Systems and methods for three-dimensional imaging - Patents.com

Abstract

To provide systems and methods for depth-resolved imaging of microscopic subjects, and volumetric three-dimensional optical imaging and / or time resolved imaging of microscopic subjects.SOLUTION: Methods, devices, and systems for up to three-dimensional scanning of target regions at high magnification are disclosed, the method comprising conveying illumination light onto and / or into a subject, and receiving image light returning from multiple depths over a surface or within the subject at a single instant.SELECTED DRAWING: Figure 10

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 194,155, filed July 17, 2015, U.S. Provisional Patent Application No. 62 / 195,729, filed July 22, 2015, and U.S. Provisional Patent Application No. 62 / 202,126, filed August 6, 2015, each of which is incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under grants R21NS053684, R01NS076628 and R01NS063226 awarded by the National Institutes of Health (NIH), 0954796 awarded by the National Science Foundation (NSF), and W911NF-12-1-0594 awarded by the Department of Defense (DoD). The Government has certain rights in this invention.

[0003] Technical Field The present invention relates to depth-resolved imaging of microscopic objects, and in some embodiments, more particularly to volumetric three-dimensional optical and / or time-resolved imaging of microscopic objects. Summary of the Invention [Means for solving the problem]

[0004] The subject matter of this disclosure includes embodiments that can transmit illumination light onto and / or into an object and simultaneously receive returned image light from multiple depths across a surface or in the object. A convenient identifier for this process is Depth Resolved Imaging (DRI).

[0005] Illumination light may include excitation light, suppression light as used in photomanipulation, and / or illumination light returning from the object as a result of reflection, scattering, or other processes to enable feature detection. Illumination light may be shaped to provide a low aspect ratio, narrow, collimated beam (pencil beam), flat beam, wide beam, or any other shape of beam suitable for the above purpose at the object. In some embodiments, illumination light (also identified herein as an illumination "beam") may aid in feature identification by optical sectioning. In some embodiments, multiple illumination beams may be projected, such as in a photomanipulation system.

[0006] In some embodiments, the position and / or orientation of the illumination beam is altered by one or more beam redirectors to aid in the formation of a two- or three-dimensional image. Additionally, the illumination beam can be repeatedly altered to generate multiple images over time, thereby forming a two- or three-dimensional moving image. The illumination beam can be progressively swept in steps, or the illumination beam can be directed in a non-continuous "scan" pattern. All of these mechanisms for altering the position of the illumination beam at the object are identified herein by the term "scanning" the illumination beam.

[0007] The scanning pattern may be continuous or non-continuous. Non-continuous scan patterns may be generated by aiming the beam with an acousto-optic deflector or spatial light modulator (SLM), e.g., the beam "hops around" rather than forming a continuous sweeping pattern. Additionally, the scanning of the illumination beam may produce continuous, but complex or irregular patterns, e.g., circles, figure eights, Lissajous patterns, or more complex chaotic patterns. With flexible control of the scanning, such as that provided by an acousto-optic deflector or SLM, the scanning may be controlled to cause the illumination beam to enter areas of greater interest more frequently, e.g., object regions with faster motion or more complex features. Possible patterns may include statistically random patterns, however, which may focus more energy on the area of ​​interest over time. The illumination beam can be projected in any manner that instantaneously projects illumination light across multiple depths of the object (again, in any manner that causes the illumination light to return), and the light returning from the multiple depths can be captured and directed so as to be usable for imaging at that instant. Thus, the illumination beam can be projected across the object in the axial direction (0°) up to the maximum angle (approximately in the long axis cross section) allowed by the numerical aperture of the objective lens.

[0008] As the area changes due to the scanning of the illumination beam, the light returning from the multiple depth regions illuminated by the illumination beam is "descanned" so that the images of the light returning from the various depths are fixedly focused or defocused on one or more photodetectors. It is also noted that the photodetectors may be zero-dimensional, one-dimensional or two-dimensional detectors. In some embodiments, the light returning from the multiple depths is directed in synchronization with the redirection of the illumination beam. In effect, both the illumination beam and the image "beam" are aimed so that the image remains fixed on the detector, and only the element or elements required for aiming are controlled during the process of forming an image covering the object area. The fixed image may be a single image representing the object at a single time point, or multiple images representing the object at multiple time points that collectively form a moving image (i.e., a time-resolved image).

[0009] It is noted that image light is sometimes referred to herein as an image "beam," but this is a figurative identifier to facilitate understanding that it is directed by imaging optics back from the object region to one or more photodetectors. The terms scanning and descanning are used to characterize the redirection of illumination and image beams to form a single static image as well as to form multiple time-resolved images. Scanning and descanning can be performed independently in multiple axes. For example, an illumination beam can be swept along one axis to illuminate a planar region, and the illuminated planar region can be swept across a volume by sweeping along a different axis.

[0010] In separate embodiments, the illumination beam and the image beam pass through a single optical assembly, the objective lens. At any one time, the optical elements that transmit the illumination beam through the objective lens, receive those lights from the object, and transmit the image beam to the photodetector are redirected by elements of a scanning / descanning assembly that is controlled to redirect the light so that the illumination beam and the image beam remain in a relationship that provides for the formation of a fixed image on one or more photodetectors. In some embodiments, a single movable element, e.g., a mirror, redirects the outgoing illumination beam and the adjacent incoming image beam through the same aperture of the scanning / descanning assembly. In some variations, the outgoing illumination beam and the adjacent incoming image beam are redirected by multiple beam redirectors that are independent of each other, but are synchronously controlled. Also, multiple independent illumination beams, e.g., from a photomanipulation illumination source, can be controlled by separate beam redirectors.

[0011] The position and orientation of the illumination beam are considered to include any position and orientation that allows light to be captured and transmitted as an image beam from multiple depths in the object by the same objective lens used to position and shape the illumination beam. The illumination beam at the object may be parallel to the optical axis of the objective lens or at an oblique angle relative to the optical axis. The angle, or position, or both of the beam at the object may vary depending on the range of patterns resulting from scanning with the illumination beam. In any of these positions and orientations, the photodetector(s) maintain an image of the illuminated area at the object on the photodetector(s).

[0012] The optical element that maintains the image on the one or more photodetectors may include imaging optics that form an intermediate image in a plane having a normal that is not perpendicular to the first imaging optical axis of the optical system on the object side at the point where the intermediate image is formed. That is, the image beam includes light from multiple depths in the area of ​​the object, so that the points where the light from different depths is focused are at different locations on the axis and define an intermediate image in a plane having a normal that is substantially oblique or perpendicular to the first imaging optical axis. In some embodiments, the photodetectors may be positioned so that the intermediate image coincides with a one-dimensional or two-dimensional detector, such as a CCD. In some other embodiments, an unfocused separate image may be formed from the intermediate image by imaging the intermediate image with another imaging optic. The optical axis of such a separate imaging optic may be aligned at a predefined angle with respect to the optical axis of the imaging optics that forms the intermediate image, but is not perpendicular to the plane of the intermediate image. The angle of alignment may be selected to be less acute than if the optical axis of the separate imaging optics was aligned perpendicular to the intermediate image plane. This can facilitate collision-free positioning and adjustment of optical elements on either side of the intermediate image and can ultimately increase the total amount of light transmitted to the photodetector or photodetectors. In some embodiments, the photodetector can be located at the location of the unfocused separate image. In other embodiments, the unfocused image can be captured by imaging optics having an axis perpendicular to the plane of the unfocused image, thereby producing a separate image on the photodetector.

[0013] In yet other embodiments where an intermediate image is formed, a beam rotating optic, e.g., an optical wedge, may be placed at or near the location of the intermediate image to substantially rotate the plane of the intermediate image. The optical axis of another imaging optic that focuses the intermediate image may be aligned parallel to the imaging optic that forms the intermediate image. Alternatively, a photodetector may be placed at the point where the rotated image is formed by the beam rotating optic.

[0014] In some further embodiments, the optical element that maintains the image on the one or more photodetectors includes imaging optics capable of forming an intermediate image in a plane where the intermediate image would be formed if no imaging optics were provided, but with a normal that is not perpendicular to the first imaging optical axis of the object-side optics where the mirror is positioned to reflect the light back to the object-side optics. As mentioned above, since the image beam contains light from multiple depths in the area of ​​the object, the points where the light from different depths is focused are different locations on the axis and define an intermediate image having a normal that is substantially oblique or perpendicular to the first imaging optical axis depending on the angle of the illumination beam. The mirror is positioned at an angle half the angle of the plane of this intermediate image, so that the light forming the intermediate image is reflected back to the imaging optics and can then be split from the incident image beam by a beam splitter and directed to the photodetector. Thus, in this embodiment, the imaging optics that forms the intermediate image is also used as the optics that captures the intermediate image and finally transmits it to the photodetector. Because the optical axis of the imaging optics is not parallel to the normal of the intermediate image plane, the imaging optics will not be focused there.

[0015] It is noted that more than one detector can be used to detect one or more different wavelengths in an image beam, which can be separated from the image beam by dichroic beam splitters, each of which selects a range of wavelengths, thereby directing selected light to each photodetector.

[0016] In DRI embodiments, it may be desirable to maximize the numerical aperture of the image beam. In some embodiments, the image beam can be spread nearly to the full aperture of the objective lens, if possible. In some embodiments, the image beam spreads across a major portion of the aperture of the objective lens. In some embodiments, the image beam covers a continuous or non-contiguous area that extends to the opposite side of the optical axis of the objective lens (i.e., is not restricted to one side of the optical axis of the objective lens). In some embodiments, the image beam is restricted to one side of the optical axis, while the illumination beam is restricted to the opposite side of the optical axis of the objective lens. In some embodiments, the image beam covers an area of ​​the aperture that is a majority of the area of ​​the objective lens aperture.

[0017] Another embodiment provides both depth-resolved image capture and confocal imaging in the same basic device, which can be configured as a multimode DRI / confocal microscope. In some embodiments, the beam from the illumination source can be shaped to have a particular shape for depth-resolved imaging and different properties for confocal imaging. Alternatively, different sources can be provided. For example, the shape and size of the beam can be changed using known techniques and devices. Here, the same imaging optics or optical redirectors used for scanning and descanning for DRI can be used for scanning and descanning the broad beam for confocal microscopy. According to some embodiments, in confocal mode, the DRI microscope can illuminate a line or spot in the long axis section by focusing the illumination broad beam to a line or spot that coincides with the front focal plane. A multimode DRI / confocal microscope can provide automated adjustability of the object (or more precisely, the support for the object), the height of the objective lens (z-axis or axial position), thereby changing the front focal plane, or can configure or move elements of the imaging device to realize focusing techniques as known to those skilled in the art for changing the focal plane height.

[0018] Objects and advantages of embodiments of the subject matter of the present disclosure will become apparent from the following description when taken in conjunction with the accompanying drawings.

[0019] Several embodiments are described below with reference to the accompanying drawings, which are not drawn to scale. Like reference numerals represent like elements throughout the drawings. Some features may not be drawn to scale or may be intentionally not drawn to scale to facilitate illustration and explanation of the underlying features. Indeed, in most drawings, only edge and center rays are shown to suggest the paths followed by the illumination and image beams to avoid unnecessarily complicating the depicted rays and because designer choices allow for variations in features such as the aperture of the optical elements. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 is a schematic diagram illustrating various high-level features of a three-dimensional imaging system in accordance with one or more embodiments of the present subject matter. [Figure 2A] 2 illustrates an objective lens that can be used as part of focusing module 108 in the schematic diagram of FIG. 1, in accordance with one or more embodiments of the presently disclosed subject matter. [Figure 2B] A gradient index (GRIN) lens that can be used as part of focusing module 108 in the simplified diagram of FIG. 1 is shown, in accordance with one or more embodiments of the presently disclosed subject matter. [Figure 2C] 1 illustrates a portion of a DRI configuration showing an objective lens associated with a GRIN lens, according to an embodiment of the disclosed subject matter. [Figure 3A] 2 illustrates an illumination setup using a cylindrical lens that can be used as part of the beam conditioning module 120 in the simplified diagram of FIG. 1, in accordance with one or more embodiments of the subject matter of the present disclosure. [Figure 3B] FIG. 1 shows an illumination setup using a single scanning mirror that can be used as part of the beam conditioning module 120 in accordance with one or more embodiments of the subject matter of the present disclosure and provides illumination scanning. [Figure 3C] 1 shows an illumination setup using a pair of scanning mirrors that can be used as part of the beam conditioning module 120 in the simplified diagram of FIG. 1 and provide illumination scanning, in accordance with one or more embodiments of the subject matter of the present disclosure. [Figure 4A] 1 shows an objective lens combined with a lens arrangement that can be used as part of a telescope module 110 in the simplified diagram of FIG. 1, according to one or more embodiments of the subject matter of the present disclosure. [Figure 4B] 1 shows a lens arrangement that can be used as part of image forming module 126 in the simplified diagram of FIG. 1, in accordance with one or more embodiments of the subject matter of this disclosure. [Figure 5A] 2 shows a schematic diagram of a first apparatus for the separation module 112, the scanning module 116 and the de-scanning module 118 of FIG. 1, in accordance with one or more embodiments of the presently disclosed subject matter. [Figure 5B] 1 shows a schematic diagram of a second apparatus for the separation module 112, the scanning module 116 and the de-scanning module 118 of FIG. 1, in accordance with one or more embodiments of the presently disclosed subject matter. [Figure 5C] 1 shows a schematic diagram of a third apparatus for the separation module 112, the scanning module 116 and the de-scanning module 118 of FIG. 1, in accordance with one or more embodiments of the presently disclosed subject matter. [Figure 5D] 1 shows a schematic diagram of an apparatus for the separation module 112, the scanning module 116 and the de-scanning module 118 of FIG. 1 for handling multiple illumination sources, in accordance with one or more embodiments of the presently disclosed subject matter. [Figure 5E] 1 shows a schematic diagram of an alternative apparatus for the separation module 112, scanning module 116 and de-scanning module 118 of FIG. 1 for handling multiple illumination sources, in accordance with one or more embodiments of the presently disclosed subject matter. [Figure 6A]1 shows an optical setup for depth-resolved imaging that can be used for the optional field rotation module 128 in the simplified diagram of FIG. 1 according to one or more embodiments of the subject matter of the present disclosure. [Figure 6B] 1 shows an alternative optical setup for depth-resolved imaging that can be used for the optional field rotation module 128 in the simplified diagram of FIG. 1 in accordance with one or more embodiments of the subject matter of the present disclosure. [Figure 6C] 1 shows an alternative multi-stage optical setup for depth-resolved imaging that can be used for the optional field rotation module 128 in the simplified diagram of FIG. 1 in accordance with one or more embodiments of the subject matter of the present disclosure. [Figure 6D] 1 shows another alternative optical setup for depth-resolved imaging that can be used for the optional field rotation module 128 in the simplified diagram of FIG. 1 in accordance with one or more embodiments of the subject matter of the present disclosure. [Figure 6E] 10 illustrates an alternative optical setup with respect to the simplified diagram of FIG. 1 without the optional field rotation module 128, in accordance with one or more embodiments of the presently disclosed subject matter. [Figure 6F] 10 illustrates an alternative optical setup using an array of optical fibers, relative to the simplified diagram of FIG. 1, without the optional field rotation module 128, in accordance with one or more embodiments of the subject matter of this disclosure. [Figure 6G] 1 shows an alternative optical setup using a spatial light modulator or custom optical components that can be used for the optional field rotation module 128 in the simplified diagram of FIG. 1 in accordance with one or more embodiments of the subject matter of the present disclosure. [Figure 6H]1 shows an alternative optical setup using a wedge component that can be used in the optional field rotation module 128 in the simplified diagram of FIG. 1, in accordance with one or more embodiments of the subject matter of the present disclosure. [Figure 6I] 1 illustrates one variation on optional field rotation module 128 that uses selectable lenses to provide different magnifications, according to one or more embodiments of the presently disclosed subject matter. [Figure 7A] 10 illustrates a detection arm setup that may be used as part of the image adjustment module 130 of FIG. 1 and that uses a movable stop to adjust the aperture, in accordance with one or more embodiments of the disclosed subject matter. [Figure 7B] 1 illustrates a detection arm setup that may be used as part of the image adjustment module 130 of FIG. 1 and that uses an emission filter, in accordance with one or more embodiments of the presently disclosed subject matter. [Figure 7C] 1 illustrates a detection arm setup that may be used as part of the image adjustment module 130 of FIG. 1 and that uses a gradient filter, in accordance with one or more embodiments of the presently disclosed subject matter. [Figure 7D] 1 illustrates a detection arm setup that may be used as part of the image adjustment module 130 of FIG. 1 and that uses an image intensifier, in accordance with one or more embodiments of the presently disclosed subject matter. [Figure 8A] 1 illustrates a detection arm setup that can be used as part of the image conditioning module 130 of FIG. 1 and that uses a spectral beam splitter, in accordance with one or more embodiments of the presently disclosed subject matter. [Figure 8B] 1 illustrates a detection arm setup that may be used as part of the image adjustment module 130 of FIG. 1 and that uses a filter wheel, in accordance with one or more embodiments of the disclosed subject matter. [Figure 8C]1 illustrates a detection arm setup that can be used as part of the image conditioning module 130 of FIG. 1 and that uses multiple wavelength-selective beam splitters, in accordance with one or more embodiments of the presently disclosed subject matter. [Figure 8D] 1 illustrates a detection arm setup that can be used as part of the image conditioning module 130 of FIG. 1 and that uses an acousto-optic tunable filter, in accordance with one or more embodiments of the disclosed subject matter. [Figure 8E] 1 illustrates a detection arm setup that may be used as part of the image adjustment module 130 of FIG. 1 and that uses a prism, in accordance with one or more embodiments of the presently disclosed subject matter. [Figure 9A] 2 shows a schematic diagram of a first arrangement for the primary illumination source 122 and the secondary illumination source 124 of FIG. 1, in accordance with one or more embodiments of the presently disclosed subject matter. [Figure 9B] 1 shows a schematic diagram of a second arrangement for the primary illumination source 122 and the secondary illumination source 124 of FIG. 1, in accordance with one or more embodiments of the presently disclosed subject matter. [Figure 9C] 1 shows a schematic diagram of a third arrangement for the primary illumination source 122 and the secondary illumination source 124 of FIG. 1, in accordance with one or more embodiments of the presently disclosed subject matter. [Figure 10] 1 illustrates a configuration of a depth-resolved imaging system using a single scanning mirror, in accordance with one or more embodiments of the presently disclosed subject matter. [Figure 11] 1 illustrates a configuration of a depth-resolved imaging system using a pair of scanning mirrors, in accordance with one or more embodiments of the disclosed subject matter. [Figure 12] 1 illustrates a configuration of a depth-resolved imaging system using a movable mirror panel, according to one or more embodiments of the disclosed subject matter. [Figure 13A] 1 illustrates a configuration of a depth-resolved imaging system that uses a motorized lens to perform scanning at an object, in accordance with one or more embodiments of the disclosed subject matter. [Figure 13B]1 illustrates a configuration of a depth-resolved imaging system using multiple objective lenses that can be selected to provide different magnifications, according to one or more embodiments of the disclosed subject matter. [Figure 14A] 1 illustrates a multi-mode confocal microscope that can be configured for conventional confocal imaging or depth-resolved imaging (DRI) according to embodiments of scanning plane microscopy, according to some embodiments of the disclosed subject matter. [Figure 14B] 1 illustrates a multi-mode confocal microscope that can be configured for conventional confocal imaging or depth-resolved imaging (DRI) according to embodiments of scanning plane microscopy, according to some embodiments of the disclosed subject matter. [Figure 15] 1 illustrates an embodiment in accordance with some embodiments of the disclosed subject matter that uses a single mirror scanning element for both scanning with illumination and de-scanning the image beam, and in which the field rotation optics and image forming optics use the same optical element. [Figure 16A] 1 illustrates an embodiment that uses a single mirror scanning element for both scanning with illumination and de-scanning the image beam, according to some embodiments of the disclosed subject matter. [Figure 16B] FIG. 16A illustrates an embodiment similar to that of FIG. 16A, but arranged such that as the illumination beam is tilted, the illumination beam moves across the front aperture of the objective lens, that uses a single mirror scanning element for both illumination scanning and image beam de-scanning, thereby improving thickness uniformity of successive 3D slices of the object, in accordance with some embodiments of the subject matter of the present disclosure. [Figure 16C] 13 illustrates another embodiment that uses a single mirror scanning element for both scanning with illumination and de-scanning the image beam, according to some embodiments of the disclosed subject matter. [Figure 17] 1 illustrates a dual-axis confocal endoscopic configuration for depth-resolved imaging, according to one or more embodiments of the disclosed subject matter. [Figure 18] 1 illustrates an objective lens configuration for depth-resolved imaging, according to one or more embodiments of the disclosed subject matter. [Figure 19] 1 illustrates a configuration using line-scan and linear detectors for depth-resolved imaging, according to one or more embodiments of the disclosed subject matter. [Figure 20A] FIG. 1 shows a mechanism for image rotation in accordance with some embodiments of the subject matter of this disclosure, where the angle between the image forming optics and the field rotation optics can be independently selected, in some embodiments even being 0°. [Figure 20B] FIG. 1 shows a mechanism for image rotation in accordance with some embodiments of the subject matter of this disclosure, where the angle between the image forming optics and the field rotation optics can be independently selected, in some embodiments even being 0°. [Figure 20C] FIG. 1 shows a mechanism for image rotation in accordance with some embodiments of the subject matter of this disclosure, where the angle between the image forming optics and the field rotation optics can be independently selected, in some embodiments even being 0°. [Figure 20D] A variation of the embodiment of FIG. 15 is shown, which uses the field rotation mechanism first described with reference to FIG. 20B. [Figure 20E] A variation of the embodiment of FIG. 15 is shown, which uses the field rotation mechanism first described with reference to FIG. 20B. [Figure 21A] 1 illustrates a multimode confocal / DRI microscope in a confocal theta configuration, according to some embodiments of the disclosed subject matter. [Figure 21B] 21B illustrates the multi-mode confocal / DRI microscope of FIG. 21A in a DRI configuration, according to some embodiments of the presently disclosed subject matter. [Figure 21C] 21A and 21B, according to some embodiments of the presently disclosed subject matter. [Figure 22A]6B and 6C illustrate specific example configuration features of the embodiment of FIG. [Figure 22B] 6B and 6C illustrate specific example configuration features of the embodiment of FIG. [Figure 23A] FIG. 11 illustrates the functionality of a DRI microscope such as the embodiment of FIG. 10, which includes an example of a detection module and exhibits a mostly rectangular profile that provides easy adjustment and configuration, e.g., by selectively inserting an angle mirror (45°) for fast switching between upright and inverted configurations. [Figure 23B] FIG. 16 illustrates the functionality of a DRI microscope such as the embodiment of FIG. 15, which includes an example of a detection module and exhibits a mostly rectangular profile that provides easy adjustment and configuration, e.g., by selectively inserting an angle mirror (45°) for fast switching between upright and inverted configurations. [Figure 23C] FIG. 23C illustrates the functionality of a DRI microscope similar to the embodiment of FIG. 23B, with the addition of a third lens assembly to recover light that may be lost due to splitting of the image light beam received from the sample. [Figure 23D] A DRI microscope is shown that illuminates a sample using light sheets coming from alternating sides of an objective lens that captures and separates the light to form an image from the alternating illumination. [Figure 23E] A DRI microscope is presented, which provides a mechanism for improving the shadowing effect of surface structures by collecting images with an illumination beam or sheet projected at multiple angles. [Figure 23F] This figure illustrates a feature of a DRI microscope that is functionally similar to the embodiments of Figures 6I, 10, and 13B, in which a turret can be used to achieve selection of a first lens assembly of a detection module to achieve adjustment of magnification by switching lens assemblies in the turret. [Figure 23G]A DRI microscope is shown that scans and descans lines at multiple depths to achieve depth-resolved point measurements of light return from a scanned line with point measurements made multiple times by multiple photomultiplier cells in a linear array with various features for high frame rate and high sensitivity. [Figure 23H] FIG. 23B illustrates structured illumination in the context of an embodiment based on the embodiment of FIG. 23A, with Ronchi rulings used to structure a beam of light. [Figure 23J] 1 shows structured illumination, where a modulatable laser is swept to produce structured illumination at the sample. [Figure 23K] 23B shows a variation of the embodiment of FIG. 23A in which the magnification in the detection arm is greater than the magnification in the illumination arm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] An embodiment of the subject matter of the present disclosure is directed to an imaging technique in which image light from an object is captured simultaneously from multiple depths to form one or more images, herein referred to as depth resolved imaging (DRI). In a particular embodiment of DRI, an illumination beam is applied to an object of interest such that the beam passes through multiple depths. The shape of the illumination beam can be selected to facilitate feature identification, such as in fluorescence microscopy using a planar illumination beam. Simultaneously with the application of the illumination beam, the resulting image light, e.g., emitted or reflected, is captured from multiple depths and used to form an image. For example, a volume can be continuously traversed and imaged at high speed by generating an illumination beam in the form of a planar beam and scanning with the planar beam while de-scanning the resulting image light. This can be repeated to capture the motion of the object and the motion in the object.

[0022] The subject matter of this disclosure includes applications and improvements of DRI devices, as well as specific DRI devices, methods, and systems for DRI. In some embodiments, DRI can be used to rapidly generate two-dimensional and three-dimensional images, particularly for generating moving images. Some embodiments relate to methods, devices, and systems with multiple modes that can achieve confocal imaging as well as DRI.

[0023] In some embodiments, a single objective lens is used for both illumination and detection. As used herein, the term "illumination" refers to any type of outgoing light used for imaging that excites or suppresses the emission of secondary radiation used to construct an image, or is returned via some physical phenomenon such as reflection, scattering, or any other means. Thus, the illumination light can generate the image light by fluorescence excitation, reflection, scattering, second harmonic generation, Raman scattering, and / or any other mechanism. Additionally, the illumination energy can suppress the image light in desired locations for improved resolution. Among other advantages, the use of a single objective lens for projection of illumination light and imaging can simplify positioning and alignment of the object compared to light sheet imaging, which uses separate optical components to project illumination and receive imaging light.

[0024] In some embodiments of DRI, a light beam is projected through an objective lens onto the object along with an elongated narrow beam or planar beam that extends to multiple depths of the object and the returning image light from the object is captured simultaneously from multiple depths and used to image features at multiple depths. This beam can be scanned across multiple locations to form a three-dimensional image. In embodiments using illumination light in the form of a planar beam, the object can be optically sectioned and the planar illumination beam swept or otherwise repositioned so that the resulting image light from the illuminated plane can be captured through the same objective lens. As the beam is moved, the returning image light is descanned and imaged onto a photodetector such as a camera. Descanning maintains the projection of the returning light from multiple depths on the photodetector. As used herein, descanning refers to maintaining a particular mapping of the light source on the object, progressively or stepwise, onto the detection area of ​​the photodetector. For example, descanning can maintain the focus of a sweeping illumination beam on a plane in the image plane of the camera. This embodiment captures or displays multiple discrete or continuous areas of a planar image to generate a 3D volumetric image without physically translating the objective lens or the object, thereby significantly increasing imaging speed while avoiding the object preparation and illumination selection associated with other types of imaging.

