers the steps of designing and configuring a microscopy system 200 to achieve (significantly higher than what is achievable with respect to either Dunsby or Yang's system). A specific embodiment for imaging a living biological sample 206 is presented below. However, the design processes and principles disclosed herein may be applied to any type of sample to be imaged.

[0031] The relationships between various important parameters of the microscopy system 200 and the constraints relating thereto can be understood by referring to Figure 3 and continuing the reference to Figure 2A. Figure 3 is a schematic diagram showing the relationship between the theoretical acquisition angles of the various microscopes in the microscopy system 200 and the resulting acquisition / emission path numerical aperture. In Figure 3, axis 302 corresponds to the primary optical axis of the microscopy system 200, which is also the primary optical axis of the first and second objective lenses 210, 230. Axis 304 corresponds to the inclined optical axis of the third objective lens 232, which is inclined by an angle α with respect to the primary optical axis 302, as discussed above. The numerical aperture of the sample / coverslip interface for each individual objective lens is given by equation (1), where x = 0, 1, 2, 3.

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[0032] The illumination light sheet 216 has a divergence angle, Φ ex The single objective lens (first objective lens 210) is used both to illuminate the sample 206 and to collect the light 220 emitted from the sample 206. Therefore, a portion of the collection angle θ1 of the first objective lens 210 is used to form the light sheet Φ, as shown in Figure 3. ex Occupied or “used up” by. Figure 3 illustrates the “best case” where the edge of the light sheet coincides with the edge of the collecting cone of the first objective lens 210 (Φ measured from the same edge line 306 in Figure 3). ex (and θ1). The light sheet is shifted inward from the collecting cone of the first objective lens 210 (i.e., Φ ex It is possible that the left edge of the cone defined by will be moved to the right or clockwise in Figure 3, but this arrangement is NA em This causes a loss (of the achievable numerical aperture NA1 of the first objective lens 210 used to collect the emission).

[0033] According to Dunsby, the maximum potential numerical aperture achievable for the overall numerical aperture resulting from the emission pathway of the Dunsby microscopy system is given by:

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[0034] The parameter Φ' used in equation (4) em It is given by the following:

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[0035] Based on the principles discussed above, embodiments of the microscopy system 200 can be designed and configured according to the following processes and considerations. Figure 4 is a process flow diagram illustrating an example of steps and considerations that may be included in a method for designing a bevel light sheet imaging microscopy system from one aspect. Those skilled in the art will understand, assuming the advantages of this disclosure, that the steps do not need to be performed in the order illustrated in Figure 4. The schematic diagram in Figure 4 is provided for clarity and convenience of explanation and is not intended to limit it. The processes described herein take advantage of the recognition that each of the three microscopes has certain properties and parameters that, when considered in the context of the overall system and in light of the properties of the light sheet 216 and sample 206 used in any given implementation, can be wisely selected and configured to provide the microscopy system 200 with imaging performance and usefulness that was previously unattainable.

[0036] Step 402 includes the step of selecting or configuring the first objective lens 210.

[0037] In certain applications, such as fluorescence microscopy through a flat coverslip, sample 206 can emit light 220 in all directions, and therefore the following condition: sinθ0 = 1, is met, and thus from equation (1), NA0 = n0, where n0 is the refractive index of sample 206. Consequently, according to one aspect, it is recognized that the first objective lens 210 can be configured to capture all the light emitted from sample 206 by setting NA1 ≥ n0. In addition, for high-quality imaging, the first objective lens should be configured such that there is a good match between the refractive index of the sample medium (n0) and the refractive index of the immersion medium of the first objective lens (n1). A mismatch in refractive index causes depth-dependent aberrations in the resulting image of sample 206. Therefore, according to one aspect, the first objective lens 210 can be configured to provide both a good refractive index match and a good collection cone angle, maximizing the light collected from sample 206 as well as the imaging resolution and quality. Ideally, the first objective lens 210 may be configured with NA1≧n0 and n1=n0. However, those skilled in the art will understand, assuming the advantages of the present disclosure, that in practice, it may be impossible to achieve both of these ideal conditions simultaneously, particularly with respect to multiple different samples. Therefore, step 402 may include selecting a configuration for the first objective lens 210 based on a compromise between these two conditions, such that the maximum practical numerical aperture is obtained while also achieving good matching in refractive index for the expected range of samples. Thus, in one embodiment, the first objective lens 210 is, [ka] and [ka] It can be constructed with the addition of [something].

[0038] Furthermore, according to one embodiment, the microscope module 202 can be configured such that the first lens 212 and the first objective lens 210 are perfect infinity corrected lenses that approximate ideal lens behavior. This arrangement provides optimal remote refocusing performance.

[0039] To re-image any plane within a limited volume in sample 206 with minimal aberration, the lateral and axial magnifications between sample 206 and the second intermediate image plane (IP2) formed at the focal plane of the second objective lens 230 must be equal. This condition is met by the magnification M RR This is achieved when n1 / n2 is equal, where n2 is the refractive index of the immersion medium of the second objective lens 232. This principle is called the refocusing rule. RR This is given by the following equation (9).

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[0040] Therefore, from equation (9) above, the refocusing rule can be defined as follows:

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[0041] Referring to Figure 4, the design process for the microscopy system 200 may include a step 406 of selecting a microscope module 202. As discussed above, a standard / existing microscope stand may be selected, preferably having a first lens 212 and a first objective lens 210, which are fully infinity-corrected lenses that approximate ideal lens behavior. Furthermore, as discussed above, the microscope module 202 in step 402 has the first objective lens 210 under conditions for a typical sample that is expected to be imaged. [ka] and [ka] It can be selected or configured to satisfy the following: Thus, the focal length f of the first objective lens Obj1 and the focal length f of the first lens TL1 This sets the primary magnification M1. As discussed above, in step 404, the parameters of the second objective lens 230 can be configured to impose refocusing conditions and minimize aberrations. For example, the focal length f of the second objective lens Obj2 and the focal length f of the second lens TL2 M BFPTo achieve a fixed value for , it can be constrained by equations (10), (11), and (15). The distance d1 can be precisely measured so that other parameters can be selected based on a known distance d1, although this may not be easily configurable, especially if the microscope module 202 is an existing microscope stand. For example, the distance d2 can be adjusted to image BFP2 from BFP1 by using the following relationship.

