Automated imaging in content screening

Abstract

A method and system for operating an optical arrangement includes a light source, an objective lens in proximity to a sample, an ocular lens in proximity to an image acquisition device, a first optical subassembly including configured to transmit a first light source signal from the light source to the sample through the objective lens, the first optical subassembly being further configured to transmit a first fluorescent signal from the sample to the image acquisition device, a second optical subassembly including a fixed portion and a movable portion and configured to transmit a second light source signal from the light source through an off-centered portion of the objective lens to the sample in an oblique illumination, the movable portion being movable with respect to an optical axis of the ocular lens and being configured to transmit therethrough a second fluorescent signal at an oblique angle to the objective lens from the sample to the image acquisition device.

Description

[0001] AUTOMATED IMAGING IN CONTENT SCREENING

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The contents of the related applications filed on even date herewith entitled “Thermal Management of Imaging System” and “Imager with Image Rotation” are hereby incorporated in their entirety by reference. Furthermore, the priority filings corresponding to these two applications, US 63 / 614,740 and US 63 / 614,743, are incorporated by reference herein in their entirety. This application claims the priority benefit of US 63 / 614,714, the contents of which are expressly incorporated herein by reference in their entirety.

[0004] BACKGROUND

[0005] High content screening (HCS) is traditionally achieved through 2D imaging of thin samples. New trends in bioimaging require 3D imaging of more complex structures like organoids and spheroids, which cannot always be imaged with sufficient quality using the existing HCS instruments on the market. High contrast 3D imaging is possible through Selective Plane Illumination Microscopy (SPIM). However, being able to obtain a good quality focus, contrast and resolution of a resulting image generated via SPIM on an image acquisition device presents challenges to be addressed in order to create a 3D HCS imaging platform.

[0006] SUMMARY

[0007] In one aspect, the technology relates to an optical subassembly having a first portion configured to receive an optical signal (e.g., an illumination light beam from an illumination source that can be a laser, a diode, or any other similar source of optical signal) along a first axis and a second portion comprising a plurality of relay lenses and a plurality of relay objectives. The second portion is configured to receive the optical signal from the first portion along a second axis and transmit the optical signal from the plurality of relay lenses to a first relay objective of the plurality of relay objectives. The second portion transmits the optical signal to the first portion from a second relay objective of the plurality of relay objectives, and the first portion transmits the optical signal received from the second relay objective to an image acquisition device. A first moving mechanism adjusts, along the second axis, a position of at least one of a first relay lens and a second relay lens of the plurality of relay lenses. Moving the at least one of the first relay lens and the second relay lens adjusts a quality of the image formed at the image acquisition device.

[0008] The position of the at least one of the first relay lens and the second relay lens can adjust a resolution of the image formed at the image acquisition device. The optical signal can include a fluorescent signal. The first portion can be a fixed portion and the second portion can be a movable portion, and the optical subassembly can further include a second moving mechanism to move the movable portion with respect to the fixed portion along a direction perpendicular to the first axis. The second axis can be perpendicular to the first axis. The plurality of relay lenses can include exactly two relay lenses. The plurality of relay objectives can include exactly two relay objectives.

[0009] In another aspect, an optical arrangement includes an optical subassembly as described above, a light source, an objective lens in proximity to a sample, a transmission subassembly comprising a plurality of optical components configured to transmit a light source signal from the sample to an image acquisition device, and a third moving mechanism configured to adjust a position of at least one of a first transmission lens of the plurality of optical components and a second transmission lens of the plurality of optical components along the first axis.

[0010] The first moving mechanism of the optical subassembly can be configured to adjust the position of at least one of the first relay lens and the second relay lens so as to increase an overlap between an optical light path and an illumination light path. Moving the first transmission lens and the second transmission lens can adjust the quality of the image formed at the image acquisition device by increasing the overlap between the optical light path and the illumination light path. The position of at least one of the first relay lens and the second relay lens and the position of at least one of the first transmission lens and the second transmission lens can be contemporaneously adjusted via the first moving mechanism and the third moving mechanism, respectively, so as to improve the quality of the image formed at the image acquisition device. The position of at least one of the first relay lens and the second relay lens and the position of at least one of the first transmission lens and the second transmission lens can be contemporaneously adjusted via the first moving mechanism and the third moving mechanism, respectively, so as to improve a resolution of the image formed at the image acquisition device.

[0011] According to another aspect, an imaging method includes receiving, at a first portion of an optical subassembly, an optical signal along a first axis, the optical subassembly further comprising a second portion that comprises a plurality of relay lenses and a plurality of relay objectives; transmitting the received optical signal from the first portion along a second axis to a first relay lens of the plurality of relay lenses in the second portion, the optical signal being transmitted through the plurality of relay lenses and to a first relay objective of the plurality of relay objectives; transmitting the optical signal to the first portion from a second relay objective of the plurality of relay objectives; transmitting the optical signal received from the second relay objective to an image acquisition device to form an image; and adjusting, along the second axis, a position of at least one of the first relay lens and a second relay lens of the plurality of relay lenses to increase a quality of the image formed at the image acquisition device.