[0025] In some embodiments described herein, scanning elements using reflective elements are described, however, other mechanisms for moving the beam progressively, incrementally, or non-continuously may be used in any of the embodiments, including those defined in the claims. Such additional embodiments may use other light redirecting devices, such as refractive or diffractive elements for scanning with a single beam or a narrow beam or a planar beam of light. These elements may include mirrors, prisms, acousto-optic deflectors, motorized lenses, spatial light modulators (SLMs), beam steering mirrors or optics, flexible light guides or optical fibers, other types of adaptive optics, or any other mechanism for controlling the direction of outgoing or incoming light. Such variations are considered to be substituted for any of the disclosed embodiments, including the claims, and the resulting embodiments are within the scope of the present disclosure herein.

[0026] In some embodiments of the subject matter of the present disclosure, the scanning and descanning optics are configured such that the illuminated path remains aligned to a fixed photodetector (e.g., a linear or two-dimensional photodetector) that is sampled to provide optical sectioning. In some embodiments, the planar illumination beam is swept across the object by moving the scanning mirror to change the angle at which the illumination light is incident on the back surface of the objective lens and thereby change the angle at which it emerges from the objective lens. It is noted that the planar illumination beam can be formed from a discrete cylindrical beam swept across the light sheet by scanning, or by an optical element that flattens the cylindrical beam. The planar illumination beam can also be formed by multiple discrete parallel cylindrical beams formed at each time. By varying the angle as described above, the planar illumination beam is swept across the object. As mentioned above, other patterns of illumination are also possible to form two-dimensional or three-dimensional images, or even linear depth resolved detection. Light from the illuminated region in the object (e.g., via fluorescence excitation, reflection, scattering, second harmonic generation, and / or Raman scattering) passes back through the same objective lens and is descanned. Even though the light is moved (or more generally repositioned) across the object by descanning, the detected light forms an image of the illuminated region that is stationary and aligned to the illuminated region in much the same way that a confocal pinhole remains aligned to the scanning illuminated focal spot in a laser scanning confocal microscope.

[0027] De-scanning can use multiple optical components to generate a still image. A camera can be focused on the image (intermediate image) to form another image at the photodetector. An unfocused camera can be aimed at the image. The image can be formed directly at the photodetector. The photodetector can include a high-speed CMOS camera, a CCD camera, a photomultiplier tube, a linear detector array, or any other light detection or imaging device. Alternatively, the imaging device, or the entrance to the imaging device, can be located at the image plane. The single fixed objective lens configuration combined with the scanning / descanning configuration in some embodiments of the disclosed subject matter allows DRI to be used in a similar manner to other upright or inverted microscopes, or DRI can be provided as an addition or modification to an existing microscope, such as a confocal microscope. Both scanning and de-scanning are understood to be changing the angle, or position, or both the angle and position, of one or more of a narrow (pencil) or planar beam of light.

[0028] With reference to FIG. 1, various high level features associated with embodiments of a DRI system 100 are illustrated. It is contemplated that an embodiment may include some or all of the features illustrated in FIG. 1, as well as other features not specifically illustrated in FIG. 1. In some embodiments, some of the features in FIG. 1 may be omitted or rearranged in consideration of other features, whether or not illustrated. Furthermore, the illustrated features are of simplified components that perform one or more functions. Based on a general discussion of FIG. 1 and the following detailed discussion of various embodiments, one of ordinary skill in the art will readily recognize numerous alternatives for performing one or more functions represented by the simplified components.

[0029] In one or more embodiments, the DRI system 100 can be used to image an object 106 microscopically or macroscopically. The DRI system 100 can include, among others, a first optical module 102 (i.e., an illumination module), a second optical module 103, and a third optical module 104 (i.e., a detection module). The illumination module 102 can provide, for example, a beam of laser light, and the light can be preconditioned to form a linear or planar beam at the object 106. The preconditioning of the beam can shape the beam to a thin or planar illumination pattern at the object. The optics themselves do not need to form the beam or the plane. An SLM is one option to precondition the beam to reduce aberrations, for example, via adaptive optics. Or Bessel beam forming, etc. are also possible options. The illumination beam can be modulated in time to produce a spatial pattern at the plane, or can be patterned to produce structured illumination, and subsequently image processing can be performed on the pattern to produce a "super-resolution" image. The illumination module 102 can provide incident light 134 for scanning to the second optical module 103 via one or more optical paths. The detection module 104 can receive light 135 reflected, scattered and / or emitted by the illuminated plane at the object 106 in response to the incident light.

[0030] The illumination module 102 may include a primary illumination source 122, e.g., a laser light source or a collimated beam source. The illumination beam generated by the primary illumination source 122 may be provided to the beam conditioning module 120, which includes one or more illumination optics and / or illumination components that condition the light beam to achieve desired illumination characteristics. For example, the one or more illumination optics and / or illumination components of the beam conditioning module 120 may include refractive, reflective, and / or diffractive optics to shape the beam into a linear beam or a planar beam at the object 106. For example, to shape into a planar illumination beam, the refractive, reflective, and / or diffractive optics may include active elements such as a scanner or passive elements such as a cylindrical lens. Alternatively or additionally, the one or more illumination optics and / or illumination components of the beam conditioning module 120 may include beam conditioning components, e.g., wavelength-selective filters, polarization-selective or polarization-changing components, graduated neutral density filters, modulators, etc. Alternatively or additionally, one or more illumination optics and / or illumination components of beam conditioning module 120 may include one or more apertures to limit the size of the beam before any conditioning of the beam, or after some or all of the conditioning of the beam, or at any other point along the optical path between the light source and the object. Alternatively or additionally, one or more illumination optics and / or illumination components of beam conditioning module 120 may include a zoom lens (not shown, but depending on the configuration of the system, may be located before focusing module 108, e.g., in telescope 110), which may be used to change the magnification of a motorized lens (not shown) and / or second module 103, which may be used to change the position of the illumination in view of focusing module 108.Alternatively or additionally, the beam conditioning module 120 may include adaptive optics, such as a spatial light modulator (SLM), that can pre-condition the beam to reduce aberrations.

[0031] It is noted that in the disclosed embodiments, one or more types of aberrations may occur due to the system's ability to obtain or maintain focus at multiple depths (multiple axial positions relative to the objective lens). These may include chromatic and spherical aberrations, depending on the configuration chosen by the designer. These aberrations may be reduced or eliminated using known techniques, such as aspheric lenses, GRIN lenses, multi-element optics, or other techniques. In some embodiments, the aberrations occurring in the objective lens are compensated for by transmitting the light through a lens assembly with properties selected to fully or substantially compensate for the aberrations introduced by the objective lens. For example, a lens assembly identical to the objective lens may be used.

[0032] In some configurations, the primary illumination source 122 may be modulated in time to form a spatial pattern in the illuminated plane at the object, or may be patterned to form structured illumination for enhanced resolution (i.e., to produce "super-resolution"), as in lattice light sheet microscopy. Alternatively or additionally, the primary illumination source 122 may generate one or more Bessel beams, which are shaped into a planar illumination beam that illuminates the object.

[0033] In some configurations, the primary illumination source 122 may include multiple light sources, e.g., multiple laser light sources having different central wavelengths. In some configurations, the beam conditioning module 120 may further include optics and / or components for selecting a wavelength for illumination, e.g., by switching to an appropriate filter from among multiple filters, or by selectively activating a particular laser, or by moving one of the multiple laser sources (or a corresponding input component) to a single input position. Alternatively or additionally, the beam conditioning module 120 may include optics and / or components for combining multiple light beams into a single beam. In such configurations, additional optics and / or components may be provided downstream of the illumination module 102, e.g., by including a multi-band dichroic as part of the separation module 112 and / or by using a notch filter as part of the image conditioning module 130, to accommodate the simultaneous multi-wavelength illumination and the resulting detection light. Alternatively or additionally, the combination of multiple light sources can achieve stimulated emission depletion (STED) imaging, for example by forming a combination of multiple planar beams that produces a narrower planar beam. Alternatively or additionally, the combination of multiple light sources can achieve pump-probe imaging, similar to stimulated Raman scattering (SRS) imaging and / or coherent anti-Stokes Raman scattering (CARS) imaging.

[0034] In any embodiment, other optics and / or components for the beam conditioning module 120 are contemplated other than those specifically discussed above. Although a laser source is described herein, any collimated light source (or light source that can be collimated) or combination of light sources, e.g., pulsed wave or continuous wave, can be used. In some embodiments, a regenerative amplifier can be used to generate the primary illumination from the primary illumination source 122.

[0035] The incident illumination 134 can be provided to a scanning module 116 of the second module 103. The scanning module 116 can include one or more optical systems and / or components for redirecting the incident illumination 134 towards the object 106 and performing a scan with the resulting planar illumination beam at the object. For example, the scanning module 116 can include one or more reflective elements (e.g., a moveable mirror attached to a galvanometer) to change the optical path of the incident illumination 134. After redirection by the scanning module 116, the illumination can proceed to a separation module 112 where the light to be directed to the object is separated from any detected light returning from the object.

[0036] In some embodiments, the separation module 112 includes one or more optical components for distinguishing between the illumination light and the detection light. For example, the separation module 112 can include a wavelength-selective beam splitter (e.g., a dichroic mirror / dichroic filter or a dielectric mirror / dielectric filter) or a polarization-selective beam splitter to direct the illumination light and the detection light along separate optical paths. In other embodiments, the separation module 112 includes an arrangement of optics or components of other modules in the system 100 that function to physically separate the illumination light and the detection light without the use of a beam splitter (see, e.g., FIG. 12). Although the separation module 112 is illustrated in FIG. 1 as a module downstream of the scanning module 116 along the incident optical path, it is also contemplated that the separation module 112, or components thereof, can be provided upstream of the scanning module 116 (and / or downstream of the de-scanning module 118 along the detection optical path), as shown, for example, in FIG. 10.

[0037] After passing through the scanning module 116 and the separation module 112, the planar incident illumination beam travels to the optional telescope module 110, which may include one or more optics or components forming a telescope for imaging the incident illumination onto the back aperture (or back incidence plane, which may be different from the back aperture) of the focusing module 108. For example, the optional telescope module 110 may image the incident illumination onto a fixed plane at any point relative to the objective lens, such as the back aperture, the back focal plane, or any other point depending on the desired scan pattern. Such a configuration may be advantageous in terms of minimizing or at least reducing the amount of light lost during scanning. Alternatively or additionally, the optional telescope module 110 may expand the location of the illumination incident on the edge of the back aperture of the objective lens (e.g., of the focusing module 108) relative to the size of the scanning mirror (e.g., of the scanning module 116), thereby allowing the use of a scanning mirror that is smaller relative to the back aperture. Similar features to the magnification results provided by telescope module 110 can be similarly applied to any de-scanning mirrors for detected light 138 (e.g., the de-scanning mirrors of de-scanning module 118). If optional telescope module 110 is provided, it can include a pair of lenses positioned along the optical path, for example, as illustrated in FIG.

[0038] The focusing module 108 (e.g., an objective lens or other reflective, diffractive, or refractive focusing optical component) receives incident illumination from the telescope module 110 and focuses the illumination into a planar beam 137 at an oblique angle (i.e., at an oblique angle with respect to the optical axis of the objective lens) onto the object 106 (or alternatively aligned with an axis (not shown) such that illumination occurs at multiple depths in the object). The focusing module 108 can also be used to receive light 138 from the object 106, for example, using the same objective lens used to form the planar incident illumination beam 137. The system 100 can be configured such that the components of the focusing module 108 and the object 106 (or at least the imaging volume (e.g., if the object moves separately from its imaging volume)) remain substantially fixed during scanning and detection.

[0039] Light 138 from the object 106 can be directed through the focusing module 108 and through multiple identical or similar modules of the second module 103 on the path to the detection module 104. For example, the focusing module 108 can direct the light 138 to the telescope module 110 where it travels through the same telescope lens group that imaged the incident illumination to the focusing module 108. The light 138 can be separated by the separation module 112 (e.g., by special optics or components for separation or by appropriate arrangement of the incoming and outgoing optical paths) and descanned by the descanning module 118. Alternatively, as noted above, descanning by the descanning module 118 can occur prior to separation by the separation module 112.

[0040] The de-scanning by module 118 can maintain a fixed detection plane of the imaging module 132 that corresponds to a plane on the object illuminated by the incident illumination, despite the movement caused by the scanning illumination. The de-scanning module 118 can include one or more optics and / or components for redirecting light from the object. The redirection by the de-scanning module 118 can be synchronized with the redirection by the scanning module 116 so that the detection plane imaged by the system 100 corresponds to the plane illuminated by the system 100. In some embodiments, the scanning module 116 and the de-scanning module 118 share some of the same components (i.e., including the scan / descan module 114, as shown in FIG. 10, for example). In such an embodiment, the scanning / descanning can be performed by the same mirror. In another embodiment, the scanning module 116 and the de-scanning module 118 use only separate components (e.g., using separate mirrors, as shown in FIG. 11, for example). In such embodiments, scanning can be performed by a first mirror that is distinct from a second mirror that performs de-scanning, but whose movements can nevertheless be coordinated to maintain the desired correspondence between the illumination plane and the detected light.

[0041] In some embodiments, the scan / descan module 114 includes one or more reflective surfaces, such as facets of a tilted macroscopic mirror or a polygon mirror. However, other reflective, diffractive, or refractive optical components for effective scanning / descanning are also contemplated according to one or more contemplated embodiments. For example, scanning / descanning can be provided by other macroscopic optical components, such as a deformable mirror or SLM, or by microscopic optical components, such as a MEMS mirror and a digital light processor (DLP).

[0042] The resulting detected light 135 from the second module 103 can be received by the detection module 104 for further optical processing and imaging. For example, the detection module 104 can include an image forming module 126, an optional field rotation module 128, an image adjustment module 130, and an imaging module 132. The image forming module 126 can be configured to form an image of the illuminated plane and / or compensate for aberrations (e.g., any aberrations that may be introduced into the second module 103). Thus, the image forming module 126 can include optics and / or components similar to or identical to those of the optional telescope module 110 and the focusing module 108.

[0043] In one or more embodiments, the optics and / or components of the image forming module 126 and the focusing module 108 may each have a magnification selected to define the angle of the intermediate image plane formed by the image forming module 126. For example, by selecting a magnification that produces an overall magnification of 1× in the intermediate image, respectively (and taking into account any immersion medium for the objective lenses of the modules 108, 126), the angle of the intermediate image plane may be the same as the angle of the illuminated plane at the object, i.e., the rotation angle may be 0°. Changing the magnifications to produce an overall magnification different from 1× may change the angle of the resulting image plane and may also introduce aberrations. Thus, the image forming module 126, in combination with the focusing module 108 and the optional telescope module 110, may be designed to produce an image of the illuminated plane at the object 106 at or near 1×. According to one or more possible embodiments, a magnification different from 1× may also be achieved, for example by rotating the image plane to increase the captured light or for any other purpose.

[0044] The resulting image formed by the image formation module 126 has an oblique angle with respect to the optical axis. If imaging were performed with the detector's detection plane about said optical axis, the image oblique angle would result in the formation of a blurred image of the illuminated plane above and below the focal plane of the focusing module 108 (e.g., primary objective lens). In some embodiments, this blurring can be tolerated and / or addressed by rotating the detector's detection plane to match or be located near the image plane. In other embodiments, an optional field rotation module 128 can be used to change the orientation of this image plane. For example, the field rotation module 128 can use one or more optical components to fully or partially rotate the image plane so that the image matches the detector's detection plane while keeping the entire image (and therefore all depths) in focus. Other configurations of the field rotation module 128 can address the formation of depth-resolved images without actually rotating the image plane, as will be described in more detail below.

[0045] Upstream or downstream of or within the field rotation module 128 (or upstream or downstream of or within the image formation module 126 if the field rotation module 128 is not provided), an image conditioning module 130 may be provided that includes one or more optics and / or components to condition the detected light (i.e., to adjust the position, magnification, and / or angle of the image plane) for eventual detection by an imaging module 132, which may be a single detection element (e.g., a photomultiplier tube (PMT)), a linear array (e.g., a linear detector array or a linear array of PMTs), or a two-dimensional array (e.g., a CCD camera). For example, the image formation module 126 may include optics or components for shaping the detected light or adjusting the numerical aperture (e.g., an adjustable aperture or iris), and / or optics or components for wavelength selection (e.g., an emission filter or polychromatic imaging component), and / or optics or components for improving the quality of the image (for image intensification or variable size adjustment). Alternatively or additionally, the image adjustment module 130 may include optics and / or components for directing light emitted from the field rotation module 128 (or the image forming module 126) to the imaging module 132, for example, by focusing the emitted light onto an incidence plane of the imaging module 132.

[0046] Alternatively or additionally, the image conditioning module 130 and / or the image forming module 126 may include one or more field lenses (not shown, but may be located at or near the intermediate image plane), one or more wedge elements, and / or one or more reflective, refractive, or diffractive elements (e.g., mirrors or spatial light modulators) to improve light collection and image formation. Rotation of the image by the field rotation module 128 may result in light loss due to the directionality of the light forming the intermediate image and the numerical aperture of the objective lens imaging the image. The field lens may redirect the light without changing the focal plane of the light. In one example, the field lens may be located at the focal plane of the relay lens set of the field rotation module.

[0047] Furthermore, according to one or more contemplated embodiments, various combinations of lenses and glass elements can be used to improve aberrations and / or change magnification and / or improve throughput, for example incorporating a combination of concave and convex lenses in image adjustment module 130. For example, image adjustment module 130 can produce a magnification different from 1×, even if the magnification produced by the combination of second module 103 and image formation module 126 forms a depth-resolved image at or near 1× magnification. In particular, image adjustment module 130 (together with optional field rotation module 128) can magnify the 1× intermediate image, so that the desired resolution in the final image corresponds to the size of the individual pixels of the detector.

[0048] The image adjustment module 130 may also use other optical systems or components that can redirect light, for example, to form an image at a particular position and orientation, such as, but not limited to, mirrors and combinations of mirrors, prisms, gratings, deformable mirrors, and spatial light modulators (SLMs). Alternatively or additionally, the image formation module 126 may include one or more zoom lens modules (not shown). The zoom lens module may also change the magnification of the detection module 104, for example, to match the pixel size of the detector of the imaging module 132 to image at a desired resolution. The zoom lens module or motorized lens may also be used to accommodate changes in the primary objective lens of the focusing module 108, for example, to change the magnification. In such a configuration, the zoom lens module or motorized lens may compensate for different back apertures (or back focal plane characteristics) by moving the position of the incident illumination thereon. Alternatively or additionally, the position of the incident illumination at the back aperture may be changed by translating the beam at a scanning mirror (e.g., of the scanning module 116).

[0049] Image adjustment module 130 may also include a displaceable component to change an objective lens (e.g., the objective lens of optional field rotation module 128) to switch between different desired magnifications, for example. Thus, instead of changing the primary objective lens for the object (e.g., the objective lens of focusing module 108), the magnification of the final image may be changed by simply activating a displaceable component (e.g., a turret of objective lenses having different magnifications) to select among various objective lenses. In such a configuration, the magnification of image formation module 126 may be changed accordingly, for example, to maintain a condition of unity or near unity magnification as described above.

[0050] According to one or more possible embodiments, the imaging module 132 can include various types of detector elements and / or configurations. In some embodiments, the imaging module 132 can include a high-speed camera (e.g., pixel rate of at least 1 MHz, e.g., 300 MHz) with a two-dimensional array of detector elements, such as a CMOS imaging element, an sCMOS imaging element, or a CCD imaging element. Alternatively or additionally, the imaging module 132 can include a linear array of detector elements. In some embodiments, the imaging module 132 can include a linear or two-dimensional array of highly sensitive detector elements, such as photomultiplier tubes, avalanche photodiodes, or single-photon avalanche diodes. Alternatively or additionally, the imaging module 132 can include one or more wave guides (e.g., optical fibers) or conduits that direct light to a series of individual detectors or an array of detector elements.

[0051] A control module 150 may be provided to process images detected by the imaging module 132 and / or to coordinate the operation of the system 100. For example, the control module 150 may control the illumination module 102 to form the incident illumination 134, for example by scanning a beam from a laser or by controlling a spatial light modulator (SLM). Alternatively or additionally, the control module 150 may control the timing of illumination from the illumination module 102 or the selection of wavelengths in the illumination module 102, for example to modulate the illumination to form structured illumination. Alternatively or additionally, the control module 150 may control the scanning module 116 to perform scanning with a planar illumination beam at the object. The control module 150 may also control the de-scanning module 118 to synchronize de-scanning of the detection light 138. Alternatively, the scanning module 116 and the descanning module 118 may be the same module (e.g., the scan / descan module 114) and the control module 150 may control the module 114 to provide scanning and descanning simultaneously.

[0052] In one or more contemplated embodiments, the control module 150 can be configured to move the object 106 (e.g., via a motorized stage) and / or to move the focusing module 108 (e.g., the primary objective lens). In such a configuration, the object 106 can be illuminated sequentially from multiple directions or from the side to provide more isotropic resolution.

[0053] Additionally, the control module 150 can be configured to construct one or more images (e.g., construct a 2D image or construct multiple 2D images into a 3D image) based on the light detected by the imaging module 132 and the angles of the scanning module 116 and the de-scanning module 118 during the corresponding detections. Alternatively or additionally, the control module 150 can correct the actual position of the planar illumination beam 137 at the object. For example, the control module 150 may use feedback signals from the scanning module 116 and the de-scanning module 118, as well as a model of the optics and / or components of the system 100, to determine the actual angles and positions of the illumination and detection light. Alternatively or additionally, the control module 150 can be configured to control the system 100 to perform two-photon imaging, optical coherence tomography (OCT), structured illumination imaging, stimulated emission suppression, or any other imaging modality.

[0054] The control module 150 can also be configured to further process the image. In particular, the control module 150 can apply a number of different analysis and image correction strategies, for example, to improve resolution, contrast and spatial linearity. In one or more embodiments, the imaging geometry (including the optics and components of the various modules from the first module 102 to the third module 104) can be modeled to map the detection elements (e.g., camera pixels) detected during the scan into a 3D Cartesian space at the object plane. This model can be stored by the control module 150 and used to generate a predicted spatially-varying point spread function (PSF) for full deconvolution of the resulting data, which can improve the resolution and sectioning of the resulting image. Alternatively or additionally, the PSF can be estimated from the data set or from calibration using standards (e.g., ghosts with fluorescent beads) or can be estimated using automated algorithms. The estimation of the PSF can be done before or after the conversion to Cartesian space. Deconvolution by control module 150 can be applied before or after lateral shift adjustment, as described below.

[0055] Alternatively or additionally, a radial transfer model of light propagation can be stored by the control unit 150 and used for further correction for the effects of scattering of light (e.g., the planar illumination beam 137 and / or the detection light 138) to reconstruct a corrected image, similar to laminar optical tomography (LOT) or diffuse optical tomography.

[0056] Alternatively or additionally, the control module 150 can be configured to process the images, for example, by applying a lateral shift adjustment to each image in editing a 3D model of the object 106. Due to the angle of illumination, stacking the acquired images into a simple cube does not correct the skew. In other words, each depth layer acquired at a single scan position of the planar illumination beam 137 is laterally shifted with respect to the layer above it depending on the angle of the planar illumination beam 137 with respect to the optical axis 139. The control system 150 can resolve this skew, for example, by shifting each layer laterally in one or multiple dimensions, and optionally by interpolating non-integer shifts. The amount of shift can be determined by simulation, estimation from the resulting images, and / or calibration using the object, and can be stored by the control module 150. In some embodiments, the reconstruction of the tilted images into a Cartesian coordinate system can be model-based or approximated by application of a lateral shift adjustment.

[0057] Alternatively or additionally, the control module 150 may be configured to use diffuse background subtraction. To compensate for any unwanted scattering, system 100 may, for example, perform imaging using two-photon imaging (e.g., using longer wavelength excitation, which reduces scattering of the excitation light, and forming a narrower planar beam due to the nonlinearity of the two-photon effect), structured illumination (e.g., by appropriate manipulation of the primary illumination source 122 and the resulting detection scheme), stimulated emission depletion (STED) imaging (e.g., by selectively deactivating fluorophores in specific regions while leaving a central focal spot active so that fluorescence is emitted in the object), HiLo imaging (e.g., by acquiring successive images with uniform structured illumination and blending high and low spatial frequencies from both images to combine into a single image), high-pass filtering (e.g., to highlight higher resolution structures), low-pass filtering (e.g., to subtract low-pass filtered data from an image to highlight higher resolution structures), and other imaging techniques (e.g., photoactivated localization microscopy (PALM), stochastic optical reconstruction (STORM ... Spatiotemporal recognition (by distinguishing features having distinct patterns of time fluctuations, similar to holographic microscopy), mathematical correction by extracting dynamic changes in the images, and / or mathematical correction using diffuse background subtraction can be used. For example, the control module 150 can identify a common pattern in each successive image acquired and the common pattern can be shifted laterally during the scan. The common pattern can be shifted, scaled, and subtracted from each image (e.g., successively acquired images) to remove differences between measurements corresponding to selective object planes.

[0058] In some embodiments, the system 100 may include an optional secondary illumination source 124. For example, the secondary illumination source 124 may be used to provide photo manipulation, stimulated emission depletion (STED), or any other desired effect on an object, either independently of or in conjunction with the light from the primary illumination source 122. The secondary illumination source 124 may include other optics or components (not shown) for manipulating (e.g., beam steering or beam shaping) the light from the secondary illumination source 124 to provide the desired illumination effect. For example, the secondary illumination source 124 may include a spatial light modulator (SLM), a beam steering mirror or optics, an acousto-optic deflector, a phase plate, adaptive optics, or any other optical component to achieve the desired effect. The control module 150 may also control the operation of the secondary illumination source 124 along with the other modules 102-104 of the system 100.

[0059] In some configurations (e.g., where photomanipulation is performed), secondary illumination from the secondary illumination source 124 can be provided directly (e.g., along beam path 136a) to the focusing module 108 for illuminating the object 106, thereby bypassing other modules of the second module 103. The photomanipulation light can be generated by any beam-shaping optical component, ranging from simple focusing of laser light at the focal plane of the focusing module to scanning with a galvanometer mirror, an acousto-optical scanner, a MEMS scanner, a DLP device, or an SLM. For example, an SLM can be used to generate any 3D pattern of photomanipulation, which can be dynamically changed during imaging. Photomanipulation includes, but is not limited to, optogenetic excitation or inhibition of cells, phototrapping, photocoagulation, photobleaching, light-induced cell death or light-induced cell damage (i.e., photothrombosis), optical cavitation for light release from microparticles or nanoparticles, photouncaging, lighting, reactive oxygen species generation, or any other manipulation using electromagnetic radiation.