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[0042] As discussed above, according to one embodiment, the third objective lens 232 provides the best potential overall numerical aperture NA for the emission / collection path represented by equation (7) above. em To maintain this, the numerical aperture NA3 of the second objective lens 230 is configured to be equal to or greater than the refractive index n2 of the immersion medium (NA3≧n2). This condition maximizes the collection efficiency of the third objective lens 232 and ensures that the third objective lens 232 is capable of capturing all the light from the second objective lens 230 (step 408 in Figure 4). However, a large numerical aperture generally results in a short working distance for the microscope due to a large associated cone angle, which can make practical implementation very difficult. In some embodiments, θ1 and θ2 can exceed, for example, 70°. Therefore, according to one aspect, the requirement regarding the numerical aperture of the third objective lens 232 can be reduced by minimizing n2 while also satisfying the condition specified in equation (15) (step 410 in Figure 4).

[0043] Referring again to Figure 2A, the slope of sample 206 is re-imaged by the combination of the third objective lens 232 and the third lens 234, forming an image of sample 206 on the final image plane IP4. In step 420, the overall system magnification M TThe focal length f of the third lens is given by the following equation (18), otherwise it is a free parameter. TL3 Through the appropriate selection of the third objective lens, the focal length f can be set to the desired value. As will be discussed further below, the focal length f of the third objective lens Obj3 This may be constrained by the optical mechanical specifications for the third objective lens 232.

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[0044] Light-sheet microscopy is generally useful for imaging living biological samples 206, which typically have an n0 value in the range of approximately 1.35–1.40 and an average refractive index of approximately 1.37. Therefore, to capture all possible light emitted within a typical living biological sample 206, [ka] Set this to 1.40 <NA oil This can be achieved by using an oil-immersion objective lens, which can be configured to have a typical numerical aperture within the range of <1.45. However, selecting an oil-immersion objective lens for the first objective lens 210 results in a refractive index mismatch (n1≠n0) between the lens immersion medium and the sample 206, which generates depth (z)-dependent spherical aberration, significantly degrading image resolution. As discussed above, the first objective lens 210 can be selected and configured on the basis of achieving a good compromise between obtaining the maximum useful numerical aperture and maintaining good refractive index matching. Therefore, in some embodiments, a silicone objective lens is selected for the first objective lens 210. In this case, NA1=NA silicone (For example, NA silicone =1.35), and n1=n siliconeIt is 1.41. For a typical living biological sample 206 with an average n0 = 1.37, this selection for the first objective lens 210 provides a delta (ΔNA) at a numerical aperture of about 0.02 (preferably, as described above, NA1 ≧ n0, and here, this condition is not very well met, but the delta is very small, meaning that almost all available light from the sample 206 can be collected by the first objective lens 210) and a delta or mismatch at a refractive index (Δn) of only 0.04 that causes minimal depth-dependent aberration. Thus, this selection may represent a good or even optimal compromise according to the above. For comparison, a good water-immersion objective lens has a value of NA water = 1.27 and n water = 1.33, with a similar depth-dependent aberration but resulting in a significantly lower collection ability (due to a lower numerical aperture). Further, as discussed above, a comparable oil-immersion objective lens maximizes the numerical aperture (e.g., with NA oil = 1.45) but causes a depth-dependent aberration (typically, n oil = 1.52, and thus, the refractive index delta is about 0.15) that significantly degrades the imaging performance at depths in the sample 206 and the associated stereoscopic information. Thus, the selection of a silicone objective lens or a similar intermediate immersion refractive index lens for the first objective lens 210 according to certain embodiments may provide significant advantages over water and oil objective lenses.

[0045] For example, in one case, a sample (such as a living biological sample) may have a refractive index within the range of 1.33 < n0 < 1.41. Thus, selecting a silicone objective lens with NA1 = 1.35 and n1 = 1.41 provides an "error" or tolerance range of 0.02 ≦ ΔNA ≦ 0.06 and 0 ≦ Δn ≦ 0.08. Therefore, in this case,

Chemical formula

[0046] Once an objective lens (e.g., a silicone objective lens) is selected for the first objective lens 210, the parameters NA1 and n1 (e.g., NA1 = 1.35 and n1 = 1.41) are determined. Therefore, in this embodiment, applying the conditions of equation (16) sets the ratio of the numerical aperture of the second objective lens to the refractive index of the second objective lens NA2 / n2 ≥ 0.96. As discussed above, it is preferable that NA3 ≥ n2 to maximize the collection efficiency, so it is desirable to minimize n2 and relax the requirements regarding the numerical aperture of the third objective lens 232. Thus, the second objective lens 230 can be an air objective lens (n2 = 1.0) with NA2 = 0.95, which is capable of re-imaging 99% of the angular range of the first (silicone) objective lens 210 (asin(0.95) / asin(0.96)).

[0047] An embodiment of the microscopy system 200, designed and configured according to the processes and principles described above, achieves a significant improvement in acquisition efficiency over that which can be achieved by the system disclosed by Dunsby and Yang. In the following embodiment, the illumination light beam 216 is Φ ex It is assumed that it has a divergence angle of 5°. For example, assuming that sample 206 has n0=1.37, an embodiment of the microscopy system 200 in which the first objective lens 210 is a silicone immersion lens with NA1=1.35 and n1=1.41, and the second objective lens is an air objective lens with NA2=0.95 and n2=1.0, is very close to the 1.37 limit achievable with conventional high-resolution microscopy, NA em =1.32 (applying equation (7) above) is achieved, and the collection efficiency (ignoring reflective loss) is 95%. In contrast, Dunsby discloses a system that achieves a much lower overall numerical aperture and a very low collection efficiency. For example, in Dunsby, both the first and second objective lenses in it theoretically have an overall numerical aperture of only 0.75 NA potDisclosed is an air objective lens with NA1=NA2=0.95 and a collection efficiency of only 63%. Another embodiment disclosed in Dunsby uses a water objective lens (NA1=1.2;NA2=0.95) for the first objective lens, but theoretically has an overall numerical aperture of only 0.74 NA pot This achieves a very poor collection efficiency of only 38%. Similarly, the embodiments disclosed by Yang do not achieve performance close to that of the embodiments of the microscopy system 200 disclosed herein. For example, Yang discloses an embodiment in which the first objective lens is a water objective lens with NA1 = 1.27 and the second objective lens is an air objective lens with NA2 = 0.9. This embodiment theoretically provides an overall system numerical aperture of 1.17 and a collection efficiency of 85%, which is significantly lower than the embodiments of the microscopy system 200 discussed above. Furthermore, Yang discloses that in practice, this embodiment actually achieved an overall system numerical aperture of only about 1.06 and a collection efficiency of only about 70%. It is noteworthy that, as demonstrated by this embodiment, Yang did not achieve the advantage of ensuring that the second objective lens is configured to collect essentially all the light from the first objective lens.