[0012] Adjusting the position of at least one of the first relay lens and a second relay lens can include adjusting a resolution of the image formed at the image acquisition device. Prior to receiving the optical signal, the method can include generating a light source signal at a light source; transmitting, via at least one objective lens and via a transmission subassembly that comprises a plurality of transmission lenses, the light source signal to a sample; transmitting, via the at least one objective and the plurality of transmission lenses, the optical signal generated by the sample to the optical subassembly; and adjusting a position of at least one of a first transmission lens of the plurality of transmission lenses and a second transmission lens of the plurality of transmission lenses along the first axis so as to increase a quality of the image formed at the image acquisition device.

[0013] The method can include adjusting the position of at least one of the first transmission lens and the second transmission lens to adjust a resolution of the image formed at the image acquisition device. The method can also include contemporaneously adjusting the position of at least one of the first relay lens and the second relay lens and the position of at least one of the first transmission lens and the second transmission lens so as to improve the quality of the image formed at the image acquisition device.

[0014] According to another aspect, an optical arrangement includes a first portion configured to receive an optical signal along a first axis; a second portion comprising a plurality of relay lenses and a plurality of relay objectives, wherein the second portion receives the optical signal from the first portion, along a second axis, and wherein the second portion transmits the optical signal from the plurality of relay lenses to a first relay objective of the plurality of relay objectives and transmits the optical signal to the first portion from a second relay objective of the plurality of relay objectives, and wherein the first portion transmits the optical signal received from the second relay objective to an image acquisition device; a light source; an objective lens in proximity to a sample stage that can hold a sample; a transmission subassembly between the light source and the objective lens, the transmission subassembly comprising an adjustable mirror coupled to a mirror adjustment mechanism and a plurality of adjustable transmission lenses, the transmission subassembly being configured to transmit a light source signal from the light source to the sample and to transmit the optical signal emitted from the sample to the optical arrangement via the plurality of adjustable transmission lenses; wherein the adjustable mirror is configured to reflect light received from the light source onto the objective lens.

[0015] The transmission subassembly can further include a beam shaping device between the light source and the adjustable mirror. The mirror adjustment mechanism can be configured to rotate the adjustable mirror around an axis of rotation thereof. The rotation of the adjustable mirror at a first angle reflects the light received from the light source at a corresponding first location of the objective lens, and a rotation of the adjustable mirror at a second angle different from the first angle reflects the light received from the light source at a corresponding second location of the objective lens, the second location being different from the first location. A rotation of the adjustable mirror at a non-perpendicular angle to reflect the light received from the light source at a corresponding location of the objective lens varies an overlap between an optical light path emitted by the sample and an illumination light path transmitted to the sample from the light source compared to a light that is reflected at a perpendicular angle. An increase in the overlap between the optical light path and the illumination light path increases a quality of the image formed at the image acquisition device. The mirror adjustment mechanism can be configured to translate the adjustable mirror along a longitudinal direction that is parallel to a direction of the light received from the light source. A longitudinal movement of the adjustable mirror at a first distance along the longitudinal direction can reflect the light received from the light source at a corresponding first location of the objective lens; and a longitudinal movement of the adjustable mirror at a second distance along the longitudinal direction different from the first distance can reflect the light received from the light source at a corresponding second location of the objective lens, the second location being different from the first location. A longitudinal movement of the adjustable mirror along the longitudinal direction to reflect the light received from the light source at a corresponding location of the objective lens can vary an overlap between a path of an optical light emitted by the sample and a path of an illumination light transmitted to the sample from the light source. An increase in the overlap between the optical light path and the illumination light path can increase a quality of the image formed at the image acquisition device.

[0016] According to another aspect, an imaging method can include generating a light source signal at a light source; directing the illumination light from the light source signal to a sample via at least one objective lens and via a transmission subassembly that comprises a plurality of transmission lenses and an adjustable mirror proximal to the light source; directing, via the at least one objective and the plurality of transmission lenses, the returned optical signal from the sample to an optical subassembly; receiving, at a first portion of the optical subassembly, an optical signal along a first axis, the optical subassembly further comprising a second portion that comprises a plurality of relay lenses and a plurality of relay objectives; transmitting the returned optical signal from the first portion along a second axis to a first relay lens of the plurality of relay lenses in the second portion, the returned optical signal being transmitted through the plurality of relay lenses and to a first relay objective of the plurality of relay objectives; transmitting the returned optical signal to the first portion from a second relay objective of the plurality of relay objectives; transmitting the returned optical signal received from the second relay objective to an image acquisition device to form an image; and adjusting a configuration of the adjustable mirror so as to increase a quality of the image formed at the image acquisition device.