[0060] Alternatively or additionally, the secondary illumination from the secondary illumination source 124 can propagate along a path that is completely or partially identical to or adjacent to the path of the primary illumination through the second module 103. For example, in a configuration where STED is performed, the secondary illumination from the secondary illumination source 124 can be directed along path 136b to the input end of the second module 103 and proceed along a beam path similar to the planar input illumination beam 134 to the object 106. STED is a super-resolution technique that uses stimulated emission suppression to reduce the size of the diffraction limiting spot or plane. In some embodiments, STED can be achieved by aligning the secondary light source 124 to form a beam that surrounds, borders, or is adjacent to the primary illumination. Thus, for point scanning, an annular beam spot of secondary illumination can be formed around the focal point of the primary illumination. For line scanning, an elongated annular shape of secondary illumination can be provided around the linear primary illumination. For planar beam scanning, two planar beams of secondary illumination can be provided on either side of the planar primary illumination beam. Other configurations of the primary and secondary illumination are also contemplated according to one or more possible embodiments. Configurations and uses of secondary illumination sources different from those for photomanipulation and STED are also contemplated according to one or more possible embodiments.

[0061] Although certain features are illustrated in FIG. 1, it is apparent that additional features may be added to those specifically illustrated features, according to one or more contemplated embodiments. Moreover, it is apparent that certain features may be omitted, substituted, or modified, according to one or more contemplated embodiments. Although a particular order and configuration of features is illustrated in FIG. 1, certain embodiments of the disclosed subject matter are not so limited. Rather, according to one or more contemplated embodiments, the order of the features may be changed to achieve a different configuration. For example, rather than the scanning module 116 being in the optical path between the illumination module 102 and the separation module 112, and the de-scanning module 118 being in the optical path between the detection module 104 and the separation module 112, as illustrated in this FIG. 1, the scanning module 116 and the de-scanning module 118 may be in the optical path between the separation module 112 and the telescope module 110. Other rearrangements and reconfigurations of the features and modules illustrated in FIG. 1 will be apparent to those skilled in the art and are within the scope of the present application.

[0062] Additionally, system 100 may be implemented in a number of different configurations, depending on the embodiment. For example, system 100 may be configured as a microscopy system, where focusing module 108 provides one or more microscope objective lenses for imaging object 106 held by a microscope stage. In another example, system 100 may be configured as a macroscopic (i.e., without magnification) imaging system, where focusing module 108 provides one or more high numerical aperture focusing lenses for imaging an object. In yet another example, system 100 may be miniaturized (e.g., by using microfabricated components, e.g., by using microelectromechanical system (MEMS) devices, and / or miniaturized imaging elements (e.g., semiconductor chip imaging elements), and / or miniaturized light sources (e.g., laser diodes or light emitting diodes (LEDs))) for a particular application. For example, the optical components can be included in an endoscope for imaging structures inside a living organism, where light acquired by the system is processed by a microscope within the endoscope or directed, for example by one or more optical fibers, to a remote detector (e.g., a PMT array).

[0063] Referring to FIG. 2A, a configuration using an objective lens 202 is illustrated. Such a configuration can form all or part of the focusing module 108 of FIG. 1. To form the geometry of the DR imaging system, the position of the illumination beam 204 can be decentered with respect to the back aperture of the objective lens 202. The position of the illumination beam 204 at the back focal plane of the objective lens 202 (or equivalently, this position can be taken with respect to the back aperture) determines the angle of the resulting planar illumination beam 210 at the object. The system can be set (i.e., via the optics and / or components of the second module 103) so that the illumination beam 204 is incident at a consistent location at or near the back focal plane of the objective lens 202, despite the scanning performed by the scanning module 116. In other words, the illumination beam 204 can be pivoted (see reference number 208) around a point at or near the back aperture of the objective lens 202 to perform a scan with the beam 210 at the object. At the same time, the objective lens 202 can be used to acquire detection light 206 from the object. For example, to minimize or at least reduce light loss during scanning, a telescope such as the telescope configuration in FIG. 4A can be used to image the illumination scanner and detection descanner to a fixed plane (e.g., back focal plane) near the back aperture of the objective lens.

[0064] 2A illustrates illumination of the object from above, the objective lens 202 (and other components of the system 100) can be maintained or repositioned in a different location with respect to the object. For example, the objective lens 202 can be in an inverted configuration so that the object is illuminated from below. Alternatively or additionally, the objective lens 202 can be such that the object is illuminated from the side.

[0065] In some embodiments, over-illumination of the object's surface (i.e., to better illuminate deeper structures in the object) can be addressed by shaping the plane of incidence so that the intensity is more three-dimensionally distributed at the object's surface. For example, this can be achieved by positioning the axial focal plane of the planar illumination beam 210 deeper in the object, thereby reducing the power density experienced by the upper layers. This may result in lower resolution of the upper layer structures, however, this may increase the resolution and signal-to-noise ratio at deeper depths, and may be particularly useful for implementing two-photon imaging. Determining the axial focal distance to deeper depths can be achieved by moving the illumination focal plane, i.e., by moving the object and / or the objective lens. Alternatively or additionally, the relative difference in energy density at the surface versus the axial focus can be increased by using a higher numerical aperture illumination beam or by more advanced beam designs, such as, but not limited to, Airy beams, Bessel beams, or combinations of multiple beamlets.

[0066] Although an objective lens 202 is illustrated in FIG. 2A, other optics and / or components for the focusing module 108 are contemplated according to one or more contemplated embodiments. For example, the objective lens 202 can be replaced with a gradient index (GRIN) lens, as illustrated in FIG. 2B, or can be used in combination with multiple objective lenses (not shown). Such a configuration can provide particular utility in deep tissue imaging, for example, by being part of a needle or endoscope that is inserted into tissue for in situ imaging. Alternatively or additionally, a GRIN lens may be used as a relay lens, especially when used as part of a needle or endoscope.

[0067] Additionally, although objective lens 202 is illustrated in Figure 2A, any other optical system or component that provides focusing may be used in focusing module 108. For example, a spatial light modulator (SLM) may be used instead of or in addition to objective lens 202. In another example, any focusing lens may be used instead of or in addition to objective lens 202. In some embodiments, objective lens 202 is not required at all, as described below with reference to, for example, Figure 19.

[0068] 2C, any of the embodiments of the microscope of the present disclosure that includes an objective lens 202 (other elements of the microscope are not shown) can perform DRI, which includes a GRIN lens arranged to extend the illumination and imaging field to locations that would otherwise be inaccessible or difficult to reach, as shown. For example, in soft tissue 255 of an animal's brain or other body part, a microscope with a GRIN lens 212 can be inserted into the tissue 255 to image deeper regions. The objective lens 202 transmits a planar illumination beam to the GRIN lens, based on which the planar illumination beam is refocused to form a planar illumination beam 256 and an image beam 258 as in any of the embodiments. According to any of the disclosed embodiments, the image beam 258 can be returned to the detection optics. By using this device and by appropriately selecting the GRIN lens, the location of the interrogated volume, planar, line or point region can be extended into the target material entity, such as a biological tissue. It is noted that in various variations, a confocal microscope or other types of microscopes can be used.

[0069] 3A-3C, a configuration of components for the beam conditioning module 120 of FIG. 1 is illustrated. As mentioned above, the beam conditioning module 120 can shape the light from the primary illumination source into a linear or planar beam for scanning at the object. With reference to FIG. 3A, the illumination source 302 generates an illumination beam 304A that is incident on a beam expander 305 that can be provided. The beam expander 305 can include one or more optical elements such as lenses 307A and 307B. The resulting expanded beam 304B can be incident on a planar beam forming element 306 to form a planar beam 308. The orientation of the cylindrical lens 306 focuses the beam 304 in the X direction. The resulting beam 308 is directed to the object through an optical system that includes an objective lens that ultimately forms a planar illumination beam focused at the object, which is at an oblique angle to the XZ plane. The second module 103 can be used to scan the object with the illumination 308, for example by translating and / or rotating the beam in the XZ plane as a result of the movement of the scanning module 116. The planar beam forming element 306 can use any suitable technique for forming a planar beam, for example a cylindrical lens or a combination of a cylindrical lens and a spherical lens, as will be well understood by those skilled in the art of optics. It is noted that other optical approaches for forming a planar beam can also be used. For example, the beam expander 305 can be omitted.

[0070] In some embodiments, scanning the object with a linear beam may be more suitable than scanning with a planar beam. For example, illumination from the illumination source can be projected as a line of illumination (e.g., along the Y direction) and then scanned laterally to illuminate an area comparable to that of the planar beam, thereby accommodating the power requirements required for two-photon imaging. Such a configuration is illustrated in FIG. 3B, where a scanning mirror 316 is used to scan the spot produced by the beam 304 to form the line of illumination 318.

[0071] In some embodiments, a regenerative amplifier or similar pulsed laser with a lower repetition rate than a standard Ti:Sapphire laser can be used to provide improved two-photon excitation capabilities. The regenerative amplifier focuses the pulses of the ~MHz laser into lower frequency pulses with higher peak energy. For example, an 80MHz Ti:Sapphire can have pulses focused into 5kHz pulses with 7W average power. As a result, more than fifty thousand (50,000) times the power per pulse can be generated for the illumination beam compared to a standard 80MHz laser. This illumination can be spread three-dimensionally across the illumination plane to provide an equivalent instantaneous excitation to a pulse from an 80MHz laser. If the image acquisition of the illumination plane is at or about 2,000 frames per second, a lower pulse rate can be accommodated, whereby each plane can be illuminated with two pulses from the regenerative amplifier. Assuming one pulse per object point, integration or dwell times are not factored into the amount of light emitted, and linear and planar beam excitation configurations are largely equivalent in a pulse rate matched scheme. In either case, a 2D detector array or a 2D camera or other configuration of image detection can be implemented. Alternatively, the plane could be descanned at a higher rate onto a linear detector object, which could have a wider bandwidth and / or better sensitivity than the camera.

[0072] Similar effects are obtained when using a linear scan type configuration to form a planar illumination beam. For example, in FIG. 3C, a separate scanner 320 can be used to scan with a low NA (or similar) single beam 304 to form a planar beam illumination 322, prior to sweeping the planar beam across the object. Such a configuration, as illustrated in FIG. 3C, can also be used to provide structured light, for example using the primary illumination, to pattern the planar illumination beam. In particular, similar to structured illumination or HiLo microscopy, structured light can be used to improve the resolution and sectioning in the plane of the DRI system. By patterning the illumination at different spatial frequencies, low and high frequency components of the image can be separated to obtain improved resolution. In some embodiments, structured illumination can be formed by modulating the intensity of the illumination beam at a particular frequency in the configuration of FIG. 3B or FIG. 3C. Alternatively or additionally, structures can be formed by using a diffraction grating or a spatial light modulator in the illumination path.

[0073] Referring to FIG. 4A, a configuration of components for the telescope module 110 of FIG. 1 is illustrated. As discussed above, the telescope module 110 can image a planar incident illumination beam onto the back aperture (or back incidence plane) of the focusing module 108 (e.g., the objective lens 202). In addition, the telescope module 110 can transmit detection light 400 from the focusing module 108 (e.g., the objective lens 202). For example, in FIG. 4A, a telescope can be formed by using a first lens 402 in combination with a second lens 406 along a common optical axis 404. The lenses 402, 406 can be positioned such that their focal points overlap at 408. The telescope can map a scanner (e.g., the scanning module 116 and / or the descanning module 118) onto the back focal plane (or back aperture) of the objective lens 202.

[0074] Referring to FIG. 4B, a configuration of components for the image forming module 126 of FIG. 1 is illustrated. As discussed above, the image forming module 126 can form an image at life-size or near life-size with the optics of the second module 103, e.g., with the objective lens 202 and the telescope lenses 402, 406. As illustrated in FIG. 4B, the image forming module 126 can include a third lens 412, a fourth lens 416, and a lens assembly 420, which can be similar to or identical to the optics and / or components of the telescope module 110 (e.g., the lenses 402, 406) and the focusing module 108 (e.g., the objective lens 202). In other words, the optical components of the image forming module 126 can be similar to or identical to the optical components of the focusing module 108 and the telescope module 110. However, if an immersion material is used for the objective lens, a different magnification may be required for the image forming module to produce the desired unity magnification of the image. Lenses 412, 416 may be positioned such that their focal points overlap at 414. Thus, light 404 (e.g., transmitted from the telescope of FIG. 4A) may be focused by third lens 412 to produce focal point 414, and light from focal point 414 may be refracted by fourth lens 416 to produce light rays 418 in the path of lens assembly 420 (or other high aperture lens) that form an image 424 of the illuminated plane originating from the object at intermediate image plane 422.

[0075] 5A-5C, configurations for the scanning module 116, the de-scanning module 118, and the separation module 112 of FIG. 1 are illustrated. As discussed above, the scanning module 116 can scan an object with a planar illumination beam, while the de-scanning module 118 can de-scan the detection light corresponding to the illuminated plane on the object. The separation module 112 can operate to separate the illumination light from the detection light, for example, using a beam splitter or by separation of the optical paths. In some embodiments, the scanning module 116 and the de-scanning module 118 can share some of the same components.

[0076] Such a configuration is illustrated in FIG. 5A, in which illumination light 504 from an illumination module 502 is directed through a beam splitter 506 (e.g., a dichroic, polarizing beam splitter, or other beam splitting element) to a common scan / descan module 510. Incident illumination light 508 is redirected by the scan / descan module 510 to perform a scan of the object. The resulting detection light 526 is redirected by the scan / descan module 510. The redirected illumination light 528 is separated from the optical path shared with the illumination light 508 by the beam splitter 506. For example, a beam splitter can be placed in the infinity space of the optical path of the second module 103, for example between the scan / descan module 510 and the detection module 104. For example, if the detection light is fluorescence excited by a planar illumination beam, the beam splitter 506 may be a long pass filter that passes the detection light 528 (e.g., having a wavelength of 525 nm) while reflecting wavelengths shorter than the wavelength of the detection light 528, and in particular the wavelength of the illumination / excitation light 504 (e.g., having a wavelength of 488 nm). Alternatively, the beam splitter 506 may be a short pass filter or a polarizing beam splitter.

[0077] In some embodiments, the scanning module 116 and the de-scanning module 118 may be separate modules for the illumination light and the detection light, respectively. Such a configuration is illustrated in FIG. 5B, where the illumination light 532 from the illumination module 502 is directed to the scan module 550. The scan beam 552 may be directed to a beam splitter 506 (e.g., a dichroic, polarizing beam splitter, or other beam splitting element) and then the object may be scanned with the illumination beam 554. For example, the beam splitter 506 may be disposed in the infinity space of the optical path of the second module 103, for example, between the focusing module 108 and the telescope module 110. The resulting detection light 556 may be received by the beam splitter 506, which separates the detection light from the optical path shared with the illumination light 554. Detection light 558 is redirected by descan module 562 to detection module 532 as descanned light 560. As with the embodiment of Figure 5A, beam splitter 506 may be a long pass filter that can pass detection light 556 while reflecting illumination / detection light 552. Alternatively, beam splitter 506 may be a short pass filter or a polarizing beam splitter.

[0078] In some embodiments, the separation of the illumination light and the detection light can be achieved by separation of the optical paths. Such a configuration is illustrated in FIG. 5C, where the illumination light 504 from the illumination module 502 is directed to a common scan / descan module 570, which can be, for example, separate mirrored facets of a polygon mirror, separate mirrored surfaces with synchronized motion, or separate mirror elements of a reflective array (e.g., a digital micromirror array or other MEMS device). The scan beam 572 can be directed to the object without passing through a beam splitter. The resulting detection light 574 can be received by the scan / descan module 570, which redirects the descanned light 576 to the detection module 532.

[0079] In some embodiments, multiple light sources having different wavelengths can be used to illuminate the object simultaneously or sequentially. To accommodate such multi-wavelength excitation and / or multi-wavelength emission, selectable splitting components can be used. For example, FIG. 5D shows a configuration for multi-wavelength excitation from multiple illumination sources 502a-502c using a single scanner 510 for both illumination and detection light. In such a configuration, the selectable splitting module 503 can be provided with individual splitting elements 506a-506c corresponding to the excitation wavelengths (and / or emission wavelengths resulting from the excitation wavelengths) of the illumination sources 502a-502c. For example, the selectable splitting module 503 can include a selectable turret or wheel of individual dichroic elements, where each dichroic has a different passband. Thus, if an illumination source 502a having a first wavelength is used, the splitting module 503 can be used to select the first dichroic 506a, which allows the first wavelength excitation to reach the object and allows the resulting emission to pass to the detector 532. Similarly, splitting module 503 can be used to select second dichroic 506b or third dichroic 506c if illumination source 502b or illumination source 502c, respectively, are desired. Optionally, a wavelength selection component 507 can be placed in the detection arm before detector 532, for example, to filter out any excitation light or other undesired wavelengths that may propagate along the detection arm.

[0080] Alternatively, Figure 5E shows a configuration for multi-wavelength excitation from multiple illumination sources 502a-502c using a scanner 550 for the illumination light and a separate scanner 562 for the detection light. In such a configuration, the selectable splitting module 503 can operate in much the same manner as in Figure 5D.

[0081] Although three illumination sources 502a-502c are illustrated in Figures 5D and 5E, fewer or additional illumination sources are contemplated according to one or more embodiments. Additionally, although only a single detector 532 is illustrated in Figures 5D and 5E, additional detectors are contemplated according to one or more contemplated embodiments. In practice, multiple detectors may be provided with separate selectable splitting modules, similar to splitting module 503, and / or selectable wavelength filters for selecting the desired detection wavelengths.

[0082] 6A-6G, for example, configurations for imaging an intermediate image plane with and without the optional field rotation module 128 of FIG. 1 are illustrated. The disclosed imaging system embodiments can simultaneously acquire data from multiple depths by illuminating an object with a narrow or planar beam of light having a depthwise extent. The focusing module can form an image of the object having an axial extent, and the formed image can also have an axial extent. In extreme cases, the image can be a completely axial planar image. However, illuminating a more oblique plane (i.e., at an angle that is not perpendicular to the optical axis) with a planar illumination beam can provide improved sectioning due to the larger angle between the illumination plane and the detection light. Due to the depth dimension of the illumination, the resulting image also extends in the depth dimension, in this example an oblique image is formed.

[0083] If the detector is positioned to have a plane of incidence perpendicular to the optical axis of the image-forming objective lens, rather than being aligned with the image plane, light from above and below the focal plane along the planar illumination beam axis will form a blurred image of the illuminated plane. Image rotation optics can be used to reorient this angled image plane so that it can be focused on the detector's plane of incidence while simultaneously keeping the entire image, and therefore all depths, in focus. The field rotation module 128 can use one or more optical components to rotate the image plane so that the image can be matched to a plane of incidence perpendicular to the optical axis while keeping the entire image (and therefore all depths) in focus, as illustrated in FIG. 6A. For example, the detection light 614 can travel along the optical axis 612 to the image-forming optics 602, which can include a fifth lens 604, a sixth lens 606, and an objective lens 608. The detection light 614 is thus focused to form an intermediate image 610.

[0084] The field rotation optics can include a lens system 616, which includes, for example, a seventh lens 624 (e.g., another objective lens) with a high numerical aperture and an eighth lens 622, disposed along a second optical axis 625 that is at an angle (i.e., not parallel or perpendicular) with respect to the optical axis 612. For example, the lens system 616 can magnify the image in contrast to the lens system 602, which in combination with a focusing module and an optional telescope module and immersion medium produces a unity magnification with respect to the intermediate image 610. For example, the lens system 616 can be used to magnify the image such that the desired resolution corresponds to the pixel size of the detector n.

[0085] Detector 618 may be located at the focal point of second lens 622, e.g., such that the plane of incidence of detector 618 is perpendicular to second optical axis 625 and / or parallel to the image plane of image 610. The focal plane of first lens 624 coincides with the image plane of image 610 so that detector 618 can form a focused image of that image plane.

[0086] Other configurations of the field rotation module 128 can address aspects of depth resolution to form an image without rotating the image plane at all. Partial image rotation may be advantageous to improve the amount of light collected and guided to the detector 618, even if image quality is potentially sacrificed. For example, FIG. 6B illustrates a configuration of the field rotation module in which the image is only partially rotated, which accepts that there is a tradeoff between the amount of light transmitted between the lens system 602 and the lens system 636 and the correction of multiple axial depths to be imaged. As in the configuration of the embodiment of FIG. 6A, the detection light 614 can travel along an optical axis 612 to the aberration correction optics 602, which can include a fifth lens 604, a sixth lens 606, and an objective lens 608. The detection light 614 is thus focused to form an intermediate image 610.

[0087] In FIG. 6B, the field rotation module can include a lens system 636, which includes, for example, a high numerical aperture seventh lens 640 (e.g., another objective lens) and an eighth lens 642, arranged along a second optical axis 644 that is at an angle with respect to the optical axis 612 (i.e., is not parallel and not perpendicular). It is noted that the angle of rotation of the second optical axis 644 with respect to the optical axis 612 is smaller in FIG. 6B than the angle of rotation of the optical axis 625 with respect to the optical axis 612 in FIG. 6A. The detector 618 can be provided at the focal point of the second lens 642, for example, such that the plane of incidence of the detector 618 is perpendicular to the second optical axis 644. However, in contrast to the embodiment of FIG. 6A, the focal plane 638 of the first lens 640 does not coincide with the image plane of the image 610. Therefore, the plane of incidence of the detector 618 does not need to be parallel to the image plane of the image 610. Despite the imperfect rotation, image 610 can be formed by detector 618 with acceptable blurring, particularly since the collection efficiency of detected light 614 can be improved. It is noted that the angle between optical axis 612 and optical axis 644 can be selected to optimize the tradeoff between the loss of captured light due to the reduction in the numerical aperture between objective lens 608 and first lens 640 caused by the larger angle between optical axis 612 and optical axis 644, and the blurring due to the axial change of the illuminated area of ​​the target (depth of object out of focus).

[0088] It is further contemplated that the image rotation by the field rotation module can be performed in multiple stages rather than in a single stage. In such a configuration, the image can be partially rotated at each stage of the field rotation module, thereby increasing the amount of light that can be collected compared to a configuration in which the rotation is completed in just one stage. Such a configuration is illustrated in FIG. 6C, where the image is only partially rotated at each stage. As in the configuration of the embodiment of FIGS. 6A and 6B, the detection light 614 can travel along an optical axis 612 to the aberration correction optics 602, which can include a fifth lens 604, a sixth lens 606, and an objective lens 608. The detection light 614 is thus focused to form an intermediate image 610.

[0089] In FIG. 6C , the field rotation module can include a first lens stage 636, which includes, for example, a ninth lens 640 (e.g., another objective lens) with a high numerical aperture and a tenth lens 642 arranged along a second optical axis 661 having a predetermined angle with respect to the optical axis 612 (i.e., having an angle that is neither parallel nor perpendicular). The field rotation module can include a second lens stage 665, which includes, for example, an eleventh lens 663 (e.g., another objective lens) with a high numerical aperture and a twelfth lens 664 arranged along a third optical axis 662 having a predetermined angle with respect to the two optical axes 612 and 661. Thus, the first lens stage 636, which has an entrance focal plane 638 defined by the first lens 640, partially rotates the image plane of the image 610 to form another intermediate image 660. A second lens stage 665, having an entrance focal plane defined by a third lens 663 that coincides with the image plane of the intermediate image 660, completes the rotation of the image, thereby enabling the image to be mapped onto the entrance plane of the detector 618.

[0090] Although two stages are illustrated in FIG. 6C, additional rotation stages are contemplated according to one or more contemplated embodiments. For example, two or more lens stages can be provided such that each stage rotates the image by the same or different amounts to perform a full or at least partial rotation of the image. Each lens stage can provide incremental magnification of the image from the 1× magnification resulting from lens system 602. This incremental magnification can minimize, or at least reduce, any aberrations that may result from stretching the image if full magnification is achieved with a single lens stage.

[0091] Although the individual lenses and lens stages are shown as separate components, it is contemplated that image rotation can be performed using a compound lens system that can also be adjusted to accommodate changes in the angle of the planar illumination beam and to optimize alignment.

[0092] As noted above, in some embodiments, incomplete image rotation may be desired to increase the amount of light collected for imaging. For example, FIG. 6D illustrates a configuration of a field rotation module in which the image is only partially rotated. As in the configuration of the embodiment of FIG. 6A, the detection light 614 can travel along an optical axis 612 to the aberration correction optics 602, which can include a fifth lens 604, a sixth lens 606, and an objective lens 608. Thus, the detection light 614 is focused to form an intermediate image 610.

[0093] Similar to the embodiment of FIG. 6C, the field rotation module can include a lens system 636 including, for example, a high numerical aperture ninth lens 640 (e.g., another objective lens) and a tenth lens 642 disposed along a second optical axis 661 having a predetermined angle with respect to the optical axis 612 (i.e., having an angle that is neither parallel nor perpendicular). However, the detector 618 can be disposed to have a plane of incidence perpendicular to a third optical axis 662 having a predetermined angle with respect to the second optical axis, and can be aligned with the image plane of the intermediate image 660. As a result, a focused image of the image 610 can be formed by the detector 618.

[0094] In another configuration, image rotation using a field rotation module can be avoided entirely. For example, as illustrated in FIG. 6E, the detection plane of detector 618 can be placed at the image plane of image 610. Such a configuration can avoid significant light loss and aberrations associated with image rotation optics. Alternatively or additionally, as illustrated in FIG. 6F, a dense fiber array 650 can be positioned with angled ends 652 that are aligned to the angle of image plane 610. Thus, the fiber array 650 relays the angled detection plane to the flat detection plane of detector 618 to map the image directly onto the detector.

[0095] In yet another configuration, the field rotation module can include custom optical components that manipulate the image optical field without using the rotation configuration of Figures 6A-6D. For example, the correction optics can be stationary during 3D scanning, since the relative position of the descanned image plane 610 does not change during scanning. As illustrated in Figure 6G, the spatial light modulator 660 can take advantage of this fixed descanned image plane to reorient the image for detection by the detector 618. For example, the spatial light modulator 660 can independently adjust the phase and / or direction of light incident on each point along its plane to refocus each point at the image plane 610 to the normal plane at the detector 618. Alternatively or additionally, once the desired pattern is optimized using the spatial light modulator 660, the spatial light modulator 660 can be replaced with a custom optical component that provides the desired refraction pattern or an equivalent reflection or transmission pattern by using the optimized spatial light modulator as the design target.

[0096] Alternatively or additionally, a mirror (not shown) can be provided at image plane 610 to reflect the image to the detector, similar to the positioning of the SLM in Figure 6G. In such a configuration, detector 618 can detect the image without rotation, similar to the embodiment described above with respect to Figure 6E.