[0048] In addition to the considerations discussed above, which may apply when selecting and configuring the first and second objective lenses, one aspect further recognizes that the inclination angle α of the third objective lens 232 can be optimized. Applying the conditions of equations (11) and (15) may result in a second objective lens 230 having a short working distance due to the large cone angle θ2, which is set by a high desired numerical aperture NA2. If the optical axis of the third objective lens 232 is parallel to the optical axis of the second objective lens 230 (α=0), the short working distance will not present a problem. However, as discussed above, the optical axis of the third objective lens 232 is inclined by an angle α (generally non-zero) with respect to the optical axes of the first and second objective lenses 210, 230 so as to effectively image a plane containing the illumination light sheet 216. Thus, one aspect of the present invention relates to these considerations and NA em The goal is to appropriately select the inclination angle α and the properties of the third objective lens 232 in light of the objective of maximizing [the specified value] (steps 412, 414, and 416 in Figure 4).

[0049] As discussed above with reference to Figure 3, the optical sheet 216 is Ф ex The divergence angle is α. The inclination angle α is related to both the optical sheet divergence angle and the collecting cone of the first objective lens 210 and can be calculated according to equation (19).

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[0050] Regarding biological imaging, a typical value for the wavelength of the optical sheet 216 is 0.5 micrometers (μm), and assuming a laser light source 214, the Rayleigh range Z R Typically, this is in the range of approximately 5–50 μm. Therefore, the beam waist ω0 can generally be in the range of approximately 1–3 μm, and the divergence angle Φ ex Therefore, [ka] Assuming (as discussed above as an example), the range may be approximately 2° to 8°. Given these parameters, as well as the considerations discussed above and the values ​​of θ1 and θ2 determined from the examples, equation (19) shows that for these examples, the inclination angle α may be in the range of approximately 20° to 26°. As an example, sample 206 among them has n0 = 1.37, Ф ex Consider the following embodiment, which involves a living biological sample with a 5° angle, and in which the first objective lens 210 is a silicone objective lens with NA1=1.35 and n1=1.41. In this embodiment, equation (3) is: [ka] This gives. Therefore, applying equation (18), this embodiment is [ka] This defines...

[0051] Based on equation (19), assuming that the light sheet 216 is used, the expected maximum divergence angle Ф exThis sets the maximum value for the inclination angle α. Using a higher inclination angle will result in a higher emission path collection angle Φ'. em Therefore, also, NA em This reduces and does not offer any obvious advantages. Therefore, according to one aspect, given the properties of the optical sheet 216 used and the collection cone angle θ1 of the first objective lens 210, the tilt angle α can be selected to optimize the collection efficiency (step 412). This aspect leverages the important realization that there exists an optimal range or value α for the tilt angle, which depends on the parameters of the optical sheet 216 and the first and second objective lenses 210, 230 (and thus can be optimized based on the sample 206 to be imaged, as discussed above), and is not utilized by either Dunsby or Yang. For example, as described above, Yang discloses that the tilt angle of the third objective lens is 30°. However, in light of the principles and aspects disclosed herein, it can be seen that Yang's recommended value for the tilt angle is too high. Applying the above equation, other parameters disclosed by Yang (e.g., Z) R =35μm;Ф ex Assuming =3°, it can be shown that the optimal value for the inclination angle is approximately 21°, which is significantly lower than the value disclosed by Yang.

[0052] As discussed above, the constraints on the second objective lens 230 result in a wide angular range (e.g., θ2 > 70°), and therefore a short working distance (for a given existing lens). For example, [ka] In this case, the working distance may be approximately 200 μm. If the third objective lens 232 also has a short working distance, which is typical for lenses with a high numerical aperture, this creates an unwieldy mechanical constraint with respect to the inclination angle α. For example, if the third objective lens 232 has the same parameters as the second objective lens 230 (e.g., [ka] If it is an air objective lens with n2=n3=1.0, then the two objective lenses are, [ka] A collision occurs in this area, which would disable the operation of the microscope. Therefore, certain aspects and embodiments relate to the step of configuring the third objective lens 232 to avoid this problem.

[0053] According to one aspect, the immersion medium of the third objective lens 232 is selected to have a relatively high refractive index in order to reduce the angular range θ3. For example, the immersion medium of the third objective lens 232 may be oily, which typically has a refractive index in the range of 1.45 to 1.65, which is significantly higher than that of air. From equation (1), with respect to any given numerical aperture, the higher the refractive index of the objective lens, the lower the angular range. Reducing the angular range can, in turn, increase the working distance and relax the mechanical constraints on the system (step 414 in Figure 4). For example, considering an oily objective lens having a slightly higher numerical aperture (NA3=1 and NA2=0.95) than the second objective lens of air discussed above, and a refractive index in the lower part of the given range above, the maximum angular range is, then, [ka] In this embodiment, the working distance can typically be in the range of approximately 4 mm to 8 mm.

[0054] According to one embodiment, the third objective lens 232 is configured to both use a material with a higher refractive index, such as glass, and have a mechanical arrangement that allows for a very short, or even zero ("contact"), or even negative working distance (step 416 in Figure 4). In one embodiment, the third objective lens 232 is configured with a solid glass front "frustum" that is shaped with a light-receiving angle that matches the applicable field of view and matches the numerical aperture required based on the condition NA3≧n2 discussed above. Thus, in one embodiment, θ frustum >θ max And θ max This is determined based on the numerical aperture NA3 of the third objective lens 232 and the refractive index n3 of the immersion medium of the third objective lens (according to equation (1)), the value of θ3 (in some embodiments, [ka] This allows the third objective lens 232 to collect essentially all the light from the second objective lens 230, while also providing the best overall emission path numerical aperture NA for the bevel light sheet microscopy system 200 without mechanical collision between objective lenses 2 and 3. em To achieve this, a significant range of adjustment of the inclination angle α is made possible, for example, up to 45° for a glass frustum. Embodiments of various configurations of the third objective lens 232, including a solid glass frustum or other shapes, are shown in Figures 6A–6C. The glass frustum can be conical in shape or, for example, have a double-chamfered chisel shape. Figure 6A illustrates an embodiment of the third objective lens 232 with a solid radial frustum. Figure 6B illustrates an embodiment of the third objective lens 232 with a chamfered solid radial frustum. Figure 6C illustrates an embodiment of the third objective lens 232 with a solid square frustum. Each of these embodiments enables efficient optical coupling over high numerical apertures, as will be further discussed below.