[0017] The transmission subassembly can further include a beam shaping device between the light source and the adjustable mirror. Adjusting the configuration of the adjustable mirror can include adjusting one of a resolution and a contrast of the image formed at the image acquisition device. Adjusting the configuration of the adjustable mirror can include rotating the adjustable mirror around an axis of rotation so as to improve the quality of the image formed at the image acquisition device. Rotating the adjustable mirror around the axis of rotation can include varying an overlap between a path of the returned optical signal from the sample and a path of the illumination light transmitted to the sample from the light source. Increasing the overlap between the returned optical signal path and the illumination light path can include increasing a quality of the image formed at the image acquisition device. Adjusting the configuration of the adjustable mirror can include translating the adjustable mirror along a longitudinal direction parallel to a direction of incidence of the light received from the light source so as to improve the quality of the image formed at the image acquisition device. Translating the adjustable mirror along the longitudinal direction can include varying an overlap between a path of the optical light emitted by the sample and a path of the illumination light transmitted to the sample from the light source. Increasing the overlap between the optical light path and the illumination light path can include increasing a quality of the image formed at the image acquisition device.

[0018] In another aspect, an optical subassembly includes a focusing module configured to receive an optical signal along an optical axis and comprising a plurality of relay objectives. The focusing module receives the optical signal at a first relay objective of the plurality of relay objectives along the optical axis, and transmits the optical signal from the first relay objective to a second relay objective of the plurality of relay objectives along the optical axis, and then to an image acquisition device. The optical subassembly includes a moving mechanism configured to adjust, along the axis, a position of at least one of the first relay objective and a second relay objective of the plurality of relay objectives along the optical axis, wherein moving the at least one of the first relay objective and the second relay objective adjusts a quality of the image formed at the image acquisition device.

[0019] The position of the at least one of the first relay objective and the second relay objective can adjust a resolution of the image formed at the image acquisition device. There can be exactly two relay objectives, or more than two relay objectives, in different aspects.

[0020] According to another aspect, a method of determining instrument positions for an oblique plane microscope includes receiving layer information from the division of a three-dimensional sample into one or more layers; for each of the one or more layers, receiving a plurality of oblique plane images of the three-dimensional sample, each image associated with a remote focus position, a pupil position or a steering position at one or more different orientations to an obliquely illuminated plane of the three- dimensional sample; determining, for each of the plurality of images, a focus score that is related to at least one of contrast, resolution, and field of view; ranking the images received for each layer based on the focus score associated with that image; storing instrument settings associated with a number of highest ranking images for each layer as candidate instrument positions, the instrument settings comprising one or more of the remote focus position, the pupil position and the steering position; and acquiring one or more final images for each of the at least one or more layers based on at least one candidate instrument position associated with each layer.

[0021] The one of more layers can include exactly two layers. The layer information can include a height of the layer within the sample. The layer information can include one or more sample features, and wherein the sample features can be input by a user at a user interface or obtained automatically using imaging techniques using the plurality of oblique plane images. The plurality of oblique plane images can be taken at a high- contrast location within each of the one or more layers. The plurality of oblique plane images can each be an average of two or more images taken at a corresponding two or more locations within each of the one or more layers. A remote focus position, a pupil orientation, or a steering position can be determined in a stepwise fashion or a continuous scan, or can be synchronized to interpolated positions from predetermined positions. The focus score can be determined by a user selecting from among the plurality of oblique plane images a number of images which have a higher contrast, resolution or field of view. The focus score can be determined automatically by a processor based on contrast, resolution, or field of view metrics. The at least one candidate instrument position associated with each layer can be used to acquire the one or more final images is selected by the user from the stored candidate instrument positions. The at least one candidate instrument position associated with each layer used to acquire the one or more final images can be selected from between at least two candidate instrument positions by selecting the two instrument positions having substantially similar scores, averaging the associated instrument settings, acquiring the one or more final images for each of the at least one or more layers comprises using the same at least one candidate instrument position to acquire each of the multiple images within the layer, and stitching the final images for each of the at least one or more layers together, wherein the multiple images within the layer differ from one another are based on the associated instrument settings for at least one of the one or more layers. The focus score can be based upon contrast, or it can be based upon field of view. The associated instrument settings can include a distance between a pair of objectives, or a distance between a pair of lenses. The associated instrument settings can include a tilt and associated pupil position of an objective, or can include a position in an X-Y plane associated with a steering position, or can be based upon an initial calibration position, or a combination thereof. The initial calibration position can be based on a known standard and the associated instrument settings include boundaries in the X-Y plane based on the known standard.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 is a schematic view of a multimode imaging system including SPIM, according to examples of this disclosure.