[0097] In yet another configuration, the field rotation module can include a light redirecting element to orient the image plane captured by the detection module 104, thereby replacing field rotation. See the discussion of Figures 20A and 20B below. With reference to Figure 6H, an optical wedge 613 can be inserted between the lens system 616 and the aberration correction optics 602, as illustrated in Figure 6H. The wedge 613 can partially or fully rotate the otherwise oblique image 610 to produce a plane of intermediate image 610' that is rotated such that the optical axis 625 of the detection module 104 (lens system 616) is parallel to the optical axis 612 of the aberration correction optics 602, thereby improving light capture efficiency and reducing image blur. Alternatively or additionally, the introduction of the wedge 613 can offset the optical axis 625 from the optical axis 612. The optical wedge 613 can be selected such that the optical axis 625 of the detection module 104 (lens system 616) is parallel to the optical axis 612 of the aberration correction optics 602. While Fig. 6H shows one axial position and extent, other embodiments discussed with reference to Figs. 20A-20D can have other axial positions. Figs. 20C and 20D show additional embodiments using one or more light redirector elements adapted to rotate the plane of the intermediate image.

[0098] In some embodiments, additional lenses (not shown) can be added to the beam path between the image plane of the image 610 and the detector 618 to adjust the magnification of the image to match the size of the detector 618. Alternatively or additionally, a selectable lens module 627 can be provided in the lens system 619 following the image 610 to switch between multiple lenses with different characteristics, as shown in FIG. 6I. For example, the selectable lens module 627 can be an objective lens turret supporting multiple objective lenses 624a-b with different magnifications, working distances, and / or numerical apertures. Although only two lenses 624a, 624b are shown in FIG. 6I, fewer or additional lenses can be provided according to one or more contemplated embodiments.

[0099] 7A-8E, configurations for image conditioning module 130 of FIG. 1 are illustrated. As discussed above, image conditioning module 130 may include one or more optics and / or components that condition detected light (i.e., adjust the position, magnification, and / or angle of the image plane) for eventual detection by imaging module 132. Such optics and / or components of image conditioning module 130 may be distributed within detection module 104, for example, upstream or downstream of field rotation module 128 and / or image formation module 126, or within field rotation module 128 and / or image formation module 126.

[0100] In some embodiments, the image / forming processing module can include an adjustable aperture 704 located upstream of the optics of the image forming module 602, as shown in FIG. 7A. Similar to the embodiment of FIG. 6A, the detection light 614 can travel along an optical axis 612 to the image forming optics 602, which can include a fifth lens 604, a sixth lens 606, and an objective lens 608. Thus, the detection light 614 is focused to form an intermediate image and directed at 702 to another module of the detection module 104. The adjustable aperture 704 located upstream of the fifth lens 604 of the image forming optics 602 can be used to adjust the numerical aperture of the detection light. Alternatively or additionally, the adjustable aperture 704 can be located between the lens 606 and the objective lens 608.

[0101] Alternatively or additionally, the image / forming processing module may include one or more imaging lenses and / or emission filters, as illustrated in FIG. 7B. The detection light 702 from the image forming module and / or field rotation module may be fed along an optical axis 710 with an image plane 706 for the image that is perpendicular to the optical axis 710 and coincides with the focal plane of a first lens 720. The light from the image plane 706 is refracted by the first lens 720 to form a beam that passes through an emission filter 722 (i.e., the emission filter 722 may pass the emission wavelengths of the detection light but block the excitation wavelengths of the illumination light). A second lens 724 may focus the filtered detection light onto the incidence plane of the detector 708 for imaging.

[0102] Alternatively or additionally, the image processing / forming processing module may include optical components to resolve intensity variations in the image. In particular, when imaging deeper structures in the object, the light detected from shallow to deep layers may be attenuated to different degrees. For example, since an image of an obliquely illuminated plane is captured in a single detection frame, the signal corresponding to the structure closest to the object's surface may be a relatively strong signal, whereas the signal corresponding to the structure deeper in the object may be a relatively weak signal. For a particular object, the detector may be saturated by this brighter signal.

[0103] To increase the dynamic range of the system, a spatially variable attenuator 726 (e.g., a ND gradation filter) can also be used, as illustrated in FIG. 7C, for example at the entrance plane of the detector 708 or at or near the intermediate image plane 706 between the second module 103 and the detector 708. Thus, the image mapped onto the detector 708 by the lenses 740, 744 can be compensated by spatially varying intensity values. Alternatively or additionally, strips of attenuators (e.g., partially reflective or absorbing glass or films) can be placed to reduce the intensity of detected light reaching the detector from upper layers of the object while allowing all or at least a large portion of the light from deeper layers to reach the detector. This allows for higher incident light illumination or detector integration times / gains without saturation.

[0104] Alternatively or additionally, a gradient attenuator may be used to achieve an effect similar to that in Fig. 7C. Such an attenuator may be physically moved (or modified using an LCD-type tunable filter or reflective surface, e.g., an SLM or digital micromirror device (DMD)) in a plane parallel to the image plane 706 to adjust the attenuation in an object-specific manner. Information about the location of the sliding attenuator, or the relative pattern of attenuation from the spatially variable attenuator, may be used during image processing to correct the data acquired by the detector 708 for its attenuation.

[0105] Alternatively or additionally, as illustrated in FIG. 7D, the image conditioning module can include an image intensifier 762 to amplify the intensity of the detected light prior to detection by the detector 708. The image intensifier can significantly increase the detection efficiency for low light levels, thereby increasing imaging speed, at the expense of potentially reducing resolution. The image intensifier 762 can be located at or slightly in front of the detection plane of the detector 708, although it is possible to locate the image intensifier 762 elsewhere, according to one or more contemplated embodiments. Alternatively, the image intensifier 762 can be an integrated part of the detector 708.

[0106] Alternatively or additionally, the image conditioning module may include one or more components for multi-wavelength imaging of the detected light. For example, multiple wavelengths or light sources may be used (in parallel or sequentially) to illuminate the object and induce different physical processes in the object. Spectral separation of the detected light may be used to achieve multi-color detection and imaging. For example, different color channels may be separated into different parts of the detector chip, and custom parts may be ordered for different wavelength channel separation. Such separation may be limited by the density of detector pixels, the frame rate, and the optics available to implement the spatial separation.

[0107] For example, as illustrated in FIG. 8A, multi-wavelength detection can be provided by using a spectral beam splitter 804. The detection light 702 from the image forming module and / or the field rotation module can be provided along an optical axis 710 with an image plane 706 for the image that is perpendicular to the optical axis 710 and coincides with the focal plane of the first lens 802. The light from the image plane 706 is refracted by the first lens 802 to form a substantially parallel beam that passes through the spectral beam splitter 804. The spectral beam splitter 804 can separate the detection light 702 into different optical paths, e.g., 808 and 812, depending on the wavelength of the detection light. Each optical path 808, 812 can be provided with a second lens 806, 810, which can focus the separated light to respective detection areas 814, 816 of the detector 818. The control module 150 can combine the images produced by the detection regions 814, 816 to form a single multi-color image.

[0108] Alternatively or additionally, multi-wavelength detection can be provided by a filter wheel 852, as illustrated in FIG. 8B. The detection light 702 from the image forming module and / or field rotation module can be provided along an optical axis 710 with an image plane 706 for the image that is perpendicular to the optical axis 710 and coincides with the focal plane of a first lens 850. The light from the image plane 706 is refracted by the first lens 850 to form a substantially parallel beam that passes through a selected first filter 854 of the filter wheel 852. The filtered light is then focused by a second lens 858 onto a detector 860 for imaging. By rotating the filter wheel so that another filter, for example filter 856, is positioned in the optical path between the lenses 850 and 858, images of different wavelengths can be acquired. In this manner, images of different colors can be acquired sequentially for the detection light 702. As with the embodiment of FIG. 8A, the control module 150 can combine the sequentially acquired images of different colors to form a single multi-color image.

[0109] In some configurations where multispectral imaging is desired, the filter wheel 852 may not be fast enough to provide real-time imaging for different wavelengths. Instead of the filter wheel 852, an acousto-optical tunable filter (AOTF) 863 can be used, as illustrated in FIG. 8D. The AOTF 863 can use acoustic waves to change the diffraction of light passing through it. Thus, using a first acoustic wave, the acousto-optical filter 863 can direct light 861a having a first wavelength to the detector 860. Light 861b and 861c having wavelengths different from the first wavelength are refracted away from the detector 860. However, by increasing or decreasing the frequency of the acoustic wave for the AOTF 863, the light 861b or 861c can be sequentially refracted to the detector 860. Because the AOTF 863 does not include moving parts, it can potentially switch between different wavelengths faster than the rotating filter wheel 852.

[0110] Alternatively, multiple detectors can be used to image different wavelengths simultaneously to provide multispectral imaging. FIG. 8C illustrates such a configuration, which uses a combination of multiple detectors and wavelength-selective beam splitters (e.g., dichroics). Detection light 702 from the image forming module and / or field rotation module can be provided along an optical axis 710 with an image plane 706 for the image that is perpendicular to the optical axis 710 and coincides with the focal plane of a first lens 880. Light from the image plane 706 is refracted by the first lens 880 to form a beam 881. The beam 881 enters a first beam splitter 882, which can pass light 894 having a first wavelength (λ1) and a second wavelength (λ2), but reflects light 884 having a third wavelength (λ3). For example, the beam splitter 882 may be a long-pass beam splitter and λ1 and λ2 may be greater than λ3. The reflected light 884 is then focused by a lens 886 onto a first detector 888 for imaging. The transmitted light beam 894 may then be incident on a second beam splitter 890, which may pass light 891 having a first wavelength (λ1) but reflect light 892 having a second wavelength (λ2). For example, the beam splitter 890 may be a long-pass beam splitter and λ1 may be greater than λ2. The reflected light 892 is then focused by a lens 896 onto a second detector 898 for imaging, whereas the transmitted light 891 is focused by a lens 893 onto a third detector 895 for imaging. In this manner, images of different colors may be simultaneously obtained for the detected light 702. As with the embodiment of FIG. 8A, the control module 150 can combine successively acquired images of different colors to form a single multi-color image.

[0111] Other configurations of beam splitters and detectors are contemplated, according to one or more contemplated embodiments. For example, a greater or lesser number of beam splitters, detectors, and / or wavelengths may be provided than those specifically illustrated in FIG. 8C. Additionally, the beam splitters may be short pass filters rather than long pass filters. Other variations and configurations will be readily apparent to those of skill in the art.

[0112] Alternatively, the multiple beam splitters 882 / 890 of FIG. 8C can be replaced with prisms as illustrated in FIG. 8E. Thus, the detection light 702 from the image forming module and / or the field rotation module can be provided along an optical axis 710 with an image plane 706 for the image that is perpendicular to the optical axis 710 and coincides with the focal plane of the first lens 850. The light from the image plane 706 is refracted by the first lens 850 and then focused by the second lens 854 onto the input face of the prism 865. For example, the prism 865 can be a dispersive prism that refracts the input light along different directions based on the wavelength of the light. Thus, the light 863a having a first wavelength can be directed to the first detector 860a, the light 863b having a second wavelength can be directed to the second detector, and the light 863c having a third wavelength can be directed to the third detector 860c. In this way, different color images can be obtained simultaneously for the detected light 702. As an alternative to a prism, a diffractive element such as a reflective grating can be considered, which can perform substantially the same function.

[0113] Although only three detectors are illustrated in Figure 8C, fewer or additional detectors may be provided in accordance with one or more contemplated embodiments. Additionally, while detectors 860a-860c are shown as separate components, the detectors may be part of a single detector unit. For example, detectors 860a-860c may be separate detector elements of a single detector or detector array.

[0114] According to one or more contemplated embodiments, other optical elements are contemplated for spatially separating different wavelengths for simultaneous detection by one or more detectors, for example, prism 865 in FIG.

[0115] 9A-9C, a configuration for the scanning module 116, the de-scanning module 118, and the separation module 112 for the primary illumination source 122 and the secondary illumination source 124 of FIG. 1 is illustrated. As mentioned above, the secondary illumination source 124 can be used to provide photo manipulation, stimulated emission depletion (STED), or any other desired effect on the object, either independently of or together with the light from the primary illumination source 122. For example, the secondary illumination from the secondary illumination source 124 can be propagated along an optical path similar to that of the primary illumination through the second module 103. Such a configuration is illustrated in FIG. 9A. The primary illumination 904 from the illumination module 902 can be directed to a beam separation module 906, which directs the primary illumination 908 to the scanning module 910. Similarly, secondary illumination 916 from secondary illumination source 914 can be directed to beam separation module 906, which can direct the secondary illumination 916 along the same optical path as the primary illumination 908, as suggested by reference numeral 918, to scanning module 910. Scanning module 910 can then direct primary illumination 912 and secondary illumination 922 to simultaneously or sequentially scan across an object.

[0116] 9B, the secondary illumination source may be provided with one or more beam steering components 930, such as an SLM, beam steering mirrors or optics, acousto-optic deflectors, phase plates, adaptive optics, or any other optical components to achieve the desired steering. Thus, a beam steering component 930 may be provided between the secondary illumination source 914 and the beam separation module 906 to steering the light prior to combination with the primary illumination light 908 and scanning 936 by the scanning module 910.

[0117] In some configurations (e.g., configurations where photomanipulation is performed), secondary illumination 916 from illumination source 914 can be provided directly to focusing module 108 (e.g., via beam separation module 958) rather than passing through scanning module 954. Thus, scanning module 954 can scan across an object with primary illumination light 960, while secondary illumination light 962 is stationary or at least unaffected by the scanning.

[0118] Referring to FIG. 10, one embodiment of a DR imaging system using a single scanning mirror 1010 is illustrated. Illumination light 1004, such as a planar illumination beam or line, can be provided from a primary illumination module 1002, such as the illumination module 102 in FIG. 1. The illumination light 1004 enters a beam splitter 1006, such as a dichroic beam splitter or a polarizing beam splitter, which functions to reflect the light along an optical path 1008. The illumination light is then redirected by the scanning mirror 1010 and directed through a scan lens 1012 and a tubular lens 1014 (the lenses 1012, 1014 together form a telescope) to an edge portion of the back aperture of an objective lens 1016. The objective lens 1016 focuses the illumination light to form a planar illumination beam 1020 at the object. The scanning mirror 1010 is configured to change the direction of the illumination light, thereby changing the angle formed by the illumination light and the edge of the rear aperture of the objective lens 1016, thereby causing scanning of the planar illumination beam 1020 at the object.

[0119] The resulting light 1022 can be collected by the objective lens 1016 based on its full numerical aperture. The detection light from the objective lens 1016 can be passed through the telescope formed by the lenses 1012, 1014 along the optical axis 1026 to be incident on the same scanning mirror 1010 as the illumination light. The scanning mirror 1010 can thus descan the detection light, i.e., by redirecting it along the optical axis 1030 to the detection module 1036 and scan it with the illumination light beam 1008. The illustrated configuration using a single mirror 1010 for scanning and descanning allows a "full aperture" of light emerging from the object to be sampled, which can improve resolution and throughput compared to a partial aperture implementation, as in the embodiment in FIG. 12.

[0120] After de-scanning by the scanning mirror 1010, the detection light can be incident on a beam splitter 1006, which is configured to pass all or a portion of the detection light (e.g., based on the wavelength or polarization of the detection light). One or more detection module optics, illustratively represented by a lens 1038 (although additional optics and / or components are contemplated, e.g., as discussed above with respect to Figures 6A-7D), can be used to form an intermediate image 1034, which can subsequently be further processed by a detection module 1036, such as detection module 104 in Figure 1, to generate an image of the illuminated region of the object.

[0121] Referring to FIG. 11, an alternative embodiment of a DR imaging system using separate scanning and de-scanning mirrors is illustrated. Illumination light 1004, such as a planar illumination beam or line, can be provided from a primary illumination module 1002, such as the illumination module 102 in FIG. 1. The illumination light 1004 enters a scanning mirror 1128, which redirects the illumination light 1104 through a scan lens 1106 and a tubular lens 1108 (the lenses 1106, 1108 together form a first telescope) to a beam splitter 1110, such as a dichroic beam splitter or a polarizing beam splitter. The beam splitter 1110 reflects the light along an optical path 1112, so that the light enters the edge of the back aperture of the objective lens 1016. The objective lens 1016 focuses the illumination light to form a planar illumination beam 1020 (oblique with respect to the axis) at the object. The scanning mirror 1128 is configured to change the direction of the illumination light, thereby changing the angle formed by the illumination light and the edge of the rear aperture of the objective lens 1016, thereby causing scanning of the planar illumination beam 1020 at the object.

[0122] Similar to the embodiment of Figure 10, the resulting light 1022 can be collected by the objective lens 1016 based on its full numerical aperture. However, rather than passing through the same telescope as the illumination light in Figure 10, the detection light from the objective lens 1016 passes through a beam splitter 1110 (either partially based on polarization or entirely based on wavelength) to travel along a different optical path 1120 than the illumination light 1104. Thus, the detection light passes from the beam splitter 1110 to its own specific tubular lens 1114 and descanning lens 1116 (which lenses 1114, 1116 together form a second telescope), which direct the detection light 1120 to a separate descanning mirror 1122. The descanning mirror 1122 can descan the detection light, i.e., by redirecting it along an optical axis 1128 to the detection module 1036. Thus, a mirror 1128 can scan with the planar illumination beam 1004, while another mirror 1122 can descan the return light. The movements of the mirrors 1122, 1128 can be synchronized, so that the image detected at the detection device corresponds to the illuminated plane on the object.

[0123] After descanning by descan mirror 1122, the detection light can be directed to one or more detection module optics, illustratively represented by lens 1124 (although additional optics and / or components are contemplated, e.g., as discussed above with respect to Figures 6A-7D), which can be used to form an intermediate image 1126. Intermediate image 1126 can be further processed by detection module 1036, such as detection module 104 in Figure 1, to generate an image of the illuminated region of the object.

[0124] Referring to FIG. 12, an alternative embodiment of a DR imaging system using separate scanning and de-scanning mirror sections with partial aperture detection is illustrated. Illumination light 1004, such as a planar illumination beam or line, can be provided from a primary illumination module 1002, such as the illumination module 102 in FIG. 1. The illumination light 1004 enters a first mirror section 1202, which redirects the illumination light 1208 through a scan lens 1210 and a tubular lens 1212 (the lenses 1210, 1212 together form a telescope), so that the illumination light 1214 enters an edge portion of the back aperture of the objective lens 1016. The objective lens 1016 focuses the illumination light to form an oblique planar illumination beam 1020 at the object. The first mirror portion 1202 is configured to change the direction of the illumination light, thereby changing the angle formed by the illumination light and the edge portion of the back aperture of the objective lens 1016, thereby causing scanning of the planar illumination beam 1020 at the object.

[0125] The resulting light 1022 can be collected by the objective lens 1016 based on its partial numerical aperture. That is, the detection light 1216 at the opposite edge of the back aperture of the objective lens 1016 is directed to the detector for imaging, whereas the other detection light 1022 does not follow a path that can be detected. The detection light can be passed through a telescope formed by the lenses 1212 and 1210 to be incident on the second mirror portion 1204, which can descan the detection light. That is, the detection light 1218 can be descanned by redirecting it along the optical axis 1222 to the detection module 1036. As with the embodiment of FIG. 11, the movements of the first mirror portion 1201 and the second mirror portion 1204 can be synchronized, so that the image detected at the detection device corresponds to the illuminated plane of the object.

[0126] After de-scanning by the second mirror portion 1204, the detection light 1222 can be directed to one or more detection module optics, illustratively represented by a lens 1224 (although additional optics and / or components are contemplated, e.g., as discussed above with respect to Figures 6A-7D), which can be used to form an intermediate image 1226. The intermediate image 1226 can be further processed by a detection module 1036, such as detection module 104 in Figure 1, to generate an image of the illuminated region of the object.

[0127] 12 as separate components, the first mirror portion 1202 and the second mirror portion 1204 may be parts of the same optical component, such as different mirror surfaces of a polygonal mirror 1206. Thus, synchronization of the movements of the first mirror portion 1202 and the second mirror portion 1204 can be achieved by simply rotating (or moving back and forth) the polygonal mirror 1206 about its axis of rotation.

[0128] The physical coupling of the scan and descan mirror parts is provided by a polygon mirror, which allows for precisely combined scanning and descanning. However, if the speed at which the planar beam must scan is relatively slow (e.g., on the order of 50-100 sweeps per second), even at a large volume rate of 50-100 volumes per second, synchronization of the two separate mirrors can be easily achieved. Furthermore, the rotation of the polygon mirror does not provide an ideal scan / descan pattern, since the mirrored facets both translate and rotate as the polygon mirror rotates about its axis of rotation. This can result in scan / descan errors that adversely affect imaging. As mentioned above, the use of a polygon mirror can limit the detected light to only half the light emitted from the back aperture of the objective lens 1016, which limits the numerical aperture (NA) of the detected light. The NA limitation can reduce the resolution of the resulting image and / or reduce the amount of emitted light reaching the detector, thereby reducing the signal-to-noise ratio. Thus, some applications may be better served by the single mirror embodiment of FIG. 10 or the two mirror embodiment of FIG.

[0129] Referring to FIG. 13A, an alternative embodiment of a DR imaging system using a motorized lens 1314 is illustrated. For example, one or more motorized lenses can be used to adjust the vertical position of the illumination plane and / or detection plane to expose the volume. Illumination light 1004 can be provided from a primary illumination module 1002, such as the illumination module 102 in FIG. 1. The illumination light 1004 is mapped by a first telescope formed by lenses 1306, 1308 to a beam splitter 1310 (e.g., a dichroic), where the illumination light 1004 is reflected through a motorized lens 1314 toward the back focal plane of the objective lens 1016, thereby forming a planar illumination beam (or illumination line) for scanning on the object. The detected light is sent via objective lens 1016 and motorized lens 1314 to beam splitter 1310 where it is mapped by a second telescope formed by lenses 1322, 1326 to an imaging lens 1330 (e.g., a second objective lens), thereby forming an intermediate image 1334. Alternatively, the first telescope and second telescope can be combined, whereby the illumination light and the detected light share one telescope (e.g., as illustrated by the telescope with lenses 1322, 1326 in FIG. 13B).

[0130] A motorized lens 1314 can be positioned between the dichroic beam splitter 1310 and the objective lens 1016 to adjust the focus of the illumination beam 1312 with respect to the back focal plane of the objective lens 1016. The motorized lens 1314 directs the illumination beam 1312 to an edge of the back aperture of the objective lens 1016a. As with the other embodiments, the objective lens 1016a focuses the illumination light to form an oblique, planar illumination beam 1020 at the object.

[0131] The position of the incident illumination beam of light 1312 at the back focal plane of the objective lens 1016a can be offset from the center with respect to the optical axis of the objective lens 1016a. This position determines the angle of the planar illumination beam at the object. Some of the objective lenses have back focal planes of different sizes and / or locations. When it is desired to change the objective lens, for example to switch from the first objective lens 1016a to the second objective lens 1016b, an objective lens turret 1328, for example as shown in FIG. 13B, is used. The turret 1328 can include multiple objective lenses with different characteristics (e.g. magnification) that can be used interchangeably.

[0132] To accommodate switching between the objective lenses 1016ab, it may be necessary to change the optical path to accommodate different back aperture geometries. For example, changing the optical path of the illumination beam can be achieved by changing the magnification of the combination of the scan lens 1306 and the tubular lens 1308, which can be done by inserting a zoom lens type geometry (not shown) in which a combination of concave and convex lenses is moved (via motorized or manual displacement) to change the position of the beam at the back focal plane of the objective lens 1016b. Alternatively or additionally, a variable focusing element 1302, which may be a zoom lens or a motorized lens, can be used to change the magnification and / or change the beam path for the illumination beam 1312, thereby accommodating the change in geometry due to the change in objective lens. It is also conceivable that this adjustment can be achieved by inserting a specially designed lens or compound lens attached to each objective lens 1016b to be used in the system. Alternatively or additionally, the location of beam 1312 can be translated across the back focal plane (eg, by a scanning mirror (not shown)) to accommodate different objective lens geometries.

[0133] Similar adjustments of the detection side lenses can also be made to provide different magnitudes of magnification depending on the configuration of the detection side optics and the method of descanning and image rotation. For example, a turret 1331 with lenses 1330a, 1330b can be provided on the detection side as the first lens of the detection module 1304. For example, when switching from the objective lens 1016a to the objective lens 1016b, a corresponding change would be made by the objective lens turret 1331 to switch from lens 1330a to lens 1330b to maintain a desired magnification (e.g., 1×) in the intermediate image. Alternatively or additionally, an optimal adjustment of the focus of the image on the detector of the detection module 1304 is achieved by translation of the detection lenses (e.g., telescope lenses 1322, 1326 and / or lens 1330a). A control module 1316 can be provided to coordinate the operation of the primary objective lens turret 1328 , the secondary objective lens turret 1331 , and / or the variable focusing element 1302 .

[0134] In one or more embodiments, the microscope can provide both DRI / depth resolved imaging and confocal imaging modes in different modes. An example of such a microscope is shown in Figures 14A and 14B, where Figure 14A shows the system in a confocal configuration and Figure 14B shows the system in a DRI configuration. An illumination source 1414 generates an illumination beam that is reflected by a beam splitter 1006, in some embodiments a dichroic beam splitter, whose reflected wavelength is selected to direct the illumination as shown. A beam translation component 1410 (e.g., a wedge or angled plate) and / or a beam expander 1411 connected to the beam translation component 1410 via an arm 1413 can be optionally provided in the beam path between the illumination source 1414 and the beam splitter 1006. In a confocal configuration (as illustrated in FIG. 14A), beam translation components 1410 and beam expanders 1411 may be provided in the beam path to move and expand the beam from the light source to illuminate the object along the optical axis of the objective lens 1016. The scanning mirror 1010 operates to scan the focal spot about an axis perpendicular to the optical axis, as suggested by the double-headed arrow. The objective lens 1016 focuses the illumination beam, thereby producing a focal spot at the object. The adjustable microscope formed by the lenses 1012 and 1014 may be moved to adjust the axial position of the focal spot 1017, or other mechanisms may be provided to axially displace the focal spot.

[0135] The image beam is captured by the objective lens 1016, transmitted through a telescope formed by lenses 1012 and 1014, and directed by the scanning mirror 1010, passes through a dichroic beam splitter 1006 and another telescope formed by lenses 1408, 1409, and is imaged by lens 1038 to an intermediate image plane. The detection arm may include a high numerical aperture lens (e.g., an objective lens) and an imaging lens 1406, which focuses the light onto a detection plane of a detector 1402, which may be a two-dimensional detector. The detection arm and / or the detection plane of the detector 1402 may be angled, for example, to an appropriate angle for image rotation when operating in DRI mode. However, when operating in confocal mode, the rotation of the detection arm (or detection plane) may be the same as in DRI mode. Alternatively or additionally, the detection arm can be rotated to align it with the optical axis of the imaging lens 1038 and the telescope lenses 1408,1409.