[0055] Therefore, in one embodiment, the third objective lens 232 may include a solid glass frustum shaped to allow a working distance of zero or near zero (e.g., about 20 μm in air). In one embodiment, the frustum has a field of view of about 200 μm and a glass tip having similar dimensions. The 20 μm working distance allows for a maximum divergence angle of about 8° Ф ex The accompanying optical sheet 216 crosses the front of the frustum of the third objective lens 232 in air, thus enabling it to be directly coupled into the second objective lens 230 at a high numerical aperture.

[0056] As discussed above, the optimal value or range for the tilt angle α may, in part, depend on the parameters of the light sheet 216. However, it may be desirable to image various different samples 206 using the same microscopy system 200, and different light sheet parameters may be preferred for imaging different types of samples. For example, large samples may be better imaged using a large field of view and weakly diverging Gaussian light sheet 216. In other words, in this case, the minimum tilt angle α min The system 200 is configured with the minimum divergence angle Ф ex_min It may be preferable to use an optical sheet 216 accompanied by [unclear]. On the other hand, the small sample 206 has a small field of view and a narrow beam waist (large inclination angle α max and large divergence angle Ф ex_max Better imaging can be achieved using a Gaussian light sheet 216 with (meaning) a Gaussian light sheet 216. It may also be desirable to use a non-Gaussian illumination beam (e.g., a Bessel beam or even a grating beam). Therefore, in one embodiment, part of the imaging module 204 can be configured with a mechanical assembly that allows at least a third objective lens 232, a third lens 234, and a detector 226 to rotate as a rigid body about the intersection of the second intermediate image plane IP2 and the third intermediate image plane IP3, so that the tilt angle α can be dynamically adjusted and optimized for samples 206 with different tilt angles (step 418 in Figure 4).

[0057] Figure 7 is a flowchart summarizing the approach to configuring the slant microscopy system 200 according to the processes and considerations discussed above. Steps 702 and 704 involve selecting / configuring the first microscope within the microscopy system 200. Step 702 corresponds to step 406 discussed above, in which the base microscope module 202 is selected. The microscope module 202 includes an embedded first lens 212, and therefore parameter f TL1 and d1 may be fixed by this selection. In step 704, the first objective lens 210 for the microscope module 202 is selected to match the type of sample expected to be imaged using the microscopy system 200. As discussed above, the refractive index of the sample 206 may vary, but assuming a known application (e.g., biological imaging), the first objective lens 210 can be selected, along with a numerical aperture, that is approximately above or equal to the average or approximate refractive index of the expected sample type, and therefore, as discussed above, [ka] Set the first objective lens 210 by parameter f Obj1 This is set, and therefore, the combination of steps 702 and 704 fixes the magnification M1 of the first microscope.

[0058] Steps 706 and 708 involve selecting / configuring a second microscope within the microscopy system 200. Step 706 includes selecting a second objective lens 230. As discussed above, the requirements for the third objective lens 232 can be relaxed by minimizing n2. The parameter n2 can vary between 1.0 for air and greater than 1.4 for oily materials or even greater than 2.0 for high refractive index glass materials. Thus, the step of selecting an air objective lens for the second objective lens minimizes n2 while also satisfying the conditions specified in equation (15) or (16). The air objective lens has an numerical aperture of less than 1.0. In practice, the best air objective lens is [ka] ( [ka] It may have (corresponding to). Selecting the second objective lens 230 within the aforementioned constraints is possible with respect to parameter f Obj2 Step 708 includes configuring a second microscope to achieve the remote refocusing condition described in equation (11) above. As discussed above, M1 is set in steps 702 and 704. From equation (10), the only free parameter that can be used to set M2 is f Obj2 Since this is set by the conditions considered in step 706, f TL2 Therefore, in step 708, f TL2 d2 can be selected to satisfy the remote refocusing condition of equation (11). As a result, the only remaining free parameter in the first and second microscopes is, here, d2. As discussed above, in some embodiments, d2 may be selected to achieve the best imaging performance according to equation (17).

[0059] Steps 710 and 712 involve steps of selecting / configuring a third microscope within the microscopy system 200. Step 710 includes the step of selecting a third objective lens 232, collecting all (or as much as possible) of the light from the second objective lens 230, and optionally, enabling an inclination α that can be dynamically adjusted as discussed above. To achieve both of these conditions, it will be ensured that the third objective lens 232 can collect all or nearly all of the light from the second objective lens 230 even with the inclination α (i.e., the third objective lens 232 can capture all or nearly all of the conical angle +α from the second objective lens 230), so NA3 ≥ n2 is set. According to an embodiment, achieving light collection using the α inclination is done by using an objective lens that is mechanically configured to capture the light cone from the second objective lens 230 while also being positioned to accommodate the rotation / inclination of α. For example, the third objective lens 232 may include a solid glass frustum with chamfers or other shaped edges as discussed above. This configuration allows the third objective lens 232 to extend into the field of view of the second objective lens (using a sharp glass tip), and thus collect all or nearly all of the light from the second objective lens 230 while also allowing a rotation (α) of up to 45° (or more depending on the exact design). Step 710 sets the parameter f Obj3 to a certain extent. Thus, step 712 sets f TL3 and may include the step of configuring the third microscope so as to define the overall system magnification M T at the correct value for the detector 226 (e.g., by achieving Nyquist sampling when using a multi-pixel detector).

[0060] According to one embodiment, the first intermediate image plane IP1 can be used as an insertion point for the optical sheet 216, as shown in Figure 2B. In addition, in one embodiment, xy scanning of the optical sheet 216 can also be introduced to provide synchronized stereo imaging. The second objective lens 230 may also be used to scan a volume in the z direction by refocusing the image onto the second intermediate image plane IP2, which is then collected on the third intermediate image plane IP3 by the third objective lens 232. Applying this approach, flexible volume options with xyz scanning can be achieved. For example, xy scanning can be performed, and adjustment in the z direction can then be used to image another volume at a different depth in the sample 206. Thus, the overall stereographic volume or flexibility of the microscopy system 200 can be increased. In one embodiment, due to the tilt angle α, the optical axis of the image of the sample 206 cannot correspond to any of the primary x, y, or z axes. Therefore, pure xy scanning or pure z scanning may result in a shift of the optical sheet beam waist ω0 relative to sample 206 as the volume is collected using these individual axes. However, in some embodiments, xy scanning may be combined with z scanning of the correct amplitude so that the volume is imaged with the optical sheet beam waist remaining centered on the field of view during scanning, giving optimal segmentation and resolution throughout the volume.

[0061] As discussed above, in one embodiment, the third objective lens 232 can be configured with a glass frustum and tip, allowing the optical sheet 216 to be directly coupled into the second objective lens 230 at high numerical aperture. This enables non-dichroic operation (i.e., the beam coupling element 218 in Figure 2B can be eliminated). In addition, in an embodiment, where the second objective lens 230 is used to provide a stereoscopic image via remote refocusing z-scanning as discussed above, this approach also provides inherent synchronization for scanning. [Examples]

[0062] The following provides an example of a module, implemented according to the aspects and embodiments discussed above, which can be coupled to a standard microscope and convert the microscope into a high numerical aperture light-sheet microscope that uses only one objective lens in the sample.

[0063] Figure 8 illustrates the concept of an embodiment of a single-objective lens light-sheet microscope according to the aspects and embodiments disclosed herein. A primary objective lens / tube lens pair (red solid / circular indicates the back focal plane (BFP) of the objective lens) images a 3D biological sample into an intermediate space. The scanning system relays this 3D image to another intermediate space, where it is collected by a second tube lens / objective lens pair (blue dashed BFP). The second objective lens creates an aberration-free 3D copy of the sample at a magnification of approximately 1. This virtual 3D sample is then re-imaged via a third tilted microscope (green dotted BFP) tilted relative to the primary image plane. The light sheet is coupled into the primary objective lens in this tilted image plane (red solid beam), resulting in a single-objective lens light-sheet microscope. Rapid movement of a single scanning mirror obtains the 3D volume. The three objective lens BFPs are imaged onto each other, as indicated by the red solid, blue dashed, and green dotted circles. The red solid disk represents the optical sheet occupied area in the BFP, and the yellow solid region represents the system's collected numerical aperture. This design achieves virtually immeasurable loss with resolution without sacrificing a perceptible amount of yellow area.

[0064] Figures 9A and 9B are schematic diagrams that further illustrate this feature by showing the ray transmission angles for the objective lenses in the system in Figure 2A. Specifically, Figures 9A and 9B illustrate a theoretical model showing the angular passbands of the primary (first) objective lens 210 (red dot), the third objective lens 232 (green dot), and the combination of the first, second, and third objective lenses (yellow dots). The angular passband of the second objective lens 230 is not visible in Figures 9A and 9B. Figures 9A and 9B are 3D equivalents of the red, blue, green, and yellow circular regions shown in the upper central portion of Figure 8. Each yellow dot on the surface of the illustrated sphere represents the ray angle that can be reached from a point source in the sample, propagating through the three objective lenses and reaching the detector. Each red dot represents a ray that is collected by the first objective lens (1.35 NA silicon in this embodiment) but clipped by the second objective lens (0.95 NA air in this embodiment). Each green dot indicates a ray that the third objective lens (1.0 NA glass in this embodiment) could collect if generated by the second objective lens. The yellow area almost completely fills the red area, demonstrating that the system transmits almost all of the ray collected by the first objective lens. In this embodiment, >95% of the ray collected by the first objective lens can pass through to the second objective lens, and >99% of the ray collected by the second objective lens can pass through to the third objective lens, providing a numerical aperture of approximately 1.33.

[0065] It should be noted that the overall numerical aperture (ANA) is influenced by the design of each objective lens in the system. For example, if the primary objective lens is selected with an NA of 1.33 instead of 1.35, >99% of the rays collected by the first objective lens may pass through the second and third objective lenses. In practice, rays at the edge of the primary objective lens's ANA may be aberrated and may not contribute to improved resolution. Therefore, it may be beneficial to distinguish between the specified ANA (rays collected) and the useful ANA (rays contributing to improved resolution). Embodiments of the system disclosed herein can capture >99% of the useful ANA of the primary objective lens. Furthermore, by replacing the second objective lens with a 0.96NA air objective lens, >99% of the specified 1.35NA rays of the primary objective lens can be collected.

[0066] Figure 10 illustrates an example of a simple, low-cost design for the system of Figure 8 with uncompromising spatiotemporal performance. Figure 10 shows an exploded view of the components of this system embodiment. Box A is a simple objective lens / tube lens pair, which may be a standard microscope stand. The primary objective lens is selected to have a high numerical aperture and intermediate refractive index, which is optimal for imaging deep into living biological samples with high resolution. Box B is a scanning repeater with a single galvanometer mirror, which can rapidly acquire volumetric images. Box C is another tube lens / objective lens pair, which, together with Box B, perfectly meets the requirements of Box A. Box D is a specially configured objective lens / tube lens pair that solves opticmechanical problems that may otherwise exist in the system.