[0136] Confocal imaging in the configuration of Figure 14A can be provided by sampling a centerline (row or column, depending on the detector orientation) of detector elements. Additionally or alternatively, a line of detector elements adjacent to the centerline (e.g., above, below, or to one side, depending on the detector orientation) can be used, for example, to deconvolve with light that would otherwise be rejected by the confocal pinhole, thus improving signal-to-noise ratio and image resolution, similar to the air scanning concept (Zeiss system).

[0137] To change from the confocal microscope configuration of FIG. 14A to the DRI configuration of FIG. 14B, the beam translation component 1410 and the beam expander 1411 can be removed from the illumination beam path, so that the illumination beam can be directed through the beam splitter 1006, the scanning mirror 1010, and the telescope lenses 1012, 1014 to the edge of the objective lens 1016. Additionally or alternatively, an aperture plate (not shown) can be provided and moved into position to adjust the numerical aperture of the illumination beam. The illumination source 1414 can be arranged in a selective configuration whereby the illumination source 1414 generates a planar illumination beam at the object. In the DRI configuration, the planar illumination beam 1020 can be scanned and the generated light 1022 can be captured. As in other DRI embodiments, the light 1022 can be descanned and transmitted to the detector 1402 via the scanning mirror 1010 to form an image.

[0138] Referring to FIG. 15, a microscope 1500A has an objective lens 1502 positioned to image an object 1524. The objective lens 1502 transmits a planar illumination beam 1529, the width of which extends across the plane of the drawing, and which is moved through a range of angles and / or positions to scan over the volume of the object 1524. The direction of the scanning movement may be parallel to the plane of the drawing and has at least a component perpendicular to the plane of the planar illumination beam 1529. An image of the object illuminated by the planar illumination beam 1529 is captured over a range of acceptance angles as suggested by reference numeral 1525. The planar illumination beam 1529 is generated using a light source 1511, which directs the illumination beam using a scanning mirror 1522. As in other embodiments, a cylindrical lens 1527 can be provided in the optical path of the illumination beam 1523 to form a planar beam. The illumination beam 1523 can be conditioned by the light source 1511 to have a planar polarization that is reflected by the polarizing beam splitter 1506 to the scanning mirror 1522. The polarization of the illumination beam can be converted to circular using a quarter wave plate 1508 before the illumination beam is reflected by the scanning mirror 1522. The illumination beam 1523 passes through the telescope 1504 to the objective lens 1502 where it is shaped into a planar illumination beam 1529 as discussed in connection with the previous and other embodiments.

[0139] The scanning mirror 1522 also descans the image beam 1519 received through the objective lens 1502 and the telescope 1504. The polarization of the image beam 1519 is changed again by the quarter wave plate 1508 to be perpendicular to the polarization of the illumination beam as it emerges from the light source 1511, and so passes through the polarizing beam splitter 1506. The image beam 1532 is circularly polarized by passing through another quarter wave plate 1531. The image beam 1532 passes through the telescope 1504 and the lens assembly 1510 and is reflected by the mirror 1520. The lens assembly 1510 can be selected to have the same properties as the objective lens 1502. The mirror 1520 is oriented at an angle half the angle of the intermediate image that would be formed at the axial position of the mirror 1520, and is positioned at an axial position where a real image will be formed with respect to an image plane at the object that is oriented perpendicular to the optical axis. This position and orientation are such that the z-axis aberration caused by the objective lens 1502 is compensated as described in "Vectorial point spread function and optical transfer function in depth-resolved imaging" J Kim, T Li, Y Wang, X Zhang, Optics Express 22(9), 11140-11151. The image beam 1535 is transformed again by the quarter wave plate 1531, which reflects it by the polarizing beam splitter 1506 to reach the imaging sensor 1512 via the imaging lens 1514.

[0140] In the configuration of FIG. 15A, a stage or other sample support may be provided to position the object 1524. The object 1524 is not part of the microscope 1500A. The telescope 1504 may or may not be provided. In some embodiments, the objective lens 1502 and the lens assembly 1510 are in a telecentric configuration. The lens 1514 is a figurative element and may include optical components made up of multiple elements. The lens assembly 1510 may be identified as a corrective lens assembly because the same optical properties as the objective lens 1502, in combination with the angular and axial positions of the mirror 1520, can compensate for aberrations in the z-axis (optical axis) by making a second pass of the image light, as described by Kim.

[0141] The elements of the telescope 1504 can be adjusted. It is noted that in the embodiment of FIG. 15, the use of the polarizing beam splitter 1506 and the quarter wave plates 1531, 1508 is believed to be for minimizing the amount of light lost during redirection through the beam splitter 1506. The polarizing beam splitter and the quarter wave plates can be used in the same manner as in any of the above embodiments where applicable. In the embodiment of FIG. 15, the single scanning mirror 1522 can be replaced by separate mirrors, one for descanning the image beam 1519 and one for scanning the illumination beam 1523. The two mirrors can be controlled or mechanically connected for synchronized movement.

[0142] Referring to FIG. 16A, a planar beam 1604 can be generated using any of the disclosed methods or other methods. The planar beam passes through a beam splitter 1612 and is used for scanning by a scanning mirror 1618, which is shown overlapping in multiple orientations. The imaging beam and the illumination beam pass through a telescope 1606. For each orientation of the scanning mirror 1618, a respective reflected beam is shown with reference number 1620. Although three orientations and beams are shown, it is contemplated that they may be examples of a continuous range of orientations and beams, or a smaller or larger set of discrete orientations and beams. The beams 1620 enter the telescope such that they are emitted to enter the area 1610 at different angles at the back aperture of the objective 1602, and the planar illumination beam 1603 is emitted from the objective to sweep over a range of angles resulting from the edge of the front aperture of the objective 1602 as suggested by reference number 1601. The image beam 1608 is captured, descanned by scanning mirror 1618 , and relayed through beam splitter 1612 to imaging device 1624 .

[0143] 16B, the introduction of focusing optics 1640 changes the angle at which illumination beam 1620 is scanned, and correspondingly, the angle at which image beam 1608 is scanned. Instead of beams 1620 entering the telescope, emerging from the telescope at regions 1610 close to one another at the back aperture of objective lens 1602, and emerging from the objective lens at the edge of the front aperture of objective lens 1602 as suggested by reference numeral 1601, the beams are emitted toward an axis at multiple points at the edge of the front aperture. The effect of this is that the scanning "walks" planar illumination beams 1648A, 1648B, 1648C along a path from the front aperture, and sweeps a narrower range of angles, resulting in more parallel scanned slices than those in the embodiment of FIG. 16B. As in the embodiment of Figure 16A, the image beam 1642 is captured, descanned by scanning mirror 1618, and relayed via beam splitter 1612 to imaging device 1624, but by passing through focusing optics 1640, the focus of imaging device 1624 is kept on the scanning planar illumination beam (1648A, 1648B, 1648C). In Figure 16C, a similar effect to Figure 16B can be achieved by moving objective lens 1602 with respect to telescope 1606, relative to its position in Figure 16A.

[0144] Although embodiments using an objective lens to focus the illumination beam on and receive light from the object are described above, embodiments of the presently disclosed subject matter are not so limited. For example, FIG. 17 illustrates a configuration for two-axis confocal imaging without an objective lens, e.g., as part of an endoscopic device 1702. The endoscopic device 1702 can be positioned adjacent to the object 106, which may be an internal tissue or structure of a patient into which the endoscopic device 1702 is inserted. Light from the illumination module 1002 can be directed to the endoscopic device 1702 via an input waveguide 1704, which may include a collimation device (e.g., a GRIN lens) at its output end. The illumination light enters a fixed optical element 1706 (e.g., a parabolic mirror), which redirects the incoming light to each scanning element 1708a of a scanning device 1708. For example, the scanning device may be a MEMS mirror array with mirror surfaces that can be individually addressed and tilted in two dimensions. Thus, the scanning element 1708a can redirect the illumination to perform a scan at the object 106 with the illumination.

[0145] A lens 1710 may be disposed in the optical path between the scanning device 1708 and the object 106 to focus the illumination light and receive the detection light. For example, the lens 1710 may be a hemispherical lens, a solid immersion lens, or a GRIN lens. The light from the object and the light received by the lens 1710 may be incident on another scanning element 1708b of the scanning device 1708. The scanning device 1708 may control the movement of the scanning element 1708b to correspond to the movement of the scanning element 1708a, so that the imaged plane corresponds to the illuminated plane. The detection light may be incident on a fixed optical element 1714 (e.g., a parabolic mirror), which redirects the detection light to each waveguide 1716, which may include a collimation device (e.g., a GRIN lens) at its incident end. 17 as a single waveguide 1716, the waveguide may be multiple waveguides, such as the angled waveguide of FIG. 6F, which provides image rotation as well as transmission of detection light to a detector in the detector module 1036. The detection light from the output waveguide 1716 may be provided to the detection module 1036 for imaging in a manner consistent with other embodiments described above. Alternatively or additionally, the waveguide 1716 may include a one-dimensional array of waveguides (e.g., a 1000×1 fiber bundle) that transmits the descanned light to a linear detector.

[0146] FIG. 18 illustrates another configuration for depth-resolved imaging without the use of an objective lens. Light from the illumination module 1002 can be directed to a scanning device 1808 via an input waveguide 1804 or other input optics. Meanwhile, detection light from the object 106 can be redirected to an output waveguide 1810 or other output optics via the scanning device 1808. The scanning device 1808 can have a single scanning element 1809 that provides both scanning and de-scanning simultaneously, as shown in FIG. 18, or can have multiple scanning elements that are synchronized, as described above with respect to FIG. 17. As with the embodiment of FIG. 17, the scanning element 1809 of the scanning device 1808 can be moved to scan the object 106 with the illumination beam, and the resulting light corresponding to the illuminated plane can be imaged by the detection module 1036.

[0147] In one or more embodiments, a linear detector can be used to perform depth-resolved imaging. In such a configuration, scanning can be performed with a linear beam (narrow beam) rather than a planar beam. Such a configuration is illustrated in FIG. 19 and may be particularly useful for two-photon imaging, for example. An excitation light source 1902 (e.g., an infrared laser source) generates an excitation beam 1901 that can pass through an aperture 1904 and can be incident on a first scanning mirror 1906 after passing through a dichroic beam splitter 1908. The first scanning mirror 1906 can be configured to sweep in the lateral Y dimension. The resulting illumination line can be directed to a second scanning mirror 1910. The second scanning mirror 1910 can be configured to sweep the illumination in the scanning dimension (e.g., the XZ plane). The resulting scanned beam can be mapped by a microscope formed by lenses 1912, 1914 to the back focal plane of objective lens 1916 and focused onto the object to form illumination beam 1920. Emitted light 1922 (e.g., fluorescent light in the 400-500 nm range produced by fluorophores in the object) can be captured by objective lens 1916 and mapped by a telescope formed by lenses 1912, 1914 onto a second scanning mirror 1910. The second scanning mirror 1910 descans the emitted light and directs the descanned emitted light towards beam splitter 1908.

[0148] After being descanned by mirrors 1906 and 1910, the image light is reflected by beam splitter 1908, and the detection light is then mapped by another telescope formed by lenses 1924, 1926 to imaging objective lens 1928 to form an intermediate image in plane 1930 at an angle to the optical axis, as in other embodiments described above. The intermediate image plane 1930 can then be focused by lenses 1932 and 1938 to a detection plane of detector 1940 (and / or filtered, for example, by emission filter 1936). However, unlike some of the embodiments described above, detector 1940 can be a linear detector array rather than a two-dimensional imaging array. In this case, the image at the image plane is a linear map of the diagonal line in the ZX plane where the emission light 1922 is captured by objective lens 1916. The scanning and descanning by mirrors 1906 and 1910 traverses the volume of the object region. In some embodiments, the linear detector 1940 is an array of PMTs. The signal generated by each element of the linear detector 1940 corresponds to a depth in the object for a given linear scan.

[0149] Referring to FIG. 20A, the imaging device 2001A has a telecentric objective lens 2022 that receives image light from a planar image area 2025 that is scanned by illuminating a planar illumination beam 2027 that coincides with the planar image area 2025. The light from the image area is descanned as described according to any of the various mechanisms described herein. This mechanism 2026 for scanning and descanning is not shown, but may include any of the applicable mechanisms described herein. As discussed elsewhere, a lens assembly 2024 with characteristics that can be selected to compensate for a particular axial focusing aberration is used to generate an image 2031 that can be captured, mapped to a sensor, and stored by an imaging device 2040 that conforms to the description of the detection module 104 and all of its modified embodiments. The imaging device 2040 can focus on the image 2031 and process the focused image on its photodetector. Imaging device 2001A is similar in schematic to most of the embodiments described herein. FIG. 20B shows imaging device 2001B, which is identical to imaging device 2001A, except that it includes an optical wedge 2032 that forms image 2033 at a different angle, e.g., perpendicular to the optical axis of lens assembly 2024 as shown. FIG. 20C shows imaging device 2001C, which uses an alternative mechanism to achieve the effect of prism 2032. Here, lens assembly 2024 is moved as suggested by arrow 2015, which corresponds to a rotation of the image beam as by prism 2032. Again, imaging device 2042 is identical to imaging device 2040, and can focus image 2033 in order to project the image of image 2033 onto a photodetector.

[0150] FIG. 20D shows an imaging arrangement 2003A, which is based on the microscope 1500A, except that instead of being reflected back through the lens assembly 1510, the image light passing through the lens assembly 1510 passes through an optical wedge 2002 oriented to change the angle of the image plane 2004. As in the configuration in FIG. 20D, the angle of the image plane 2004 may be any angle that can be obtained by imaging optics 2007 having an optical axis parallel to the axis of the lens assembly 1510. FIG. 20E has a similar function to the imaging arrangement 2003A, except that instead of optical wedges, a set of reflectors 2008 are used, in which the angle of the reflectors 2008 can be selected so that the optical axis of the imaging optics 2021 is parallel to the optical axis of the lens assembly 1532. The lens assembly 1532 has the same function as the lens assembly 1510 of the imaging arrangement 2003A. In imaging devices 2003A and 2003B, the imaging light is redirected by refraction through a wedge prism or by folding using a reflector after passing through an optical assembly to correct axial aberrations. This can be done so that the image plane is tilted to a position that reduces the angle required between the axis of the image forming optics and the field rotation optics. See, for example, field rotation optics 609 (including lens system 616 and camera 618) and image forming optics 602 in FIG. 6A. Acute angles can cause valuable image light to be lost due to low aperture between the image forming optics and the field rotation optics. Again, imaging device 2042 is identical to imaging device 2040 and can focus on image 2033 to project an image of image 2033 onto a photodetector.

[0151] It is noted that in the embodiment of Figures 20B and 20C, the imaging device 2042 may be a photodetector having a detection surface that is aligned and positioned to coincide with the image 2031 or 2033, as discussed with reference to the embodiment of Figure 6E. It is also noted that an additional advantage of the embodiment of Figures 20B and 20C is that the optical elements of the imaging device 2042 can be moved closer to the lens assembly 2024 without collision, thereby allowing the optics of the imaging device 2042 to be more parallel to the optical axis of the lens assembly 2024, thereby allowing a higher numerical aperture for the optics of the imaging device 2042. Furthermore, the rotation of the image plane 2031 or 2033 can reduce the complexity of the mechanical alignment, for example by providing an alignment table that provides only relative translation of the optical elements of the imaging device 2042 and the lens assembly 2024, without the need to provide both relative angular and positional adjustments.

[0152] FIG. 21A illustrates a multimode confocal / DRI microscope 2100 in a confocal theta configuration, according to some embodiments of the disclosed subject matter. An illumination module 2102, having the functionality of providing a beam expander, conforming to the description of the illumination module 102 in FIG. 1, is positioned at a collimated illumination source. The illumination module 2102 can generate a collimated narrow beam 2131, which can be selectively widened by a beam expander 2135, which can be as discussed with reference to the beam expander 305 in FIG. 3A. The beam expander 2135 can be selectively positioned in the path of the collimated beam 2131 under the control of one or more controllers and / or actuators, indicated by reference numeral 2160. In the confocal mode, the beam expander 2135 can be positioned in the path of the collimated beam 2131 to provide a broad beam 2108 after reflection by a prism mirror element 2130 used for scanning with the beam. Scanning by the prismatic mirror element 2130 may be performed under the control of one or more controllers and / or actuators 2160. The prismatic mirror element 2130 redirects the illumination beam 2108 via a scan lens 2110 and a tubular lens 2112 (which together form a telescope) such that the illumination beam 2114 is incident on the back aperture of the objective lens 2116 after passing through a cylindrical lens 2136 that is selectively positioned in the path of the illumination light 2114 under the control of one or more controllers and / or actuators 2160.

[0153] The cylindrical lens shapes the illumination beam 2114 into a focused line at the focal plane 2140 at the location labeled 2152 after being focused by the objective lens 2116. The image beam 2115 returning through the objective lens 2116 passes through the scan lens 2110 (forming a telescope) and the tubular lens 2112 and lens 2124, and is finally focused as a line and detected by the detection module 2137. An intermediate real image can be generated at the location labeled 2123. The prism mirror element 2130 descans the image beam 2115. The detection module 2137 of any of the disclosed DRI embodiments includes those described with reference to Figures 6A-6I.

[0154] FIG. 21B illustrates the multimode confocal / DRI microscope of FIG. 21A in a DRI configuration, according to some embodiments of the disclosed subject matter. The parallel beam 2131 is directly incident on a beam realigner 2134, e.g., an optical slab or other type of beam deflector, which is selectively placed in its path to place the multimode confocal / DRI microscope 2100 in DRI mode. A cylindrical lens 2132, selected to form a planar beam after being focused by the objective lens 2116, is selectively placed in the path of the offset beam 2131 to place the multimode confocal / DRI microscope 2100 in DRI mode. Mode switching can be performed under the control of one or more controllers and / or actuators 2160 in response to commands via a user interface or lab computer interface (not shown). The scanning of the prism mirror element 2130 redirects the planar illumination beam 2128 through the scan lens 2110 and the tubular lens 2112 (which together form a telescope) so that the planar illumination beam 2128 is incident on the back aperture of the objective lens 2116. The cylindrical lens 2136 is positioned out of the path of the planar illumination beam 2128 by one or more controllers and / or actuators 2160 when the multimode confocal / DRI microscope 2110 is in DRI mode. The planar illumination beam 2128 forms a planar illumination beam 2129 used for scanning when the prism mirror element 2130 is pivoted.

[0155] After passing back through objective lens 2116, image beam 2115 passes through scan lens 2110 (forming a telescope) and tubular lens 2112 and lens 2124 to be focused and detected. In some embodiments, an intermediate image 2126 can be generated, which is imaged by detection module 2137 according to some of the embodiments described herein. As mentioned above, prismatic mirror element 2130 descans image beam 2115. Detection modules 2137 of any of the disclosed DRI embodiments include those described with reference to Figures 6A-6I.

[0156] Referring now to FIG. 21C, a line can be projected onto an angled detector 2170, which may be, for example, a CCD camera. An image can be formed using selected pixels of the detector to eliminate unwanted light to form an image at a selected focal plane 2140. A deconvolution algorithm can be applied to selected pixels on either side of the image that are aligned along the line to improve resolution. This can avoid the need for a slit. If the cylindrical lens 2136 is positioned out of the path of the image beam 2115, the illumination beam 2114 can be focused to a point, in which case the need for a pinhole can be avoided by selecting one or more pixels of the CCD camera required to measure the light level from the collimated focal region 2152. Instead of a CCD camera, the detector 2170 of the detection module 2137 can be a linear array of photomultiplier elements oriented to be aligned to the linear one-dimensional image generated by the multimode confocal / DRI microscope 2100 when in DRI mode.

[0157] 22A, there is shown a microscope for illumination and imaging of a plurality of elements of the imaging device 2200A, for example a continuous planar illumination beam or a series of discontinuous planar illumination beams, as suggested by reference numeral 2230, at an object volume 2201, by which a two-dimensional or three-dimensional image of the object volume is generated at a photodetector in one of the secondary imaging optical component 2204 or the tertiary imaging optical component 2205, described below with reference to FIG. 22A or FIG. 22B. The primary imaging optical component 2202 may have an objective lens (not shown separately, but integrated in the primary imaging optical component 2202), which may be positioned relative to the object support 2241, for imaging a portion of the object illuminated by the planar illumination beam 2230. The first optical component 2202 comprises optical elements selected to cooperate with the objective lens to generate a first intermediate image 2206 of a portion of the object illuminated by the planar illumination beam 2230 in the area in front of the objective lens. In order to move the planar illumination beam and thereby perform a scan over a predefined volume of the object area, a light source and a scanning / descanning element comprising a movable light redirecting element may be included in the primary imaging optical component 2202 to direct the planar illumination beam generated by the light source via the primary imaging optical component to a location labeled with reference number 2230. The movable light redirecting element of the scanning / descanning element further directs the image light received from the first optical component to maintain the focus of the planar illumination beam at the plane 2206 of the first intermediate image as it is swept by the planar illumination beam. Details included in the previous description will not be repeated here for clarity. The secondary imaging optical component 2204 has an optical axis 2209 located at a first angle between the optical axis 2207 of the primary imaging optical component and a normal 2210 to the plane of said first intermediate image.As a result, the magnitude of the first angle is not such that it can be focused by the secondary imaging optical component 2204 onto said first intermediate image plane 2206. The secondary imaging optical component may include a photodetector such as a CCD chip onto which an image of the intermediate image is projected by its optical system.

[0158] Referring to FIG. 22B, the components of FIG. 22A are depicted where identical reference numbers are used, however, it will be understood that the secondary imaging optical component 2204′ differs in that it generates a second intermediate image that can be imaged by the tertiary imaging optical component 2205. The tertiary imaging optical component 2205 has an optical axis 2211 and is positioned to focus the second intermediate image 2206′ formed by the secondary imaging optical component 2204′. The tertiary imaging optical component 2205 forms an image that is focused on a photodetector, such as a CCD sensor. The tertiary imaging optical component 2205 can have an optical axis that is located at a second angle 2211 between the first angle 2209 and the optical axis 2207 of the primary imaging optical component.

[0159] It is noted that in any embodiment, rather than an optical wedge, a similar element performing the above-mentioned functions may be used. For example, a GRIN element may provide the same or similar functionality of tilting the image plane, for example, in a direction perpendicular to the optical axis of the lens assembly 2024.

[0160] FIG. 23A shows the functionality of a DRI microscope such as the embodiment of FIG. 10, which includes an example of a detection module and shows a mostly rectangular profile that provides easy adjustment and configuration, for example by selectively inserting an angle mirror (45°) to rapidly switch between upright and inverted configurations. Illumination light from a light source such as a laser 2300 is directed by a number of mirrors 2315 and shaped by a cylindrical lens 2310, so that the illumination light forms a sheet of illumination at the sample. Optionally, the alignment can include an adjustment on the last mirror 2315. The same adjustment can also be used for switching between confocal and DR modes. A beam splitter such as a dichroic beam splitter 2320 redirects the illumination beam along a first optical path to a scan / descan assembly that includes a scanning element such as a galvo mirror 2325 or other type of light redirector. The light emitted from the galvo mirror then passes through optical elements 2331, 2332 defining a microscope for, for example, directing the illumination beam to a peripheral region of an objective 2340, e.g., a telecentric objective. The beam is emitted as an oblique sheet of light 2342 towards the sample 2345, which is moved (i.e., moved depending on the orientation of the galvo mirror, etc. 2325) by a scan / descan assembly as described with respect to other embodiments, to multiple illuminated regions of the sample 2345, each illuminated at multiple depths.

[0161] Image light from the oblique imaging plane is captured by the same objective lens 2340 and passes through optical elements 2331, 2332 along a second optical path. This image light is then descanned by the same galvo mirror 2325 and passes through the beam splitter 2320. A tilted intermediate image 2370 is then formed by the image forming optics, e.g., a telescope including lenses 2351, 2355 and a second objective lens 2360. Notably, this tilted intermediate image remains fixed regardless of the orientation of the galvo mirror 2325. In some preferred embodiments, the optical properties of the telescope and objective lens used to form the intermediate image (i.e., telescopes 2351, 2355 and objective lens 2360) are matched to the optical properties of the corresponding items in the illumination arm (i.e., telescopes 2331, 2332 and objective lens 2340) to eliminate some distortion in the tilted intermediate image 2370. De-scanning, rotation, and imaging can then be performed as discussed with respect to Figure 10. For example, rotation and imaging can be performed by an imaging module including an objective lens 2380 followed by a long pass filter 2381, a lens 2382, and a camera 2390 including a photodetector (e.g., a CCD).

[0162] A 45° angle mirror (not shown) can be selectively inserted between the distal telescope element 2332 and the objective lens 2340 to provide fast switching between upright and inverted configurations.

[0163] In the embodiment of FIG. 23A, a single mirror 2325 is used for scanning and descanning. Also, in the layout shown, the alignment of the orthogonal arms facilitates precise image setup and adjustment. The overall configuration of this layout is similar to that of a typical confocal microscope, and allows for switching between confocal and DRI modes as described above. In some embodiments, as discussed above, there is a problem with light loss due to alignment and interference between the imaging module and the optics forming the intermediate image, which arises due to the image rotation approach. Solutions to this problem are discussed above and in the embodiments below.

[0164] FIG. 23B shows the functionality of a DRI microscope like the embodiment of FIG. 15, which includes an example of a detection module as described above in connection with FIG. 23A, and which shows a mostly rectangular outline, offering easy adjustment and configuration, for example by selectively inserting an angle mirror (45°) for fast switching between upright and inverted configurations. Components in FIG. 23B that share reference numbers with components in FIG. 23A function in the same way as their counterparts in that figure. Here the principles explained in the discussion of FIG. 15 can be applied. Here again, image rotation is achieved by two passes through a single objective lens 2360a, using the principle of remote focusing. Angle adjustment of the illumination sheet becomes simpler and also realizes a higher numerical aperture (i.e. light transmission at the intermediate image rotation stage with a shorter working distance for improved collection and resolution). An adjustable mirror 2365 is used for imaging, which reflects the image light back to the objective lens. Again, a quarter wave plate 2357 can be used to ensure that the image beam passing through the polarizing beam splitter 2356 is reflected upon a second pass. A long pass filter 2381 is used to select the image light originating from fluorescing (or multiphoton) objects from the reflected or scattered light.

[0165] In one variation of this Figure 23B embodiment, the magnification of the second objective lens 2360a is selected to be greater than the magnification of the first objective lens 2340. For example, the magnification of the second objective lens 2360a may be 60x, while the magnification of the first objective lens 2340 is 20x. If this magnification mismatch exists, the angle of the turning mirror 2365 can be made small (i.e., flat), which can minimize light loss and simplify alignment of the system.