[0067] Figure 11 illustrates an embodiment of a specially configured objective lens / tube lens pair in box D with a glass tip (which is also an embodiment of the third objective lens discussed above). Figures 12A–C further illustrate a side view of an embodiment of a specially configured objective lens with a glass tip, configured to have optical performance and geometric shape that enables the extraction of high-resolution images of nearly 100 μm from a flat surface with negligible loss in resolution and high transmittance over a tilt angle range of 0–45 degrees. Figure 12A is a drawing showing the photomechanically coupled design considerations of this embodiment of an objective lens specially configured to collect tilted air space images of only about 100 μm from a flat surface with a tilt angle range of 0–45 degrees. Figure 12B is a corresponding CAD rendering of the embodiment of the specially configured objective lens, and Figure 12C is a photograph of the embodiment of the lens. Table 1 below provides specifications for the embodiment of the lens corresponding to Figures 12A–C. Table 1 [Table 1]

[0068] An embodiment of the emission path of the system shown in Figure 10 was constructed, harmonized, and tested, and the test data demonstrate that the system can image a 30-degree tilted plane without measurable loss in resolution compared to a conventional microscope with the same primary objective lens. The embodiment of the system shown in Figure 10 used to obtain these results was configured according to the optical system specifications listed in Table 2 below. The optical elements are listed in Table 2 in order of physical arrangement (from sample to camera). The system was used to image an Argolight SIM slide using 488 nm laser excitation in a simple reflected-emission configuration. Coherent interference / speckle from the laser was acceptable. An additional Chroma ET525 / 50 emission filter was added immediately before the quad-band filter (item 12 in Table 2) to narrow the emission bandwidth, which would otherwise be very wide. The image stack was obtained by using a closed-loop piezoelectric actuator in the second objective lens. The xy stage, primary objective lens, galvanometer, and third objective lens remained constant throughout the process, and the camera exposure time and laser power were kept constant throughout. Table 2 [Table 2-1] [Table 2-2]

[0069] To benchmark the emission path and evaluate the implementation of a specially configured third objective lens (hereinafter referred to as the AMS-AGY v1.0 lens), data was obtained for the following three configurations. 1. Nikon 40x 0.95NA, tilt=0°: The third objective lens in the emission path was a Nikon 40x 0.95NA air objective lens in a standard remote refocusing array (axially aligned without tilt), providing a reliable system benchmark. 2. AMS-AGY v1.0, tilt = 0°: The third objective lens of Nikon in configuration 1 was replaced with an embodiment of a third objective lens specially configured without tilt to directly correspond to configuration 1. 3. AMS-AGY v1.0, tilt = 30°: For comparison with configurations 1 and 2, an embodiment of this system was configured using an AMS-AGY v.1.0 lens as a third objective lens with a 30° tilt.

[0070] The following four different patterns on the Argolight SIM slide were used for imaging. Target: A set of 240 μm diameter concentric rings spaced 10 μm apart. Each ring consists of two pairs of lines separated by 750 nm (good for viewing at maximum field of view). Grid: 110 × 110 μm with 10 μm spacing 2 Square grid. Each line pair is separated by 750nm (good for viewing the higher quality portion of the center of the field of view and for checking field flatness and distortion). SIM lines: 14 line pairs ranging from complete overlap to 390nm separation in 30nm increments (good for evaluating resolution). 3D ring: A submicron diameter ring of a 9×9×9 3D cubic array separated by 5 μm in the X, Y, and Z directions (good for evaluating 3D field of view).

[0071] The data was collected for each of the three system configurations identified above, by imaging the patterns and features described above.

[0072] Test data generated by imaging the four patterns described above for each of the three system configurations identified above demonstrated that Nikon-based remote refocusing provides the same resolution as conventional microscopes. Using all Nikon objective lenses, the remote refocusing portion of the emission path delivers at least 270 nm resolution at the center of the field of view. This is the same resolution as that provided by standard commercial-based primary objective lenses. Remote refocusing configured with an AMS-AGY v1.0 lens as the third objective lens also provides the same resolution as conventional microscopes. Substituting the AMS-AGY v1.0 lens for the third Nikon objective lens preserves the 270 nm resolution in non-tilted remote refocusing. The target pattern images for both configurations 1 and 2 cover 240 × 240 μm, as captured by the camera sensor. 2 The system demonstrated the ability to image the field of view, with line pairs visible down to approximately 200 μm, far exceeding the system's designed 100 μm diameter field of view. Grid pattern imaging was achieved with high-quality 110 × 110 μm grids. 2 The field of view was shown, and the line pairs were visible throughout with minimal distortion. SIM line imaging demonstrated a 270nm resolution at the center of the field of view. Image stacks of the 3D ring pattern showed no qualitatively distinguishable difference in imaging performance between configurations 1 and 2 across the 3D field of view.

[0073] Furthermore, remote refocusing, configured using the AMS-AGY v1.0 lens, still provides the same resolution as a conventional microscope with a 30-degree tilt of the third microscope. Using the AMS-AGY v1.0 lens, the third microscope was tilted to an angle of 30 degrees, and the acquired image data demonstrated that the system retained a 270nm resolution on both the parallel and vertical axes. By the Rayleigh criterion, a 270nm resolution corresponds to an effective numerical aperture of 1.2. After rescaling of ycos(α) (where α is the tilt angle) was applied to compensate for the "stretching" along the image y-axis caused by the tilt, the acquired image stacks of target and grid patterns demonstrated the ability to produce high-quality images across the field of view that are essentially the same quality as those achieved using configurations 1 and 2. Similarly, the SIM line imaging and 3D ring pattern imaging performance was qualitatively indistinguishable from the untilted configuration after applying y-axis rescaling and zsin(α) shear correction.

[0074] Tables 3-5 below provide specifications for several alternative optical configurations for parts of the system in Figure 10 based on the specifications provided in Table 1. For example, Table 3 below provides an alternative embodiment for the primary objective lens. Table 3 [Table 3]

[0075] In some embodiments and applications, galvanometric scanning may not be required. Table 4 below provides embodiments that may be used for high-content screening systems (or a combination of both), for example, using a piezoelectric element on a second objective lens to capture volume, using stepped scanning or a system. Table 5 below provides another embodiment in which galvanometric scanning is eliminated, using the alternative primary objective lens described in Table 3. Table 4 [Table 4] Table 5 [Table 5] [Examples]

[0076] Example 2: Design Options Many applications will benefit from different choices of magnification, numerical aperture, field of view (FOV), and immersion medium (e.g., water). Table 6 below provides additional examples of primary objective lens options. Various scanning regions and fields of view may also be implemented.