[0166] A drawback of the configuration of FIG. 23B may be the loss of image light due to passing through the polarizing beam splitter. FIG. 23C illustrates the functionality of a DRI microscope similar to the embodiment of FIG. 23B, with the addition of a third lens assembly to recover light that may be lost due to splitting of the image light beam received from the sample. Components in FIG. 23C that share reference numbers with components in FIG. 23A and FIG. 23B function similarly to their counterparts in those figures. Here, a third objective lens 2360b captures light that, in the configuration of FIG. 23B, was lost due to reflection by the polarizing beam splitter (PBS) 2356 and additional mirror 2365. The light captured by the third objective lens 2360b is reflected back to the objective lens 2360b by an adjustable mirror 2365b, and passes through a quarter wave plate 2357b and a polarizing beam splitter 2356 using a quarter wave plate-based capture technique described elsewhere. Other features are as described elsewhere herein.

[0167] FIG. 23D shows a DRI microscope that illuminates a sample from different angles using light sheets 2342, 2342' coming from alternating sides of an objective lens 2340 that capture and separate light to form an image from the alternating illumination. Components in FIG. 23D that share reference numbers with components in FIG. 23A-C function similarly to their corresponding parts in those figures. This embodiment has a similar layout to the embodiment of FIG. 23C, however, the angle of the mirror 2365b' of the third objective lens 2360b is angled in the opposite direction to pass image light from the opposite side of the primary objective lens 2340, which corresponds to the depth resolved imaging light received from the alternating illumination beam or light sheet. Here, a laser is adjusted such that a sheet of light is formed by a cylindrical lens 2310, and an automated redirector 2316 is introduced to selectively displace the illumination beam as suggested by the solid and dotted lines (represented by reference numbers 2317 and 2318, respectively). During a first moment or first cycle, the illumination beam is directed from one side of the primary objective lens 2340 to form an angled illumination sheet 2342 at the sample, and during a next moment or next cycle, the incident beam is switched by the redirector 2316 to the opposite side of the primary objective lens 2340, thereby switching the orientation and angle of the illumination beam or illumination sheet to the orientation and angle represented by reference number 2342'. Image light received primarily from the opposite side of the primary objective lens 2340 is relayed via the dichroic beam splitter 2320 to the polarizing beam splitter 2356, where both the second objective lens 2360a and the third objective lens 2360b are used to form and rotate intermediate images of the light resulting from illumination directed in each direction during each moment or cycle.

[0168] During a cycle, scanning / descanning can be performed with the illumination beam 2342 directed in one direction. In a subsequent cycle, scanning / descanning can be performed with the illumination beam 2342' directed in the opposite direction. During each cycle, the redirector 2316 switches the path of the illumination beam to the other side of the primary objective lens 2340, thereby changing direction. In some embodiments where the beam direction is changed each time, the redirector 2316 changes the path of the illumination beam multiple times during one scan / descan cycle. In any embodiment where the illumination beam is switched each cycle or each time, the polarizing beam splitter 2356 transmits each rotated depth resolved image to the polarizing image splitter 2383. For image light received from the second objective lens 2360a, the illumination energy is polarized in one direction, and for image light received from the third objective lens 2360b, the illumination energy is polarized in the other direction, so that the image beams can be mapped by polarizing image splitter 2383 to respective portions (or respective imaging devices) of imaging device 2390, such as one or more cameras. In some embodiments, an image from illumination on one side can be presented to the photodetector array adjacent to an image from illumination on the other side.

[0169] FIG. 23E shows a DRI microscope that provides a mechanism for improving the shadowing effect of surface structures by collecting images with illumination beams or sheets projected at multiple angles. Components in FIG. 23E that share reference numbers with components in FIG. 23A-D function the same as their counterparts in those figures. Here, the illumination sheet is angled using an additional galvo mirror 2319 such that it is pivoted in a plane parallel to the plane of the light sheet at the sample (i.e., pivoted towards and away from the plane of the drawing). As a result, the beam arrives at the back focal plane of the objective lens at different positions 1, 2, 3 in the peripheral region of the objective lens 2340 as shown in inset 23E1. This changes the orientation of the light sheet to each position 1, 2, 3, as shown in inset 23E2, which in turn changes the location of the shadow, thereby revealing features that may otherwise be under-illuminated. Image processing can be used to select optimal regions of each image (each image being formed by light being differently directed around the sheet by galvo mirror A) to form a single optimized image. Of course, many such optimized images can be combined to form a single 3D image. This embodiment can be described as a "rocking sheet" configuration.

[0170] FIG. 23F illustrates features of a DRI microscope that is functionally similar to the embodiments of FIG. 6I, FIG. 10, and FIG. 13B, using a turret 2380T. Components in FIG. 23F that share reference numbers with components in FIG. 23A function similarly to their counterparts in that figure. The turret 2380T can provide for selection of a first lens assembly of the detection module to provide adjustment of magnification by switching lens assemblies in the turret. More specifically, rotation of the turret 2380T inserts one of a number of third objective lenses 2380a, 2380b, etc. into the optical path to the camera 2390. Optionally, an image splitter can be included in front of the camera 2390.

[0171] FIG. 23G shows a DRI microscope that scans and descans lines at multiple depths to achieve depth-resolved point measurements of light return from a scanned line with multiple point measurements made by a linear array of multiple photomultiplier cells with various features for high frame rate and high sensitivity. Components in FIG. 23G that share reference numbers with components in FIG. 23A function similarly to their counterparts in that figure. The operation of this embodiment is similar to the embodiment of FIG. 23A, except that instead of a sheet of light 2342 projected onto the sample 2345, a pencil beam of light 2342a is projected onto the sample. This pencil beam 2342a is then swept so that a substantial sheet of light is formed. This is accomplished by removing the cylindrical lens 2310 in FIG. 23A and replacing the galvo mirror 2325 in the embodiment of FIG. 23A with a pair of galvo mirrors 2325a. The galvo mirror pair 2325a operates to sweep the pencil beam from end to end to form a substantially reconstructed sheet of light, and in directions towards and away from the primary objective lens to completely sweep the volume, the first direction of sweep therefore being out of the plane of the drawing.

[0172] A broadband linear detector array 2392, such as one with multiple photomultiplier elements (e.g., 10 elements), receives the depth-resolved light as the pencil beam is scanned and descanned by the galvo mirror pair 2325a. Although one mechanism for image rotation (using components 2380-2382) is illustrated, any of the alternative mechanisms for image rotation described herein may be used instead. For example, a linear detector array (with fewer pixels) may be placed directly at the intermediate image plane, in which case the magnification associated with components 2370-2382 is eliminated from the system. Each detector (i.e., each pixel) of the linear detector receives light from a different depth in the sample. To increase throughput and scanning speed, each light may be processed by a separate signal channel clocked in parallel. Additionally, each pixel may have an independently selected gain that allows for optimization of the variable illumination received for each depth for a fully linear depth-resolved "image." That is, light from greater depths has a lower intensity and can therefore be compensated for with a higher gain compared to light from shallower depths.

[0173] This embodiment extends the depth-dependent detection architecture to be more compatible with both two-photon and endoscopy. The linear detector resolves different depths in the sample by simply focusing obliquely on the sample, and avoids many of the problems associated with traditional multi-layer two-photon and confocal approaches. For example, this embodiment avoids the need for piezo scanners and objective lens movement / synchronization, and also avoids the use of temporal or spectral encoding, which adds cost and complexity.

[0174] The XY (or Lissajous, AOD, MEMS or equivalent) scanner moves at the standard two-photon frame rate, but produces as many depths as elements in the linear detector array. This embodiment can provide improved detection bandwidth compared to conventional volume scanning, since each detector can have a high bandwidth for a single depth. This embodiment also allows the use of higher bandwidth and sensitivity detectors, such as APD and PMT arrays, compared to conventional camera technology. It can also provide improved dynamic range, since each detector element can have a fixed gain for its depth position (although the detectors at deeper depths have higher gain / sensitivity to compensate for the attenuated return from the deeper depths). In these embodiments, the linear array acts as a slit, which reduces the effect of detection light scattering compared to sheet illumination (similar to rotating shutter type SPIM). Also, the configuration in this embodiment can be easily adjusted to achieve conventional confocal microscopy using a z-stage.

[0175] FIG. 23H illustrates structured illumination in the context of an embodiment based on that of FIG. 23A, with Ronchi rulings used to structure the beam of light. Components in FIG. 23H that share reference numbers with components in FIG. 23A function the same as their counterparts in that figure. In the embodiment of FIG. 23H, a Ronchi ruling element 2316 is inserted in the path of the illumination beam before it reaches the beam splitter 2320. This creates a parallel grid of illumination beams for emission from the primary objective lens 2340 to the sample. Optionally, an additional galvo mirror 2312 can be added to the illumination beam before it reaches the Ronchi ruling element 2316, which allows the grid of light to wobble at the sample (similar to the wobbly sheet configuration of FIG. 23E), thereby providing improved out-of-plane rejection and / or super-resolution imaging.

[0176] FIG. 23J shows an alternative approach to implementing structured illumination. Components in FIG. 23J that share reference numbers with components in FIG. 23A function in the same way as their counterparts in that figure. In the embodiment of FIG. 23J, a modulatable laser 2302, e.g., a coherent OBIS laser, is used (instead of the continuous laser 2300 from the embodiment of FIG. 23A). The illumination light is rapidly switched on and off, and a galvo mirror 2312 is added and swept to create structured illumination at the sample. It is noted that the galvo mirror 2312 only needs to perform line scans at the image frame rate of the camera. This approach can improve sectioning at the expense of significant speed reduction, and would provide advantages for imaging in scattering tissues such as mouse brain tissue. This could also be implemented with a two-photon embodiment to improve the effects of light scattering on the detection.

[0177] In an alternative embodiment, the same effect can be achieved by replacing the standard galvo mirror 2325 with a bidirectional galvo mirror (not shown) instead of the addition of galvo mirror 2312 shown in Figure 23J. The on-off state of the laser during the bidirectional sweep creates a grating pattern at the sample, thereby providing structured illumination.

[0178] FIG. 23K shows a variation of the embodiment of FIG. 23A in which the magnification in the detection arm is greater than the magnification in the illumination arm. Components in FIG. 23K that share reference numbers with components in FIG. 23A function the same as their counterparts in that figure. In this embodiment, the magnification of the second objective lens 2360c in the detection arm is greater than the magnification of the first objective lens 2340 in the illumination arm. This mismatch reduces the tilt of the intermediate image plane 2370'. This reduction in magnification of the intermediate image plane can provide easier alignment of the camera, higher throughput, higher NA detection, use of shorter WD, and higher NA lenses. Optionally, additional magnification can be added after image rotation without affecting the angle of tilt of the intermediate image plane.

[0179] In some embodiments using a confocal microscope configuration, the detector can be simply exposed as a line is swept to form a planar illumination beam that fills the surface and forms a single 2D image. This strategy can be combined and synchronized with a rolling shutter type acquisition at the detector, which can be thought of as similar to having a detection slit (e.g. in line scanning confocal) that can improve optical sectioning and resolution.

[0180] In some embodiments, the detection light can be descanned in a conventional manner (e.g., along a first direction) and again (e.g., along a second direction orthogonal to the first direction) to compensate for the linear beam motion that forms the plane. This two-dimensional descanning can reduce the detection side image to a single line corresponding to the line of light illuminating the object (in the oblique z direction) at a given time. This incident beam scanning, and / or additional descanning, can be at a line scan rate equal to the volume rate (e.g., ∼20 volumes per second) multiplied by the y sweep rate (e.g., ∼200 y pixels per plane), with an achievable 4 kHz scan / descan rate. The detection descanning of the light along the second direction can also be combined with a conventional cylindrical lens or similar formation of a fully excited planar illumination beam, or it can be any other combination of planar formation and descanning.

[0181] In some embodiments, the linear detector used in such implementations may be a line scan camera or a linear array (e.g., photomultiplier tubes or avalanche photodiodes) with a number of elements corresponding to the number of depths along the z-direction to be acquired, or a linear array or even a linear fiber bundle guided to individual detectors. Such a configuration may be advantageous for endoscopic implementations, since the linear fiber bundle may be used to relay imaging data to an external linear imaging array.

[0182] Throughout the specification and claims, the terms "actuator" and "motor" should be understood to include any type of final control element capable of generating a motive force. Additionally, in any of the embodiments using a light redirector, any type of controllable beam deflector may be used, including reflectors, or any type of device depicted or described with respect to scanning and / or descanning functions, such as acousto-optic deflector(s) and spatial light modulator(s). Such variations are considered additional embodiments of the subject matter of this disclosure.

[0183] Although multiple separate mirrors are shown in Figures 10-22, it is contemplated that each of the scan mirrors and / or descan mirrors may be a mirror, another type of reflective optical element, a refractive element, a diffractive element, an acousto-optic modulator, a MEMS device, an SLM, or any other optical component.

[0184] In all embodiments where a narrow beam of light is scanned laterally to form a plane, it is recognized that the scan can be performed with different illumination patterns, including regular progressive patterns, non-linear patterns such as Lissajous shapes, or even irregular and random patterns. Various other illumination beam shapes can be provided. It is clear that the key features of the subject matter of this disclosure are the ability to perform depth resolved detection of features of the target object, and that the angle of the illumination beam at the object can be parallel to the optical axis of the objective lens or angled with respect to the optical axis of the objective lens. These possible variations apply to all embodiments and all claims. The embodiments and claims in which the illumination light is projected from the edge of the objective lens can be modified so that the light beam is projected from the center and aimed parallel or nearly parallel to the optical axis, in which case there is a sufficient optical aperture from the high numerical aperture objective lens to collect the light emitted from the illuminated area to form an image.

[0185] As used herein, "depth resolved" is intended to indicate that the imaged light comes from multiple axial positions in front of the objective lens.

[0186] In one or more first embodiments, an imaging device, e.g., a microscope, includes an objective lens and another lens assembly, a light source, a scanning / descanning optical assembly, and a light sensor. The objective lens and the other lens assembly are of identical configuration, at least with respect to axial focusing aberration. The objective lens and the other lens assembly are arranged such that image light received by the objective lens passes through the other lens assembly. The scanning / descanning optical assembly has a movable light redirecting element that directs light from the light source, thereby causing a planar illumination beam to emerge obliquely from the objective lens and sweep the volumetric region with the illumination beam. The planar sweeping illumination beam scans over the volumetric region. The movable light redirecting element redirects the image light received from the volumetric region to maintain the focus of light returned from the region illuminated by the planar illumination beam on the light sensor as the sheet of light is swept.

[0187] In the first or any other embodiment, the imaging arrangement further includes a beam splitter arranged to form a branch point where the image light and the light from the light source are positioned to follow adjacent paths to and from the objective lens, and to follow split paths away from the objective lens.

[0188] In the first or any other embodiment, the imaging arrangement further includes a fixed reflector positioned to reflect the image light back through the further lens assembly. In the first or any other embodiment, the fixed reflector is oriented at an angle relative to the optical axis of the further lens assembly, the angle being selected according to the angle of the intermediate image to be formed at the mirror by the objective lens and the further lens assembly.

[0189] In this or any other embodiment, the movable light redirecting element is a motor-controlled reflective element. In this or any other embodiment, the reflective element has reflective properties that make the angular change in direction of the light from the light source and the angular change in direction of the light received from the volumetric region the same as if it were a single, planar reflective surface. In this or any other embodiment, the reflective element has a single, planar reflective surface onto which both the light from the light source and the light received from the volumetric region are incident.

[0190] In this or any other embodiment, the light source generates light selected to cause fluorescence in an object containing a fluorescent dye, and in this or any other embodiment, the light source includes beam flattening optics that flatten a round beam of light into said planar beam.

[0191] In the first or any other embodiment, the optical sensor includes a camera, a linear array of photomultiplier elements, and / or a single photomultiplier element.

[0192] In the first or any other embodiment, the scanning / descanning optical assembly includes a movable optical element that can be selectively positioned in the path of the light from the light source to vary the illumination beam between a planar beam of light directed to the edge of the back aperture of the objective lens (which can be referred to as a scan mode) and a wide illumination beam that spreads across the back aperture (which can be referred to as a confocal mode). In the first or any other embodiment, the movable optical element is refractive and expands the width of the light from the light source. In the first or any other embodiment, the movable optical element is refractive and moves the centerline of the light from the light source. In the first or any other embodiment, the scanning / descanning assembly is configured to control the position of the light redirecting element over a predefined range when the movable optical element is selectively positioned to provide a wide illumination beam that spreads across the back aperture (confocal mode). In the first or any other embodiment, the scanning / descanning assembly is configured to control the position of the light redirecting element over a predefined range in response to confocal mode control commands that selectively position the movable optical element.

[0193] In the first or any other embodiment, the scanning / descanning assembly includes an image forming optics having an optical axis for generating an intermediate real image of image light returned from the planar illumination beam in a scan mode, and a detection module for imaging the intermediate real image, and a photosensor includes a linear or two-dimensional array of pixels onto which an image of the intermediate real image is focused. The detection module has an optical axis that is oblique to the optical axis of the image forming optics, such that a pixel or a selected pixel among the pixels can function as a confocal pinhole or slit in a confocal mode, and such that in a scan mode, the angle between the optical axis of the detection module and the optical axis of the image forming optics is effective for correcting axial positional changes of points along the planar illumination beam imaged by the detection module.

[0194] In one or more second embodiments, an imaging device, e.g., a microscope, includes an objective lens, another lens assembly, a light source, and a scanning / descanning optical assembly. The objective lens and the another lens assembly are arranged such that image light received by the objective lens passes through the another lens assembly. The scanning / descanning optical assembly has one or more movable light redirecting elements that direct light from the light source to generate a planar swept beam of light that is emitted obliquely from an edge of the objective lens, the planar swept beam of light moving across the volume. The scanning / descanning optical assembly includes an imaging light sensor. The one or more movable light redirecting elements redirect the image light received from the volume region to maintain the focus of the planar illumination beam on the light sensor as said sheet of light is swept. The scanning / descanning optical assembly includes a movable optical element that can be selectively positioned in the path of light from the light source to change the illumination beam between a sheet of light directed to the edge of the back aperture of the objective lens (which can be referred to as a scan mode) and a wide illumination beam that spreads across the back aperture (which can be referred to as a confocal mode).

[0195] In the second or any other embodiment, the imaging arrangement further includes a beam splitter arranged to form a branch point where the image light and the light from the light source are positioned to follow adjacent paths to and from the objective lens, and to follow split paths away from the objective lens.

[0196] In the second or any other embodiment, the imaging arrangement further includes a fixed reflector positioned to reflect the image light back through the other lens assembly. In the second or any other embodiment, the fixed reflector is oriented at an angle relative to the optical axis of the other lens assembly, the angle being selected according to one or more of the angles of the sheet of light obliquely swept from the objective lens.

[0197] In the second or any other embodiment, the movable light redirecting element is a motor-controlled reflective element.

[0198] In the second or any other embodiment, the optical sensor includes a camera, a linear array of photomultiplier elements, and / or a single photomultiplier element.

[0199] In the second or any other embodiment, the light source includes beam flattening optics that flatten a round beam of light onto said sheet.

[0200] In the second or any other embodiment, the movable optical element is refractive and expands the width of the light from the light source.In the second or any other embodiment, the movable optical element is refractive and moves the centerline of the light from the light source.

[0201] In the second or any other embodiment, the scanning / descanning assembly is configured to scan with a focused spot or line in a volumetric region when the movable optical element is selectively positioned to provide a wide illumination beam (confocal mode) that spreads across the back aperture.

[0202] In the second or any other embodiment, the scanning / descanning assembly is configured to scan with a focused spot or line in a volumetric region in response to confocal mode control commands that selectively position the movable optical element described above.

[0203] In this or any other embodiment, the scanning / descanning assembly is configured to adjust a range of positions of the light redirecting element in response to selectively positioning the movable optical element to provide a wide illumination beam (confocal mode) that spreads across the back aperture. In this or any other embodiment, the scanning / descanning assembly is configured to adjust a position of the light redirecting element over a corresponding range of positions in response to confocal mode control commands that selectively position the movable optical element.

[0204] In the second or any other embodiment, the scanning / descanning assembly includes an image forming optics having an optical axis for generating an intermediate real image of image light returned from the planar illumination beam in a scan mode, and a detection module for imaging the intermediate real image, and a photosensor includes a linear or two-dimensional array of pixels onto which an image of the intermediate real image is focused. The detection module has an optical axis that is oblique to the optical axis of the image forming optics, such that a pixel or a selected one of the pixels can function like a confocal pinhole or slit in a confocal mode, and such that in a scan mode, the angle between the optical axis of the detection module and the optical axis of the image forming optics is effective for correcting axial positional changes of points along the planar illumination beam imaged by the detection module.

[0205] In one or more third embodiments, a microscope illuminates and images a target volume with a continuous or discontinuous planar illumination beam to generate a three-dimensional view. The microscope includes a primary imaging optical component, a light source, a scanning / descanning element, and a secondary imaging optical component. The primary imaging optical component includes an objective lens, which can be positioned relative to an object support. The first optical assembly includes optical elements selected to cooperate with the objective lens to generate a first intermediate image of the planar sub-area in an area in front of said objective lens. The scanning / descanning element includes a movable light redirecting element that directs illumination light from the light source through the first optical assembly to generate a swept sheet of light emitted obliquely from the objective lens, which scans the volume of said area. The movable light redirecting element of the scanning / descanning element further directs the image light received from the first optical assembly to maintain a focus of the planar illumination beam at the plane of said first intermediate image as said sheet of light is swept through. The secondary imaging optical component has an optical axis that forms an oblique first angle with respect to an optical axis of the primary imaging optical component and an angle having a normal to the plane of said first intermediate image, whereby the magnitude of the oblique angle is less than an angle that would be required to focus the secondary imaging optical component on said plane of said first intermediate image.

[0206] In the third embodiment or any other embodiment, the secondary imaging optical component includes a tertiary imaging optical component having an optical axis, the tertiary imaging optical component positioned to focus a second intermediate image formed by the secondary imaging optical component onto the imaging light sensor.

[0207] In the third embodiment or any other embodiment, the apparatus includes a tertiary imaging optical component having an optical axis that is located at a second angle between the first angle and the optical axis of the primary imaging optical component.

[0208] In the third embodiment or any other embodiment, the primary imaging optical component includes another component that causes the first intermediate image to have zero magnification.

[0209] In the third or any other embodiment, the primary imaging optical component includes another component having axial aberration characteristics identical to those of the objective lens and arranged such that the axial aberration of the first intermediate image is compensated.

[0210] In the third embodiment or any other embodiment, the secondary imaging optical component includes an imaging light sensor having a sensor array defining a plane, the normal to the plane of the sensor array being at an angle with respect to the optical axis of the secondary imaging optical component.

[0211] In one or more fourth embodiments, an imaging device, such as a microscope, for imaging an area in a target volume includes a primary imaging optical component, a secondary imaging optical component, and an angle correction element. The primary imaging optical component has an objective lens that can be positioned relative to an object support, and the first optical assembly itself has an optical element effective for cooperating with the objective lens to generate a first intermediate image of a planar area in a region in front of said objective lens. The secondary imaging optical component has an optical axis parallel to the optical axis of the primary imaging optical component and has a back focal plane in which an imaging light sensor can be arranged. The angle correction element is provided between the primary imaging optical component and the secondary imaging optical component and focuses the first intermediate image onto the back focal plane of the secondary imaging optical component.

[0212] In the fourth embodiment or any other embodiment, the angle correcting element comprises an optical wedge. In the fourth embodiment or any other embodiment, the angle correcting element comprises a refractive element.

[0213] In the fourth embodiment or any other embodiment, the optical axis of the secondary imaging optical component is not collinear with the optical axis of the primary imaging optical component.

[0214] In the fourth embodiment or any other embodiment, the imaging arrangement further includes a camera sensor positioned at the back focal plane.

[0215] In the fourth or any other embodiment, the imaging apparatus further includes a light source and a scanning / descanning element comprising a movable light redirecting element that directs illumination light from the light source through the primary imaging optical component to generate a swept sheet of light that emerges obliquely from the objective lens and is scanned across the volume of said region by said swept sheet of light. The movable light redirecting element of the scanning / descanning element further directs the image light received from the primary imaging optical component to maintain the focus of the planar illumination beam at said back focal plane as said sheet of light is swept.

[0216] In one or more fifth embodiments, an imaging device, such as a microscope, includes a controllable light redirecting element and a beam splitter. The controllable light redirecting element is positioned to sweep the light beam received from the light source across the object volume to illuminate the volumetric region. The light redirecting element is further positioned and configured to receive light from multiple depths in the object volume illuminated by the light received from the light source and maintain a focus of the received light on the imaging sensor to form an image of the volumetric region. The beam splitter is positioned and configured to separate the light received from the light source from the light received from the multiple depths in the object volume. The light redirecting element has a single planar reflective surface on which both the light received from the light source and the light received from the multiple depths in the object volume are incident.

[0217] In the fifth embodiment or any other embodiment, the beam splitter is a polarizing beam splitter or a dichroic beam splitter.

[0218] In the fifth embodiment or any other embodiment, the imaging device further comprises a beam flattening element forming a sheet of light from the light source, the light being swept across the object volume with a sweep direction having a substantial component perpendicular to a major plane of said sheet of light.

[0219] In the fifth or any other embodiment, the light redirecting element is controlled to form a fixed image of the object volume on the imaging sensor, whereby changes in the characteristics of the object volume are received by the imaging sensor.

[0220] In the fifth or any other embodiment, the microscope includes an element configured to receive light from different depths in the object volume and to compensate for a fixed oblique orientation of the image relative to the optical axis of the primary objective lens.

[0221] In one or more sixth embodiments, an imaging device, e.g., a microscope, includes an objective lens and a light source. The objective lens captures and transmits image light received by the objective lens to a scanning / descanning optical assembly. The scanning / descanning optical assembly directs light from the light source such that a swept sheet of light emerges obliquely from the objective lens. The swept sheet of light scans across a volumetric region. The scanning / descanning optical assembly includes a light sensor. A movable light redirecting element redirects the image light received from the volumetric region to maintain a focal point of the planar illumination beam on the light sensor as the sheet of light sweeps. The focal image light includes light passing through the objective lens on both sides of the optical axis of the objective lens, whereby a central portion of the objective lens aperture is captured and used to form an image of the target.

[0222] In the sixth embodiment or any other embodiment, the focused image light includes light passing through a major portion of the objective lens aperture.

[0223] In the sixth or any other embodiment, the imaging apparatus further includes another lens assembly, the other lens assembly being of identical configuration at least with respect to axial focusing aberration, and the objective lens and the other lens assembly are arranged such that image light received by the objective lens passes through the other lens assembly and a fixed reflector positioned to reflect the image light back through the other lens assembly. In the sixth or any other embodiment, the fixed reflector is oriented at an angle with respect to an optical axis of the other lens assembly, the angle being selected in response to one or more of the angles of the sheet of light obliquely swept from the objective lens.

[0224] In the sixth embodiment or any other embodiment, the scanning / descanning optical assembly includes a movable light redirecting element. In the sixth embodiment or any other embodiment, the movable light redirecting element is moved by an actively controlled light redirecting element of the scanning / descanning optical assembly. In the sixth embodiment or any other embodiment, the reflective element has reflective properties that make the angular change in the direction of the light from the light source and the angular change in the direction of the light received from the volumetric region the same as if it were a single planar reflective surface. In the sixth embodiment or any other embodiment, the reflective element has a single planar reflective surface on which both the light from the light source and the light received from the volumetric region are incident.

[0225] In the sixth embodiment or any other embodiment, the light source generates light selected to cause fluorescence in an object containing a fluorescent dye. In the sixth embodiment or any other embodiment, the light source includes beam flattening optics that flatten a round beam of light into said sheet of light.

[0226] In the sixth embodiment or any other embodiment, the optical sensor includes a camera, a linear array of photomultiplier elements, and / or a single photomultiplier element.

[0227] In the sixth embodiment or any other embodiment, the scanning / descanning optical assembly includes a movable optical element that can be selectively positioned in the path of the light from the light source to change the illumination beam between a sheet of light directed to the edge of the back aperture of the objective lens (which can be referred to as a scan mode) and a wide illumination beam that spreads across the back aperture (which can be referred to as a confocal mode). In the sixth embodiment or any other embodiment, the movable optical element is refractive and expands the width of the light from the light source. In the sixth embodiment or any other embodiment, the movable optical element is refractive and moves the centerline of the light from the light source.

[0228] In the sixth or any other embodiment, the scanning / descanning assembly is configured to scan with a focused spot or line in a volumetric region when the movable optical element is selectively positioned to provide a wide illumination beam (confocal mode) that spreads across the back aperture.

[0229] In the sixth embodiment or any other embodiment, the scanning / descanning assembly is configured to scan with a focused spot or line in a volumetric region in response to confocal mode control commands that selectively position the movable optical element described above.

[0230] In the sixth embodiment or any other embodiment, the scanning / descanning assembly includes an image forming optics having an optical axis for generating an intermediate real image of image light returned from said planar illumination beam in a scan mode, and a detection module for imaging said intermediate real image, and an optical sensor includes a linear or two-dimensional array of pixels onto which an image of said intermediate real image is focused. The detection module has an optical axis that is oblique to the optical axis of said image forming optics, such that a pixel or a selected pixel among said pixels can function as a confocal pinhole or slit in a confocal mode, and such that in a scan mode, the angle between the optical axis of said detection module and the optical axis of the image forming optics is effective for correcting for axial positional changes of a plurality of points along said planar illumination beam imaged by said detection module.

[0231] In one or more seventh embodiments, an imaging device, e.g., a microscope, includes one or more lenses, an optical deflection element, and a scanning / descanning device. The one or more lenses include an objective lens and are arranged to transmit an illumination beam from an illumination source through the objective lens to form a planar illumination beam located in a plane that is oblique with respect to the optical axis of the objective lens. The objective lens has a front end at which an object to be imaged can be positioned and a rear end opposite the front end. The objective lens has an aperture through which an image beam passes from the front end to the rear end adjacent to said illumination beam, the image beam having a cross section that covers a major portion of the aperture and extends through the optical axis of the objective lens. The optical deflection element is positioned along a first path of said illumination beam extending in a direction away from the rear end of the objective lens, and the illumination beam and the image beam are along said first path. The optical deflection element is shaped, sized and positioned relative to the objective lens to deflect the image beam along said first path away from the illumination beam, whereby the image beam follows a second path that travels away from a third path followed by the illumination beam. A scanning / descanning device is positioned to scan and descan the image beam and illumination beam, respectively, such that the planar illumination beam sweeps a volume near the front end of said objective lens, and the focal point of the planar illumination beam is maintained on the detector at all times during the sweep.

[0232] In the seventh embodiment or any other embodiment, the scanning / descanning device includes a motor for pivoting an optical deflection element or an acousto-optic deflector.

[0233] In the seventh embodiment or any other embodiment, the light deflecting element has a V-shaped reflective surface.

[0234] In one or more eighth embodiment, a multimode microscope includes a microscope objective, an illumination component, a receiving component, a scanning / descanning element, and a light detection module. The microscope objective has an aperture and an optical axis. The illumination component is configured to selectively generate a first beam and a second beam, the first beam having a width that spans a majority of the aperture of the objective and being centered on or offset with respect to said optical axis, and the second beam being restricted to a smaller area of ​​the aperture and to one side of the aperture of the objective than the first beam. The receiving component is positioned to receive an image beam that constitutes the light received through said aperture, propagating in a direction opposite to the direction of propagation of the illumination beam, the image beam spanning a majority of said aperture. The scanning / descanning element includes a movable light redirector in the path of the first beam, the second beam, and the image beam, the movable light redirector positioned to scan the illumination beam and descan each of the first and second beams and the image beam. The light detection module is positioned to receive light from the light redirector, and the light detection module has an imaging element, the imaging element having a plurality of pixels extending along one or two axes. The light detection module has optics adapted to form and rotate an image from the image beam and map the image onto the imaging element, the image including development of light returned from the target at a plurality of depths along the optical axis of the objective in a volumetric imaging mode.

[0235] In the eighth embodiment or any other embodiment, the image includes a two-dimensional image of a plane in the volume imaged by the objective lens, the plane being oblique relative to the optical axis of the objective lens. In the eighth embodiment or any other embodiment, the image includes a two-dimensional image of a line in the volume imaged by the objective lens, the line being oblique relative to the optical axis of the objective lens.

[0236] In the eighth embodiment or any other embodiment, the movable light redirector is adapted to be pivoted about two orthogonal axes to scan and descan the illumination beam and the image beam over a stereoscopic angular range. In the eighth embodiment or any other embodiment, the movable light redirector is adapted to be pivoted to scan and descan the illumination beam and the image beam over a predetermined angular range.

[0237] In one or more ninth embodiments, a method of using the microscope of any of the eighth embodiments includes configuring an illumination component to generate a first beam, and using light from a selected subset of pixels from the plurality of pixels to discriminate light in the image beam arising from a single point during confocal mode.

[0238] In one or more tenth embodiments, a method of using the microscope of any of the eighth embodiments includes configuring an illumination component to generate a first beam, and using light from a single pixel selected from the plurality of pixels to discriminate light in the image beam arising from a single point during confocal mode.

[0239] In one or more eleventh embodiments, a method of using the microscope of any of the eighth embodiments includes configuring an illumination component to generate a second beam and using light from all pixels of the plurality of pixels to generate an image of a volume of an object.

[0240] In one or more twelfth embodiments, a method of using the microscope of any of the eighth embodiments includes using light from a selected subset of pixels from the plurality of pixels to discriminate light in the image beam arising from a single point during confocal mode.

[0241] In one or more thirteenth embodiments, a method of using the microscope of any of the eighth embodiments includes using light from a single pixel selected from the plurality of pixels to discriminate light in the image beam arising from a single point during confocal mode.

[0242] In one or more fourteenth embodiments, a method of using the microscope of any of the eighth embodiments includes using light from all pixels of the plurality of pixels to generate an image of a volume of an object.

[0243] In one or more fifteenth embodiments, a multimode microscope includes a microscope objective, an illumination component, a receiving component, a scanning / descanning element, and a light detection module. The microscope objective has an aperture and an optical axis. The illumination component is configured to selectively generate a first beam and a second beam, the first beam having a width that spans the aperture of the objective and being centered on said optical axis, and the second beam being restricted to a smaller area of ​​the aperture on one side of the aperture of the objective. The receiving component is positioned to receive an image beam that constitutes the light received through said aperture, propagating in a direction opposite to the propagation direction of the illumination beam, the image beam spanning a majority of said aperture. The scanning / descanning element includes a movable light redirector in the path of the first beam, the second beam, and the image beam, the movable light redirector being positioned to scan the illumination beam and descan each of said first beam, the second beam, and said image beam. The light detection module is positioned to receive light from the light redirector, and the light detection module has an imaging element having a plurality of pixels extending along one or two axes, and the light detection module has optics adapted to form and rotate an image from said image beam and map the image onto said imaging element, the image including a development of light returned from a target at a plurality of depths along the optical axis of the objective in a volumetric imaging mode.

[0244] In the fifteenth embodiment or any other embodiment, the image includes a two-dimensional image of a plane in the volume imaged by the objective lens, the plane being oblique relative to the optical axis of the objective lens. In the fifteenth embodiment or any other embodiment, the image includes a two-dimensional image of a line in the volume imaged by the objective lens, the line being oblique relative to the optical axis of the objective lens.

[0245] In the fifteenth embodiment or any other embodiment, the movable light redirector is adapted to be pivoted about two orthogonal axes to scan and descan the illumination beam and the image beam over a stereoscopic angular range. In the fifteenth embodiment or any other embodiment, the movable light redirector is adapted to be pivoted to scan and descan the illumination beam and the image beam over a predetermined angular range.

[0246] In one or more sixteenth embodiments, a method of using the microscope of any of the fifteenth embodiments includes configuring an illumination component to generate a first beam, and using light from a selected subset of pixels from the plurality of pixels to discriminate light in said image beam arising from a single point during confocal mode.

[0247] In one or more seventeenth embodiments, a method of using the microscope of any of the fifteenth embodiments includes configuring an illumination component to generate a first beam, and using light from a single pixel selected from the plurality of pixels to discriminate light in the image beam arising from a single point during a confocal mode.

[0248] In one or more eighteenth embodiments, a method of using the microscope of any of the fifteenth embodiments includes configuring an illumination component to generate a second beam and using light from all pixels of the plurality of pixels to generate an image of a volume of an object.

[0249] In one or more nineteenth embodiments, a method of using the microscope of any of the fifteenth embodiments includes using light from a selected subset of pixels from the plurality of pixels to discriminate light in the image beam arising from a single point during confocal mode.

[0250] In one or more twentieth embodiments, a method of using the microscope of any of the fifteenth embodiments includes using light from a single pixel selected from the plurality of pixels to discriminate light in the image beam arising from a single point during confocal mode.

[0251] In one or more twenty-first embodiments, a method of using the microscope of any of the fifteenth embodiments includes using light from all pixels of the plurality of pixels to generate an image of a volume of an object.

[0252] In one or more twenty-second embodiments, an imaging device, for example a microscope, includes an objective lens, a further optical element, and a detection module. The objective lens is arranged to receive light from the volumetric region at a front end of the objective lens. The further optical element is arranged to receive image light from the objective lens and form a first image of a planar region in the volumetric region, the plane of the first image and the plane of the planar region being oblique relative to the optical axis of the objective lens. The detection module has a first detection optics with an optical axis positioned at a first angle relative to the optical axis of the further optical element at the point where the first image is formed, the first angle being selected to be smaller than a second angle of the first image relative to the optical axis of the further optical element, whereby the further optical element is focused on a plane forming an angle relative to said plane of the first image. The detection module includes a photodetector positioned to receive image light from said first image and form an unfocused image of the first image.

[0253] In the twenty-second embodiment or any other embodiment, the further optical element comprises an optical element substantially identical to the objective lens described above.

[0254] In the twenty-second embodiment or any other embodiment, the imaging arrangement further includes a scanning / descanning element adapted to vary a position of the planar illumination beam passing through the planar area, thereby moving the planar area. In the twenty-second embodiment or any other embodiment, the imaging arrangement further includes a scanning / descanning element adapted to vary a position of the planar area by redirecting the image light received from the objective lens.

[0255] In one or more twenty-third embodiments, an imaging device, for example a microscope, includes an objective lens, a further optical element, and a detection module. The objective lens is arranged to receive light from the volumetric region at a front end of the objective lens. The further optical element is arranged to receive the image light from the objective lens and form a first image of a planar region in the volumetric region, the plane of the first image and the plane of the planar region being oblique relative to the optical axis of the objective lens. The detection module has a first detection optics with an optical axis positioned at a first angle relative to the optical axis of the further optical element at the point where the first image is formed, the first angle being selected to be smaller than a second angle of the first image relative to the optical axis of the further optical element, whereby the further optical element is focused on a plane that forms an angle relative to said plane of the first image and forms the second image. The detection module includes a second detection optics focused on said second image and a photodetector positioned to have a detection surface coincident with the second image.

[0256] In the twenty-third embodiment or any other embodiment, the further optical element comprises an optical element substantially identical to the objective lens described above.

[0257] In the twenty-third embodiment or any other embodiment, the imaging arrangement further includes a scanning / descanning element adapted to vary a position of the planar illumination beam passing through the planar area, thereby moving the planar area. In the twenty-third embodiment or any other embodiment, the imaging arrangement further includes a scanning / descanning element adapted to vary a position of the planar area by redirecting the image light received from the objective lens.

[0258] In one or more twenty-fourth embodiments, an imaging device, e.g., a microscope, includes an objective lens, another optical element, a detection module, and an optical prism. The objective lens is arranged to receive light from the volumetric region at a front end of the objective lens. The other optical element is arranged to receive image light from the objective lens and form a first image of a planar region in the volumetric region, the plane of the first image and the plane of the planar region being oblique relative to the optical axis of the objective lens. The detection module has a first detection optics with an optical axis positioned parallel to and offset relative to the optical axis of the other optical element at a point where the first image will be formed by the other optical element. The optical prism is positioned between the other optical element and the detection module to rotate the first image orthogonal to the optical axis of the first detection optics. The detection module includes a photodetector positioned to receive image light from said first image, and the first detection optics is adapted to form a final image of the first image on the photodetector.

[0259] In the twenty-fourth embodiment or any other embodiment, the further optical element comprises an optical element substantially identical to the objective lens described above.

[0260] In the twenty-fourth embodiment or any other embodiment, the imaging arrangement further includes a scanning / descanning element adapted to vary a position of the planar illumination beam passing through the planar area, thereby moving the planar area. In the twenty-fourth embodiment or any other embodiment, the imaging arrangement further includes a scanning / descanning element adapted to vary a position of the planar area by redirecting the image light received from the objective lens.

[0261] In one or more twenty-fifth embodiments, a multimode microscope includes a microscope objective, an illumination component, a receiving component, a scanning / descanning element, and a light detection module. The microscope objective has an aperture and an optical axis. The illumination component is configured to selectively generate a first beam and a second beam, the first beam being confined to one side of said optical axis and the second beam being confined to the other side of said optical axis. The receiving component is positioned to receive an image beam that constitutes the light received through said aperture and propagates in a direction opposite to the direction of propagation of the illumination beam, the image beam extending across a majority of said aperture. The scanning / descanning element includes a movable light redirector in the path of the first beam, the second beam, and the image beam, the movable light redirector being positioned to scan the illumination beam and descan each of said first beam, the second beam, and said image beam. The light detection module is positioned to receive light from the light redirector, and the light detection module has an imaging element having a plurality of pixels extending along one or two axes, and the light detection module has optics adapted to form and rotate an image from said image beam and map the image onto said imaging element, the image including a development of light returned from a target at a plurality of depths along the optical axis of the objective in a volumetric imaging mode.

[0262] In the twenty-fifth embodiment or any other embodiment, the image includes a two-dimensional image of a plane in the volume imaged by the objective lens, the plane being oblique relative to the optical axis of the objective lens. In the twenty-fifth embodiment or any other embodiment, the image includes a two-dimensional image of a line in the volume imaged by the objective lens, the line being oblique relative to the optical axis of the objective lens.

[0263] In the twenty-fifth embodiment or any other embodiment, the movable light redirector is adapted to be pivoted about two orthogonal axes to scan and descan the illumination beam and the image beam over a stereoscopic angular range. In the twenty-fifth embodiment or any other embodiment, the movable light redirector is adapted to be pivoted to scan and descan the illumination beam and the image beam over a predetermined angular range.

[0264] In one or more twenty-sixth embodiments, a method of using the microscope of any of the twenty-fifth embodiments includes configuring an illumination component to generate a first beam, and using light from a selected subset of pixels from the plurality of pixels to discriminate light in said image beam arising from a single point during confocal mode.

[0265] In one or more twenty-seventh embodiments, a method of using the microscope of any of the twenty-fifth embodiments includes configuring an illumination component to generate a first beam, and using light from a single pixel selected from the plurality of pixels to discriminate light in the image beam arising from a single point during a confocal mode.

[0266] In one or more twenty-eighth embodiments, a method of using the microscope of any of the twenty-fifth embodiments includes configuring an illumination component to generate a second beam and using light from all pixels of the plurality of pixels to generate an image of a volume of an object.

[0267] In one or more twenty-ninth embodiments, a method of using the microscope of any of the twenty-fifth embodiments includes using light from a selected subset of pixels from the plurality of pixels to discriminate light in the image beam arising from a single point during a confocal mode.

[0268] In one or more thirtieth embodiments, a method of using the microscope of any of the twenty-fifth embodiments includes using light from a single pixel selected from the plurality of pixels to discriminate light in the image beam arising from a single point during a confocal mode.

[0269] In one or more thirty-first embodiments, a method of using the microscope of any of the twenty-fifth embodiments includes using light from all pixels of the plurality of pixels to generate an image of a volume of an object.

[0270] In one or more thirty-second embodiments, an imaging device, e.g., a microscope, includes one or more lenses, an optical deflection element, and an adjustment mechanism. The one or more lenses, including an objective lens, are arranged to transmit an illumination beam from an illumination source through the objective lens to form a planar illumination beam located in a plane that is oblique with respect to an optical axis of the objective lens. The objective lens has a front end at which an object to be imaged can be positioned and a rear end opposite the front end. The objective lens has an aperture through which an image beam passes from the front end to the rear end adjacent to the illumination beam. The optical deflection element is positioned along a first path of the illumination beam extending away from the rear end of the objective lens, and the illumination beam and the image beam are adjacent along the first path. The optical deflection element is shaped, sized and positioned relative to the objective lens to deflect the image beam along said first path away from the illumination beam, whereby the image beam follows a second path that travels away from a third path followed by the illumination beam. The scanning / descanning device is positioned to scan and descan the image beam and the illumination beam, respectively, such that the planar illumination beam sweeps a volume near the front end of said objective lens and the focus of the planar illumination beam is maintained on the detector, whereby a plurality of planar illumination beam planes are defined by the sweep, the planes being either infinitely continuous instances or discrete instances. An adjustment mechanism can adjust the spacing of one or more lenses to adjust the proximal spacing and angle between adjacent instances of said planes.

[0271] In one or more thirty-third embodiments, an imaging device, such as a microscope, includes a light redirecting element and a beam splitter. The light redirecting element is positioned to sweep the light beam received from the light source across the object volume to illuminate a line along the axial extent of the object volume. The light redirecting element is further positioned and configured to receive light from multiple depths in the object volume illuminated by the light received from the light source and maintain a focus of the received light on the imaging sensor to form an image of the volumetric region. The beam splitter is positioned and configured to separate the light received from the light source from the light received from the multiple depths in the object volume. The light redirecting element has one or more planar reflective surfaces on which both the light received from the light source and the light received from the multiple depths in the object volume are incident.

[0272] In the thirty-third embodiment or any other embodiment, the beam splitter is a polarizing beam splitter or a dichroic beam splitter.

[0273] In the thirty-third embodiment or any other embodiment, the imaging device further includes a beam flattening element forming a sheet of light consisting of light originating from the light source, the light used to sweep across the object volume being swept with a sweep direction having a substantial component perpendicular to a major plane of said sheet of light.

[0274] In the thirty-third embodiment or any other embodiment, the light redirecting element is controlled to form a fixed image of the object volume on the imaging sensor, whereby changes in the characteristics of the object volume are received by the imaging sensor.

[0275] In the thirty-third embodiment or any other embodiment, the microscope includes an element configured to receive light from different depths in the object volume and to compensate for a fixed oblique orientation of the image relative to the optical axis of the primary objective lens.

[0276] In one or more thirty-fourth embodiments, a method for imaging an object includes scanning an illumination beam and de-scanning an image beam, respectively, by redirecting an illumination beam from a light source using a light redirector to provide light to an object volume, and by redirecting an image beam containing light from different depths in the object volume to the light redirector to provide said light to a light detector that can detect individual measurements of light from different locations along a first direction, using a beam splitter to separate the illumination beam provided to the object volume from the image beam, and controlling the light redirector during said scanning and de-scanning to provide a fixed image of the illuminated area of ​​the object volume to the light detector.

[0277] In the thirty-fourth embodiment or any other embodiment, the illumination beam comprises a planar beam that illuminates a plane at the object.

[0278] In the thirty-fourth embodiment or any other embodiment, the method includes refracting light from a light source to be provided to the object volume, or refracting light from different depths in the object volume to be provided to a light redirector.

[0279] In the thirty-fourth embodiment or any other embodiment, the method includes refracting light received from a light source to illuminate a plane in the object volume.

[0280] In a thirty-fourth embodiment or any other embodiment, the method includes filtering light received from different depths in the object volume and providing fixed filtered images to the light detector during the scan.

[0281] In a thirty-fourth embodiment or any other embodiment, the method includes converting the scan data detected by the optical detector into two-dimensional (2D) or three-dimensional (3D) image data at appropriate image pixel locations corresponding to a plurality of locations in the object volume.

[0282] In the thirty-fourth embodiment or any other embodiment, the method includes compensating light received from different depths in the object volume for a fixed image orientation.

[0283] In a thirty-fourth embodiment or any other embodiment, the method includes varying a beam width of light from different depths in the object volume using an aperture.

[0284] In one or more thirty-fifth embodiments, a method for imaging includes passing an illumination beam through an objective lens toward an object and through a plurality of depths along an optical axis of the objective lens, receiving image light returned from the object through the objective lens, the image light including light returned from the plurality of depths, controlling the illumination beam to move the illumination beam laterally of the optical axis of the objective lens through a plurality of locations, and controlling the image light returned through the objective lens to generate a fixed image of the light returned from the plurality of depths at each portion of the photodetector as the illumination beam is moved. The controlling is such that the illumination beam and the light returned through the objective lens follow adjacent paths propagating through a common aperture. The method further includes separating the image light from the illumination beam and the image light at the common aperture.

[0285] In the thirty-fifth embodiment or any other embodiment, the separation is performed using a beam splitter. In the thirty-fifth embodiment or any other embodiment, the beam splitter is a dichroic beam splitter, and the image light and the illumination beam have different wavelength characteristics.

[0286] In the thirty-fifth embodiment or any other embodiment, controlling the light returning through the objective lens includes passing the image light through an optical assembly in a first direction and reflecting the image light off a fixed mirror to direct the image light back through the same optical assembly in an opposite direction. In the thirty-fifth embodiment or any other embodiment, the optical assembly includes focusing optics. In the thirty-fifth embodiment or any other embodiment, the optical assembly is effective to form an intermediate real image of a feature in the object illuminated by the illumination beam. In the thirty-fifth embodiment or any other embodiment, the optical assembly is identical to the objective lens described above.

[0287] In the thirty-fifth embodiment or any other embodiment, controlling the image light returned through the objective lens is effective to generate a fixed image of light returned from multiple depths at each position of the photodetector at each instant of movement of the illumination beam.

[0288] In the 35th embodiment or any other embodiment, controlling the illumination beam includes directing an illumination beam from a light source to a movable mirror. In the 35th embodiment or any other embodiment, controlling the image light includes directing the image light to the same movable mirror. In the 35th embodiment or any other embodiment, the illumination beam and the image light pass through a common aperture toward the same movable mirror. In the 35th embodiment or any other embodiment, the same movable mirror has a single continuous planar reflective surface that redirects both the illumination beam and the image light. In the 35th embodiment or any other embodiment, the fixed mirror is oriented at an angle with respect to an optical axis of the optical assembly.

[0289] In a thirty-fifth embodiment or any other embodiment, the wavelength characteristics of the illumination beam are selected to cause fluorescence of a predefined dye, and the method further includes incorporating the predefined dye into a composition of the object.

[0290] In the thirty-fifth embodiment or any other embodiment, controlling the illumination beam comprises actuating the movable mirror by a motor. In the thirty-fifth embodiment or any other embodiment, controlling the illumination beam comprises actuating the movable mirror by a galvo motor. In the thirty-fifth embodiment or any other embodiment, controlling the illumination beam comprises actuating the movable mirror by a motor controlled by a processor that performs a predefined scan sequence.

[0291] In the thirty-fifth embodiment or any other embodiment, controlling the illumination beam includes directing the illumination beam from the light source to a processor-controlled light redirector. In the thirty-fifth embodiment or any other embodiment, the light redirector includes one of a motor-actuated reflective element, a motor-actuated refractive element, a digital micromirror device (DMD), a microelectromechanical system (MEMS) light guide, a mirror, or other suitable MEMS device, a fiber optic bundle, an acousto-optic deflector (AOD), a spatial light modulator (SLM), or other technology, such as a MEMS, DMD, or SLM device that can be used as a light director that can be controlled to provide a specific spatial relationship between the first light redirector and the illumination light and the image light. In the thirty-fifth embodiment or any other embodiment, the light redirecting element includes a reflective element with reflective properties that cause an angular change in the direction of the illumination beam from the light source to be the same as an angular change in the direction of the image light received from the object. In the thirty-fifth embodiment or any other embodiment, the light redirector causes the same angular change in both the incident image light and the illumination beam.

[0292] In the thirty-fifth embodiment or any other embodiment, the light detector includes a camera, a CCD sensor, and / or a linear photomultiplier array.

[0293] In the thirty-fifth embodiment or any other embodiment, the method further includes adjusting the illumination beam such that the illumination beam has a flat cross-section.

[0294] In the thirty-fifth embodiment, or any other embodiment, controlling the illumination beam is effective to move the illumination beam in a non-continuous pattern. In the thirty-fifth embodiment, or any other embodiment, controlling the illumination beam is effective to move the illumination beam in a pattern that includes a non-linear portion.

[0295] In the thirty-fifth embodiment or any other embodiment, the method further includes varying a width of the illumination beam and processing image data received from the photodetector to distinguish light returned from a focal point and a focal line at the object. In the thirty-fifth embodiment or any other embodiment, the processing includes deconvolving the image data to remove blur in the image. In the thirty-fifth embodiment or any other embodiment, the processing includes selecting data from predefined pixels and making the photodetector act like a slit diaphragm or pinhole. In the thirty-fifth embodiment or any other embodiment, the illumination beam is controlled to move the focal point or focal line in a plane at a single axial distance from the objective lens as the width is varied. In the thirty-fifth embodiment or any other embodiment, the change is in response to a command to change from a depth-resolved imaging mode to a confocal mode.

[0296] In one or more thirty-sixth embodiments, a method for imaging includes passing an illumination beam through an objective lens toward an object, passing an illumination beam through a plurality of depths along an optical axis of the objective lens, receiving image light returned from the object through the objective lens including light returned from the plurality of depths, controlling the illumination beam to move the illumination beam laterally of the optical axis of the objective lens through a plurality of locations, and controlling the image light returned through the objective lens to generate a fixed image of the light returned from the plurality of depths at each portion of a photodetector as the illumination beam is moved. Controlling the image light includes passing the image light through focusing optics to generate an intermediate image and focusing optics that are not in the plane of the intermediate image but are in a plane that intersects the plane of the intermediate image, and generating the fixed image from the intermediate image at the photodetector using the focusing optics.