[0077] Embodiments without galvanometer scanning may be less expensive due to fewer optical systems, higher efficiency, and easier maintenance. Galvanometer-free systems may be attractive for high-content screening systems using stepwise scanning. Sample scanning may be a good option for wide-area exploration, stepwise scanning, or high-throughput screening (i.e., multi-well plates). The z-range may be limited by the working distance of objective lens 1. High-speed motion may be coupled to the sample via the immersion medium. Objective lens 1 scanning may be good for conventional focusing using the working distance of the primary objective lens. Objective lens 2 scanning may be good for high-speed scanning isolated from the sample using remote space. The z-range may be optically limited by either the mechanics of objects 2 and 3, or by the remote refocusing range. Objective lens 3 scanning may be similar to objective lens 2 scanning, but in a tilted array.

[0078] In embodiments with a single "scanning" galvano, the field of view (FOV) may be limited by objective lens 3 on only one axis. The primary objective lens may limit the field of view on the scanning axis. Adding a galvano scanning unit provides ultrafast scanning and increases the FOV. Typical galvano steps and settling times are shorter than the rotation time of modern sCMOS chips, and therefore the stereoimaging rate is limited only by the camera's data rate (or available photons).

[0079] In embodiments with two "XY" galvanometers, the field of view (FOV) is not limited by objective lens 3. The primary objective lens can determine the field of view. A second (orthogonal) galvanometer scanner is used as a tilting device to access the full field of view of the primary objective lens. This is an attractive and rapid method for accessing the FOV of the primary objective lens when using optical arrays or camera chips that do not support a full field of view within a single frame. Table 6 [Table 6] [Examples]

[0080] Example 3: Alignment targets for good performance The following guidelines may be applicable to various optical configurations.

[0081] XY and Tip / Tilt Alignment: The primary objective lens (O1) sets the optical axis of the system. Each subsequent lens must then be aligned so that its optical axis is collinear with the primary axis. In a system including one or more galvanometers, each galvanometer mirror should be aligned on the optical axis, and the rotation axis of each galvanometer should coincide with and be perpendicular to the optical axis. This defines the xy (translation) and tip / tilt (rotation) of each element, which in practice can be achieved using a suitable laser beam.

[0082] Z-matching: In principle, lens pairs should be axially separated so that they preserve collimation. That is, for any pair of lenses, a collimated beam input should give a collimated beam output. This is the simplest way to match the system and define the z position of each lens, once the primary objective lens position is fixed. The galvanometer mirror should be conjugated to the back focal plane of the primary objective lens (BPF1) so that the rotation of the mirror results in pure translation in image space (for small angles).

[0083] Commercial Base: When using a commercial base, the primary objective lens (O1) and the first tube lens (TL1) typically do not preserve collimation (usually the tube lens is too close). This can be corrected by shunting the entire optical column after the first scanning lens (SL1) to restore collimation. For systems without a galvanometer, the correction can be made after the second tube lens (TL2). The following equations describe the required displacement for each case and can be used as an index of magnitude (in practice, the correct displacement must be found by aligning with the appropriate laser beam). • No galvanic: [ka] During the ceremony, [ka] • One or more galvanos: [ka] During the ceremony, [ka] Note: In addition to the above, when using a commercial base for best performance, O1 must ensure good back focal plane mapping at its nominal focal position (for the intended sample).

[0084] Back focal plane mapping: If the above goals are achieved, the back focal plane (BFP1) of the primary objective lens will be effectively imaged throughout the system, i.e., from BFP1 to G1, from G1 to BFP2, and from BFP2 to BFP3 (and the same concept also applies to non-galvano or two-galvano systems). This is similar to relaying from image plane IP0 to IP4, but if not done well enough, it will be accompanied by less (but still significant) results. Errors in image plane alignment are usually obvious from the resulting focus shift. Errors in the BFP relay are more difficult to notice and can result in the following symptoms.

[0085] Error from galvano to BFP1: It can introduce angular motion, which should be a pure parallel translation of the image plane. This can modulate the tilt of the light sheet as a function of the scan and confuse image processing and data interpretation.

[0086] Error from BFP1 to BFP2: An easy mistake if attention is not paid during alignment, which can result in clipping of the image of BFP1 at BFP2. The result is a reduced effective aperture and impaired remote refocusing performance. If the point spread function measurement is good at IP1 and unexpected in the far space at IP2, this alignment should be checked.

[0087] Error from BFP2 to BFP3: Similarly, an easy mistake if attention is not paid during alignment, which also results in reducing the effective aperture of the system. If the point spread function measurement is good in the far space at IP2 and unexpected at IP4, this alignment should be checked.

[0088] Note: When O1 has a z range (e.g., about 10 mm for a commercial base), it should be set to its nominal focal position during the alignment phase for best performance (with respect to the intended sample).

[0089] Optical sheet coupling and tilt angle "α": Any given optical sheet should be coupled into the primary objective lens (O1) such that its marginal rays coincide with the marginal rays of the objective lens (i.e., the edge of the optical sheet should touch the maximum collection cone angle of the objective lens). For a given option of the optical sheet, the third microscope system should then be tilted so that the optical sheet is uniformly focused. Together, there are many options of primary objective lenses and many optical sheet options that will result in a tilt angle α being determined, and thus an exact alignment target cannot be set without further defining the system. However, by following this basic alignment protocol, unnecessary losses in resolution and efficiency can be avoided.

[0090] Galvano flatness: If the above alignment goals are achieved but non-acceptable PSF results are presented, check the galvano flatness (e.g., using a shearing interferometer). It is desired to be specified better than λ / 10 PV or λ / 14 RMS across the aperture (image of BFP1 in the galvano).

Example

[0091] Example 4: Configuration indicators for a high NA single objective lens optical sheet The following non-limiting guidelines may facilitate the configuration of a single objective lens optical sheet microscope according to one or more embodiments.

[0092] 1. Select the primary objective lens (O1): Use the table below to select the primary objective lens based on the intended sample type. With O1 selected, use the corresponding manufacturer's tube lens (TL1) for commercial base compatibility. Select the remote refocusing module by selecting the recommended secondary objective lens (O2) and corresponding tube lens (TL2). Note: This selection is independent of the base microscope; for example, a Nikon base (O1+TL1) can be matched with Olympus remote refocusing (O2+TL2). Oil immersion with coverslip: [Table 7] Silicone immersion with coverslip: [Table 8] Submersion in water with coverslip: [Table 9] Water immersion: [Table 10]

[0093] Choosing a 2.0, 1, or 2 galvanometer scanner: While galvanometer scanners offer ultrafast scanning across the full field of view of the primary objective lens, they may not be ideal for all systems as they add surplus optics, etc. The following (1 magnification) repeaters are a good starting point. [Table 11] Considerations for galvanometer scanners:

[0094] Magnification: The galvanometer scanner must be a repeater with a magnification of 1. f SL2 / fSL1 = 1

[0095] Scanning lens performance: It is desired to confirm that the scanning lens is capable of handling the pupil size, field of view, and color range that it is desired to adapt. For example, the above CLS-SL scanner can deliver the following (diffraction-limited) field diameters over (400 - 700) nm with a given pupil size. Field of φ26 mm, pupil of φ2.5 mm Field of φ22 mm, pupil of φ3 mm Field of φ16 mm, pupil of φ4 mm Field of φ13 mm, pupil of φ5 mm Field of φ11 mm, pupil of φ6 mm Field of φ9 mm, pupil of φ7 mm Note: The pupil diameter must not exceed the image of the back focal plane of the primary objective lens. Scanning pupil ≥ BFP1 (f SL1 / f TL1 ), where BFP1 = 2f TL1 (NA1 / M1) (and NA1 and M1 are the numerical aperture and magnification of O1)

[0096] Galvano size: It is desired to confirm that the galvano diameter is large enough to avoid clipping of the image of the back focal plane of the primary objective lens.

Number

[0097] 3. Camera Selection: High quantum efficiency (QE) and low readout noise sCMOS chips are recommended. For example, the PCO edge 4.2 is a good choice for most configurations. When selecting a camera, consider the following:

[0098] Pixel count: The AMS-AGYv1.0 objective lens can deliver a diffraction-limited field of view of φ150 μm and up to φ250 μm at lower NAs. λ ave Regarding Nyquist sampling at approximately 0.55 μm, this is comparable to the highest quality at approximately 900 pixels and up to approximately 1,500 pixels, and the imaging is still very good.

number

[0099] Pixel size: The AMS-AGYv1.0 objective lens has an effective focal length of 5mm. The last tube lens, TL3, can be used as a free parameter to adjust the magnification for Nyquist.

number

[0100] Example 5: Expanded microscopy examination of a relatively large (aqueous) sample at high resolution. The following provides an example of a module implemented according to the aspects and embodiments discussed above, which can be coupled to a standard microscope to represent high-resolution images of weakly fluorescent samples. The system uses an optical sheet with minimal optics on the emission path. Nikon-based and Olympus objective lenses are used.

[0101] For large samples in this embodiment, a galvanometer repeater will not be used. Instead, the sample will be scanned at a lower rate, but without size limitations, except for the relatively large working distance of the primary objective lens (approximately 600 μm in this case). An AR-coated window will be used in the second objective lens for maximum transmittance. If sample scanning is problematic, a galvanometer scanner can be added later.

[0102] Table 7 below provides an example of optical arrays and imaging components for a given configuration, taking these criteria into consideration. Table 7 [Table 12-1] [Table 12-2] [Examples]

[0103] Zebrafish immersion system with large field of view and long working distance. The following provides an example of a module implemented according to the aspects and embodiments discussed above, which can be coupled to a standard microscope to optimize the field of view and speed. Thus, two galvanometer systems would be selected to maximize the data rate on the sCMOS camera at the expense of optical performance.

[0104] Please note the following regarding the system. BFP1=2 * 180 * (1 / 20) = 18mm • Scanning pupil = 18 * (70 / 180) = 7 mm ·d G1 =√2 * Scanning pupil = 9.9 mm The 7mm scanning pupil is large for the CLS-SL scanning repeater, and therefore the diffraction-limited field of view will be about 9mm at IP1 (or about 450μm in the sample), but will still image up to 22mm (the field of view of the primary objective lens) at lower numerical apertures. The 10mm galvanometer is large enough for the pupil, but this is a very large mirror, and therefore its flatness should be checked. Here, a standard 25mm coverslip is used to compensate for O2 to save costs. Taking all criteria into consideration, the optical array system settings can be found in Table 8 below. Table 8 [Table 13-1] [Table 13-2] [Examples]

[0105] Example 7: Tube Lens Assembly One of the challenges in designing a single objective lens optical sheet is to create good remote refocusing. A key requirement for air-based remote refocusing is to match the magnification of the remote image to the refractive index of the primary objective lens. As the primary objective lens changes, the magnification and refractive index often change as well, which can make it difficult to find the correct set of optical systems. Table 9 below provides examples of various tube lens assemblies. Table 9 [Table 14]

[0106] While several aspects of at least one embodiment have been described above, it should be understood that various modifications, alterations, and improvements will be readily conceivable to those skilled in the art. Such modifications, alterations, and improvements are intended to be part of this disclosure and within the scope of the invention. It should be understood that the embodiments of the methods and apparatus discussed herein are not limited in this application to the details of the structure and arrangement of components described in the foregoing description or illustrated in the accompanying drawings. The methods and apparatus are implementable in other embodiments and can be practiced or carried out in various ways. Specific implementation examples are provided herein for illustrative purposes only and are not intended to limit the scope of this invention.

[0107] Furthermore, the terminology and grammar used herein are for illustrative purposes only and should not be considered limiting. In this specification, the use of “including,” “comprising,” “having,” “containing,” “involving,” and their variations is intended to encompass the items listed thereafter, their equivalents, and additional items. A reference to “or” may be interpreted as inclusive, such that any term described using “or” may refer to a single term, more than one, or all of the terms described. Any references to front and back, left and right, top and bottom, upper and lower, and vertical and horizontal are for illustrative purposes only and are not intended to limit the system and method or its components to any single position or spatial orientation. The terms “light,” “optical signal,” and “optical signal” may be used synonymously herein and generally refer to electromagnetic signals propagating through a given medium, which may be empty space, e.g., a vacuum, or the atmosphere, e.g., consisting of air, or other medium such as a fiber or other optical system component. The terms “light,” “optical signal,” and “optical signal” are not intended to imply any particular properties of light such as frequency or wavelength, bandwidth, coherence, spectral density, quality factors, and may include infrared, visible, and / or ultraviolet electromagnetic radiation, or other non-ionized electromagnetic radiation conventionally handled in the field of optics.

[0108] Therefore, the above description and drawings are merely examples of embodiments.

Claims

[Claim 1] The invention as described in the drawings of the present application.

Images