[0297] In the thirty-sixth embodiment or any other embodiment, the first control step and the second control step are to cause the illumination beam and the light returning through the objective lens to follow adjacent paths propagating through a common aperture.

[0298] In the thirty-sixth embodiment or any other embodiment, the method further comprises separating the image light from the illumination beam and the image light at the common aperture. In the thirty-sixth embodiment or any other embodiment, the separation is performed using a beam splitter. In the thirty-sixth embodiment or any other embodiment, the beam splitter is a dichroic beam splitter, and the image light and the illumination beam have different wavelength characteristics.

[0299] In the 36th embodiment or any other embodiment, controlling the illumination beam includes directing an illumination beam from a light source to a movable mirror. In the 36th embodiment or any other embodiment, controlling the image light includes directing the image light to the same movable mirror. In the 36th embodiment or any other embodiment, the illumination beam and the image light pass through a common aperture toward the same movable mirror. In the 36th embodiment or any other embodiment, the same movable mirror has a single continuous planar reflective surface that redirects both the illumination beam and the image light.

[0300] In a thirty-sixth embodiment or any other embodiment, the wavelength characteristics of the illumination beam are selected to cause fluorescence of a predefined dye, and the method further includes incorporating the predefined dye into a composition of the object.

[0301] In the thirty-sixth embodiment or any other embodiment, controlling the illumination beam comprises actuating the movable mirror by a motor. In the thirty-sixth embodiment or any other embodiment, controlling the illumination beam comprises actuating the movable mirror by a galvo motor. In the thirty-sixth embodiment or any other embodiment, controlling the illumination beam comprises actuating the movable mirror by a motor controlled by a processor that performs a predefined scan sequence.

[0302] In the thirty-sixth embodiment or any other embodiment, controlling the illumination beam includes directing the illumination beam from the light source to a processor-controlled light redirector. In the thirty-sixth embodiment or any other embodiment, the light redirector includes one of a motor-actuated reflective element, a motor-actuated refractive element, a digital micromirror device (DMD), a microelectromechanical system (MEMS) light guide, a mirror, or other suitable MEMS device, a fiber optic bundle, an acousto-optic deflector (AOD), a spatial light modulator (SLM), or other technology, such as a MEMS, DMD, or SLM device that can be used as a light director that can be controlled to provide a particular spatial relationship between the first light redirector and the illumination light and the image light.

[0303] In the thirty-sixth embodiment or any other embodiment, the light redirector includes a reflective element with reflective properties that cause the angular change in direction of the illumination beam from the light source to be the same as the angular change in direction of the image light received from the object. In the thirty-sixth embodiment or any other embodiment, the light redirector causes the same angular change in both the incoming image light and the illumination beam.

[0304] In the thirty-sixth embodiment or any other embodiment, the light detector includes a camera, a CCD sensor, and / or a linear photomultiplier array.

[0305] In one or more thirty-seventh embodiments, a method for imaging includes passing an illumination beam through an objective lens toward an object, passing through a plurality of depths along an optical axis of the objective lens, receiving image light returned from the object through the objective lens, the image light including light returned from the plurality of depths, controlling the illumination beam to move the illumination beam laterally of the optical axis of the objective lens through a plurality of locations, and controlling the image light returned through the objective lens to generate a fixed image of the light returned from the plurality of depths at each portion of the photodetector as the illumination beam is moved. Controlling the image light includes passing the image light through focusing optics configured to generate an intermediate image of the light returned from the plurality of depths. The method may further include receiving the light at the focusing optics and rotating a beam of light in the received light, the focusing optics forming the intermediate image when no light is received.

[0306] In the thirty-seventh embodiment or any other embodiment, rotating the beam includes passing the light beam through an optical wedge.

[0307] In one or more thirty-eighth embodiments, a method for forming an image of an object includes projecting light onto the object through a first lens having a predetermined magnification along a predetermined path such that the light illuminates an area having a major dimension that forms a first angle relative to an optical axis of the first optical assembly; capturing image light from the area of ​​the object through the first optical assembly and relaying the image light through a back aperture of the first optical assembly to an aperture of a second optical assembly having a second magnification greater than the magnification of the first optical assembly to focus the captured image light; capturing light from the second optical assembly using a third optical assembly positioned such that a point where the captured image light is focused by the second optical assembly is located between the second optical assembly and the third optical assembly and forming an image of the illuminated area from the captured light. As a result, the angle formed by the intermediate image relayed between the second optical assembly and the third optical assembly relative to the optical axis of the second optical assembly is reduced compared to the angle of the same image if the magnification of the second optical assembly was the same as the magnification of the first optical assembly.

[0308] In the 38th embodiment or any other embodiment, the second optical assembly and the third optical assembly are the same optical assembly. In the 38th embodiment or any other embodiment, using the third optical assembly includes reflecting the image light from a mirror back to the second optical assembly, whereby the second optical assembly operates as the third optical assembly. In the 38th embodiment or any other embodiment, the second optical assembly and the third optical assembly are physically different devices and have different magnifications. In the 38th embodiment or any other embodiment, an angle is formed between the optical axis of the second optical assembly and the optical axis of the third optical assembly. In the 38th embodiment or any other embodiment, capturing the light from the second optical assembly includes passing the light through a prism, whereby light from different depths in the object has different path lengths in said prism. In the 38th embodiment or any other embodiment, the combination of the larger magnification and the multiple path lengths of the prism results in a rotation of the intermediate image between the second optical assembly and the third optical assembly.

[0309] In one or more thirty-ninth embodiments, a method for forming an image of an object includes projecting light through a first lens along a predetermined path onto the object such that the light illuminates an area having a major dimension that forms a first angle relative to an optical axis of a first optical assembly; capturing image light from the area of ​​the object through the first optical assembly and relaying the image light through a back aperture of the first optical assembly and a polarizing beam splitter to an aperture of a second optical assembly to focus the captured image light; reflecting the image light back to the second optical assembly and the polarizing beam splitter so that the image light is reflected to an imaging device; relaying the image light through the back aperture of the first optical assembly by reflecting the image light from the polarizing beam splitter to an aperture of a third optical assembly to focus the captured image light; and reflecting the image light through the third optical assembly and the polarizing beam splitter to the imaging device. As a result, the imaging light that is first transmitted through and first reflected from said polarizing beam splitter is directed towards said imaging device.

[0310] In the thirty-ninth embodiment or any other embodiment, the optical properties of the second optical assembly and the optical properties of the third optical assembly are identical.

[0311] In one or more fortieth embodiment, a method for forming an image of an object includes projecting light through a first lens along a predetermined path onto the object such that at a first time, the light illuminates a first area having a major dimension that forms a first angle relative to an optical axis of a first optical assembly; projecting light through the first lens along a predetermined path onto the object such that at a second time, the light illuminates a second area having a major dimension that forms a second angle relative to the optical axis of the first optical assembly; capturing image light from said first area of ​​the object through the first optical assembly; and focusing said captured image light through a back aperture and a polarizing beam splitter of the first optical assembly onto an aperture of a second optical assembly to focus said captured image light. relaying the image light from the second region of the object through the back aperture of the first optical assembly by reflecting the image light from the polarizing beam splitter to an aperture of a third optical assembly to focus the captured image light; and reflecting the image light from the second region through the third optical assembly and polarizing beam splitter to an imaging device, whereby imaging light that first passed through and was first reflected from the polarizing beam splitter is directed to the imaging device.

[0312] In the 40th embodiment or any other embodiment, the first angle and the second angle are equal and directed in opposite directions. In the 40th embodiment or any other embodiment, the first region and the second region intersect. The 40th embodiment or any other embodiment may include splitting the image light to form an image from the image light from the first region and the second region at each portion of the light detection array. In the 40th embodiment or any other embodiment, the optical characteristics of the second optical assembly and the optical characteristics of the third optical assembly are identical.

[0313] In one or more forty-first embodiments, a method of imaging an object includes, at each instant in time, projecting light as a sheet at multiple depths in the object, tilting the sheet of light at multiple angles in the plane of the sheet so that the same feature in the object is shadowed in different directions, capturing image light from the multiple depths, forming multiple images from the captured image light, each image corresponding to a different one of the multiple angles and each image having a portion corresponding to a depth, and combining the multiple images to form a single image.

[0314] In a forty-first embodiment or any other embodiment, the combination includes compensating for a shadowed area in one image using an area in another image. In a forty-first embodiment or any other embodiment, the projecting includes forming a light sheet that forms an angle relative to an optical axis of an objective lens, and the capturing includes passing light received from the object through the same objective lens, and the method further includes repeating the projecting, tilting and capturing steps for multiple positions or angles of the light sheet relative to the optical axis, thereby scanning with the light sheet, and the forming of the multiple images includes descanning the image light, thereby fixing the multiple images between the repetitions.

[0315] In one or more forty-second embodiments, an optical imaging device includes a beam splitter, a first optical scanning element, a second optical scanning element, an objective lens, an illumination source, and a photodetector. The illumination source transmits illumination light to the objective lens via a first optical path including the beam splitter and the first optical scanning element. The beam splitter and the first optical scanning element redirect the illumination light to a peripheral region of the objective lens, whereby the illumination light passes through the objective lens and forms an oblique imaging plane at the tissue. The position of the oblique imaging plane at the tissue varies depending on the orientation of the first optical scanning element. The objective lens receives light returning from the oblique imaging plane and transmits the returning light to a second optical path including the beam splitter and the second optical scanning element, and the beam splitter and the second optical scanning element direct the returning light along the second optical path to form a fixed oblique intermediate image plane. A photodetector captures an image of a fixed tilted intermediate image plane.

[0316] In the forty-second embodiment or any other embodiment, a single reflective surface is used as both the first and second optical scanning element. In some of those embodiments, the illumination light reaches a beam splitter before reaching the single reflective surface, and the returned light reaches the single reflective surface before reaching the beam splitter. In some of those embodiments, the apparatus further includes a telescope disposed in both the first optical path and the second optical path. The illumination light reaches the single reflective surface before reaching the telescope, and the illumination light reaches the telescope before reaching the objective. The returned light reaches the objective before reaching the telescope, and the returned light reaches the telescope before reaching the single reflective surface.

[0317] In the forty-second embodiment or any other embodiment, the first light scanning element and the second light scanning element use different reflective surfaces that move synchronously with each other. In some of those embodiments, the illumination light reaches the first light scanning element before reaching the beam splitter, and the returned light reaches the beam splitter before reaching the second light scanning element. In some of those embodiments, the device further includes an illumination telescope disposed in the first light path and a return light telescope disposed in the second light path. The illumination light reaches the first light scanning element before reaching the illumination telescope, and the illumination light reaches the illumination telescope before reaching the objective lens. The returned light reaches the objective lens before reaching the return light telescope, and the returned light reaches the return light telescope before reaching the second light scanning element. In some of those embodiments, the first light scanning element and the second light scanning element are mechanically independent of each other.

[0318] In the 42nd embodiment or any other embodiment, the photodetector is positioned directly at the fixed tilted intermediate image plane. In the 42nd embodiment or any other embodiment, the photodetector is positioned away from the fixed tilted intermediate image plane and the optical imaging arrangement further includes at least one telescope that directs light from the fixed tilted intermediate image plane to the photodetector. In the 42nd embodiment or any other embodiment, the photodetector is positioned away from the fixed tilted intermediate image plane and the optical imaging arrangement further includes a fiber optic bundle that directs light from the fixed tilted intermediate image plane to the photodetector.

[0319] In the 42nd embodiment or any other embodiment, the illumination source includes a laser generating a pencil beam and an auxiliary optical scanning element that expands the pencil beam into a sheet of light, and the photodetector includes a linear image sensor. In the 42nd embodiment or any other embodiment, the illumination source includes a laser generating a pencil beam and a cylindrical lens that expands the pencil beam into a sheet of light, and the photodetector includes a 2D image sensor.

[0320] In the forty-second embodiment or any other embodiment, the returned light includes fluorescence emitted from tissue in the oblique imaging plane in response to illumination of the tissue in the oblique imaging plane by the illumination light.

[0321] In one or more forty-third embodiments, the optical imaging device includes a beam splitter, an optical scanning element, an objective lens, an illumination source, and an optical detector. The illumination source transmits illumination light to the objective lens via a first optical path including the beam splitter and the optical scanning element. The beam splitter and the optical scanning element redirect the illumination light to a peripheral region of the objective lens, whereby the illumination light passes through the objective lens and forms an oblique imaging plane at the tissue, the position of the oblique imaging plane at the tissue varying depending on the orientation of the optical scanning element. The objective lens receives light returning from the oblique imaging plane and sends it to a second optical path including the beam splitter and the optical scanning element, and the beam splitter and the optical scanning element direct the returning light along the second optical path to form a fixed, oblique intermediate image plane. The optical detector captures an image of the fixed, oblique intermediate image plane.

[0322] In the forty-third embodiment or any other embodiment, the light scanning element has only a single planar reflective surface.

[0323] In the 43rd embodiment or any other embodiment, the illumination light reaches the beam splitter before reaching the optical scanning element, and the returned light reaches the optical scanning element before reaching the beam splitter. In some of those embodiments, the apparatus further includes a telescope disposed in both the first optical path and the second optical path, where the illumination light reaches the optical scanning element before reaching the telescope, the illumination light reaches the telescope before reaching the objective lens, the returned light reaches the objective lens before reaching the telescope, and the returned light reaches the telescope before reaching the optical scanning element.

[0324] In one or more forty-fourth embodiments, a method for optical imaging includes sending illumination light to an objective lens via a first optical path including a beam splitter and a first optical scanning element. The illumination light is redirected to a peripheral region of the objective lens, whereby the illumination light passes through the objective lens and forms an oblique imaging plane at the tissue, the position of the oblique imaging plane at the tissue varying depending on the orientation of the first optical scanning element. The returning light travels from the oblique imaging plane along a second optical path including a beam splitter and a second optical scanning element to form a fixed oblique intermediate image plane. An image of the fixed oblique intermediate image plane is captured.

[0325] In the 44th embodiment or any other embodiment, a single reflective surface is used as both the first light scanning element and the second light scanning element, in some of these embodiments, the illumination light reaches a beam splitter before reaching the single reflective surface, and the returning light reaches the single reflective surface before reaching the beam splitter.

[0326] In the 44th embodiment or any other embodiment, the first light scanning element and the second light scanning element use different reflective surfaces that move synchronously with each other. In some of these embodiments, the illumination light reaches the first light scanning element before reaching the beam splitter, and the returning light reaches the beam splitter before reaching the second light scanning element. In some of these embodiments, the first light scanning element and the second light scanning element are mechanically independent of each other.

[0327] In one or more forty-fifth embodiments, an imaging device includes an objective lens and another optical assembly, a light source, a scanning / descanning optical assembly, a light sensor, a light redirecting element, and a beam splitter. The objective lens and another optical assembly are arranged such that image light received by the objective lens passes through the another optical assembly. The scanning / descanning optical assembly has a light redirecting element that directs illumination light from the light source and projects the illumination beam through the objective lens and at multiple depths across the volumetric region and at multiple positions and / or angles of the volumetric region, thereby scanning the volumetric region with the illumination beam. The light redirecting element redirects the image light received from the volumetric region to the light sensor. In cooperation with the optical properties of said another optical assembly, it is effective to maintain the focus of light returning from the region illuminated by the illumination beam on the light sensor when scanning with said illumination beam. The beam splitter is arranged to form a junction where the image light and the illumination light follow adjacent paths through the objective lens, but are positioned to follow separate paths away from the objective lens.

[0328] In the forty-fifth embodiment or any other embodiment, the apparatus further includes a fixed reflector positioned to reflect the image light back through the further optical assembly, in some of these embodiments, the fixed reflector is oriented at an angle relative to the optical axis of the further optical assembly, the angle being selected according to the angle of the intermediate image formed at the mirror by the objective lens and the further optical assembly.

[0329] In the forty-fifth embodiment or any other embodiment, the beam splitter is a dichroic beam splitter and the wavelength of the light source is selected to excite fluorescence in a predefined fluorescent material selected by the properties of the dichroic beam splitter.

[0330] In the forty-fifth embodiment or any other embodiment, the light redirecting element includes one of a motor-actuated reflective element, a motor-actuated refractive element, a digital micromirror device (DMD), a microelectromechanical system (MEMS) light guide, a mirror, or other suitable MEMS device, an optical fiber bundle, an acousto-optic deflector (AOD), a spatial light modulator (SLM), or other technology, such as a MEMS, DMD, or SLM device that can be used as a light director that can be controlled to provide a particular spatial relationship between the first light redirector and the illumination light and the image light.

[0331] In the forty-fifth embodiment or any other embodiment, the light redirecting element includes a reflective element with reflective properties that equalize the angular change in direction of the illumination light from the light source with the angular change in direction of the image light received from the volumetric region, hi some of these embodiments, the reflective element has a single planar reflective surface on which both the illumination light from the light source and the light received from the volumetric region are incident.

[0332] In the 45th embodiment or any other embodiment, the light source generates light selected to cause fluorescence in an object containing a predefined fluorescent dye. In the 45th embodiment or any other embodiment, the light sensor includes a camera, a linear array of photomultiplier elements, or a single photomultiplier element.

[0333] In the forty-fifth embodiment or any other embodiment, the light source includes a beam flattening optical element that flattens a cross section of said illumination beam.

[0334] In the forty-fifth embodiment or any other embodiment, the scanning / descanning optical assembly includes a configurable optical element positioned in the path of the light from the light source to change the path and shape of the cross section of the illumination light to switch between depth-resolved and confocal modes, where in the confocal mode the cross section of the illumination beam has a lower aspect ratio and follows a different path than it follows in the depth-resolved mode. In some of these embodiments, the configurable optical element includes a refractive component that is movable relative to the light source and is shaped to expand the width of the incident light beam. In some of these embodiments, the configurable optical element includes a refractive component that is movable relative to the light source and is shaped to move the centerline of the incident light beam. In some of these embodiments, the scanning / descanning assembly changes the position of the focused spot or line in the volumetric region in the confocal mode. In some of these embodiments, the scanning / descanning assembly is configured to scan with a focused spot or line in a volumetric region in response to confocal mode control commands that selectively position the movable optical element.

[0335] In some of these embodiments, the scanning / descanning assembly includes image forming optics having an optical axis for generating an intermediate real image of image light returned from the illumination beam in a scan mode, and a detection module for imaging the intermediate real image, and an optical sensor includes a linear or two-dimensional array of pixels onto which an image of the intermediate real image is focused. The detection module has an optical axis that is oblique to the optical axis of the image forming optics, such that a pixel or a selected one of the pixels can function like a confocal pinhole or slit in a confocal mode, and such that in a scan mode, the angle between the optical axis of the detection module and the optical axis of the image forming optics is effective for compensating for axial position changes of points along the illumination beam imaged by the detection module.

[0336] In any of the embodiments defined above, the listed features of a microscope can be used in any other type of imaging device, since the optical functions can be applied to features of objects of other sizes.

[0337] In any embodiment, including claims where an illumination beam, a sheet of light, illumination light or other type of outgoing excitation light or outgoing illumination light is recited, structured illumination can be substituted to form additional embodiments. Any embodiment can be provided with image processing to increase the resolution of the image as would be known to one skilled in the art. Image processing where it is known that structured illumination should be used to obtain super-resolution is considered inherently disclosed in the context of structured illumination. In any device, method or system using structured illumination, the movement of the illumination beam can be controlled so that multiple images can be obtained at a given location by controlling the illumination beam to enter a particular area multiple times, each time with a different illumination pattern. This can be done in any suitable order.

[0338] In all embodiments where a light redirector is described, the light redirector may include a mirror, a prism, an acousto-optic deflector, a motorized lens, a spatial light modulator (SLM), a beam steering mirror or optics, a flexible light guide or optical fiber, other types of adaptive optics, or any other mechanism for controlling the direction of outgoing or incoming light, and may have associated actuators. Furthermore, the associated actuators may be controlled by any of the control or computer technologies listed below, based on techniques known to those skilled in the art of processors, computers, smart controllers, embedded systems, or programmable control systems.

[0339] It is understood that the control system and / or image processing described herein can be implemented by hardware, software programmed hardware, software instructions stored on a non-transitory computer readable medium, or a combination thereof. For example, the disclosed control method and / or image processing technique can be implemented using a processor configured to execute a set of programmed instructions, e.g., stored on a non-transitory computer readable medium. For example, a processor may include, but is not limited to, a personal computer, a workstation, or other such computing system that includes a processor, microprocessor, microcontroller device, or the processor is comprised of control logic including an integrated circuit, e.g., an application specific integrated circuit (ASIC). The instructions can be compiled from source code instructions provided according to a programming language, e.g., Java, C++, C#.net, etc., and the instructions can be compiled from source code instructions provided according to, e.g., Visual Basic TM The program may include code and data objects provided in accordance with the programming language, Lab VIEW, or other structured or object-oriented programming language. The programmed sequence of instructions and their associated data may be stored in a non-transitory computer-readable medium, such as a computer memory or storage device, which may be any suitable memory device, such as, but not limited to, a read-only memory (ROM), a programmable read-only memory (PROM), an electrically erasable programmable read-only memory (EEPROM), a random access memory (RAM), a flash memory, a disk drive, etc.

[0340] Further, the control system and / or image processing can be implemented as a single processor or as a distributed processor. Furthermore, the steps referred to herein can be performed on a single processor or a distributed processor (single-core and / or multi-core). Also, the processes, modules, and sub-modules illustrated in the embodiments herein and in the various figures relating to those embodiments can be distributed across multiple computers or systems or located on a single processor or system. Below are alternative embodiments of exemplary structures suitable for implementing the control system and / or image processing described herein.

[0341] The control system and / or image processing described above can be implemented, for example, as a programmed general-purpose computer, an electronic device programmed with microcode, a hardwired analog logic circuit, software stored on a computer-readable medium or signal, an optical computing device, a networked system of electronic and / or optical devices, a special purpose computing device, an integrated circuit device, a semiconductor chip, and software modules or objects stored on a computer-readable medium or signal.

[0342] Embodiments of the control system and / or image processing (or other subcomponents or modules thereof) may be implemented in a general purpose computer, a special purpose computer, a programmed microprocessor or microcontroller and peripheral integrated circuit elements, an ASIC or other integrated circuit, a digital signal processor, hardwired electronic or logic circuitry, e.g., discrete element circuitry, programmed logic circuitry, e.g., programmable logic devices (PLDs), programmable logic arrays (PLAs), field programmable gate arrays (FPGAs), programmable array logic (PAL) devices, etc. In general, any process capable of implementing the functions or steps described herein may be used to implement embodiments of a method, system, or computer program product (a software program stored on a non-transitory computer readable medium).

[0343] Furthermore, embodiments of the control system and / or image processing can be easily implemented in software, fully or partially, for example, using object-oriented software development environments or objects that provide portable source code that can be used on a variety of computer platforms. Alternatively, embodiments of the control system and / or image processing can be implemented in hardware, partially or fully, for example, using standard logic circuits or very-large-scale integration (VLSI) designs. Other hardware or software can be used to implement the embodiments depending on the system, the specific functionality, and / or the speed and / or efficiency requirements of the specific software or hardware system, microprocessor, or microcomputer used. Embodiments of the control system and / or image processing can be implemented in hardware and / or software using any known or future developed system or structure, device, and / or software that will be apparent to one of ordinary skill in the art from the functional description provided herein and from a general basic knowledge of optical systems, image processing, and / or computer program techniques.

[0344] In this application, unless otherwise stated, the use of the singular includes the plural and the use of "or" means "and / or". Furthermore, the terms "including" or "having" or other forms such as "including", "included", "has" or "had" are not meant to be limiting. Any ranges described herein are intended to include the endpoints and all values ​​between the endpoints. Any details herein of positional or angular relationships between optics, components, beam paths, and / or light rays are intended to include a variation of 10% or less. In general, whenever "substantially", "about", "approximately" or similar terms are used herein, a variation of 10% or less is intended.

[0345] Additionally, the above description may in some cases be applied to examples that have been produced in a laboratory, however, those examples may be extended to production techniques, and when applying, for example, quantities, techniques, or configurations to the laboratory examples, they should not be construed as limiting.

[0346] Features of the disclosed embodiments may be combined, rearranged, omitted, etc., to produce additional embodiments within the scope of the present invention. Furthermore, it may be advantageous to use certain features without the corresponding use of other features.

[0347] It is therefore apparent that there have been provided, in accordance with the present disclosure, systems, methods, and apparatus for imaging. Numerous alternatives, modifications, and variations can be realized by this disclosure. While particular embodiments have been illustrated and described in detail to demonstrate application of the principles of the invention, it is understood that the invention can be otherwise embodied without departing from such principles. Accordingly, applicants intend to include all such alternatives, modifications, and equivalents, and variations that are within the spirit and scope of the invention.

Claims

1. In an optical imaging device, a first optical scanning element; and a second optical scanning element; and An objective lens; an illumination source that delivers illumination light to the objective lens via a first optical path that includes the first optical scanning element; A photodetector; Contains the first optical scanning element redirects the illumination light to a peripheral region of the objective lens such that the illumination light passes through the objective lens and forms an oblique imaging plane at the tissue, the position of the oblique imaging plane at the tissue varying with an orientation of the first optical scanning element; the objective lens receives the returning light from the oblique imaging plane and directs the returning light along a second optical path that includes the second optical scanning element, the second optical scanning element directing the returning light along the second optical path to form a fixed oblique intermediate image plane; the photodetector captures an image of the fixed tilted intermediate image plane; both the first light scanning element and the second light scanning element are comprised of a single reflective surface; the optical imaging device further includes a telescope disposed in both the first optical path and the second optical path; the illumination light reaches the single reflective surface before reaching the telescope; the illumination light reaches the telescope before reaching the objective lens; the returning light reaches the objective lens before reaching the telescope; the returning light reaches the telescope before reaching the single reflective surface; Optical imaging device.

2. the photodetector is positioned directly at the fixed tilted intermediate image plane; 2. The apparatus of claim 1.

3. the photodetector is positioned at a distance from the fixed tilted intermediate image plane; the optical imaging device further includes at least one telescope that directs light from the fixed tilted intermediate image plane to the photodetector.

2. The apparatus of claim 1.

4. the photodetector is positioned at a distance from the fixed tilted intermediate image plane; the optical imaging device further includes a fiber optic bundle that directs light from the fixed tilted intermediate image plane to the photodetector.

2. The apparatus of claim 1.

5. the illumination source includes a laser that generates a pencil beam and an auxiliary optical scanning element that expands the pencil beam into a sheet of light; The photodetector includes a linear image sensor.

2. The apparatus of claim 1.

6. the illumination source includes a laser that generates a pencil beam and a cylindrical lens that expands the pencil beam into a sheet of light; The photodetector includes a 2D image sensor.

2. The apparatus of claim 1.

7. the return light includes fluorescent light emitted from the tissue in the oblique imaging plane in response to illumination of the tissue in the oblique imaging plane by the illumination light.

2. The apparatus of claim 1.

Images