[0024] FIGS. 2 A and 2B are schematic views of movable SPIM focusing modules in a multimode imaging system, according to various examples of this disclosure.

[0025] FIGS. 3A-3F are images formed via confocal imaging and light sheet imaging at a plurality of magnifications. FIGS. 4 A and 4B are images formed via SPIM imaging in various focus positions, in accordance with examples of the disclosure.

[0026] FIGS. 4C and 4D depict adjustable focus configurations of optical systems according to examples of this disclosure.

[0027] FIG. 5 depicts a method of performing multimode imaging, in accordance with various examples of the disclosure.

[0028] FIG. 6 depicts a block diagram of a computing device.

[0029] FIGS. 7A-7D are schematic views of a multimode imaging system including SPIM, according to examples of this disclosure.

[0030] DETAILED DESCRIPTION

[0031] SPIM, also referred to as light sheet microscopy, is a technique for obtaining optically sectioned images of a sample. SPIM uses two objective lenses, separated by an angle of 90° or some other oblique angle relative to one another and used to view the same sample. One lens is used to illuminate only a thin “sheet” within the sample and the second lens is used to produce a diffraction-limited image of this sheet. A drawback of SPIM is that two objective lenses are required and this gives rise to disadvantages at least because the alignment of the two objective lenses is typically difficult to achieve and to maintain. Another disadvantage is that it is mechanically difficult to arrange for the two objectives to be placed close enough to one another so that a high numerical aperture lens can be used to collect the light while still being able to produce a thin sheet of illumination, and that a special sample holder is required to be used. Other challenges include the fact that improving the image quality, e.g., improving the image focus, contrast and / or resolution, is difficult to achieve in oblique plane imaging in its current configuration.

[0032] The use of oblique plane microscopy in large, complex 3D samples, may be substantially limited due to the impact of the refractive index and the sample structure on the illumination angle when imaging deep in a vertical, or “Z” direction.

[0033] There is therefore a need to be able to use a technique similar to SPIM, but having the possibility of focusing the image formed at an image acquisition device such as a detector or camera. In order to address the above drawbacks, examples of the disclosure provide a SPIM imaging platform that allows for adjustment of the distance between various lenses along the light path of the signal between the sample and an image acquisition device.

[0034] An “analyzer / controller” refers to a system to facilitate data acquisition and control of the imaging systems described herein. The controller may be a single integrated electronic enclosure or may comprise multiple distributed elements. The control elements may provide control for positioning and / or scanning of the optical components used herein, or of the sample. They may also collect data about the sample or optical components’ deflection, motion or other response, and provide control over radiation source power, polarization, steering, focus, and / or other functions. The control elements etc. may include a computer program method or a digital logic method and may be implemented using any combination of a variety of computing devices (computers, Personal Electronic Devices), analog and / or digital discrete circuit components (transistors, resistors, capacitors, inductors, diodes, etc.), programmable logic, microprocessors, microcontrollers, application-specific integrated circuits, or other circuit elements. A memory configured to store computer programs may be implemented along with discrete circuit components to cany out one or more of the processes described herein.

[0035] A “camera” refers to an array-based photodetector comprising a plurality of photosensitive pixels. A camera may comprise one or more technology types including but not limited to CCD, EM-CCD, CMOS, s-CMOS, and / or other photosensitive array technologies. The camera may support frame rates from a few frames per seconds, hundreds of frames per second, or even thousands of frames per second or higher.

[0036] The phrase “image forming” or “image acquisition” refers to collecting light that has interacted with a sample to form a 2D or 3D representation of that sample. The light can be collected after reflection, scattering, transmission, evanescent wave coupling, and / or transmission by or through the sample and collection by a detector or camera.

[0037] “Confocal microscopy” refers to a form of optical microscopy in which the light collected at a detector is confined to light that passes through a small volume within the 3D focus volume of an optical objective on a sample. Confocal microscopy is often performed by placing a “confocal aperture” at a focal plane that is equivalent with the focal plane of the sample, thus blocking stray light that does not pass through the focus volume on the sample.

[0038] A “detector” refers to a device that produces a signal indicative of the power, intensity and / or energy of light / radiation incident on the detector surface. The signal will generally be an electrical signal, for example a voltage, current and / or an electrical charge. The detector may be a photodiode, a photo-transistor, a charge coupled device (CCD). In some cases, a detector may be a semiconducting detector, for example a silicon PIN photodiode. A detector may also be an avalanche photodiode, a photomultiplier tube, or any other device that produce a change in current, voltage, charge, conductivity or similar upon incidence of light. A detector may comprise a single element, multiple detector elements, for example a bi-cell or quad-cell, a linear or two dimensional array of detector elements, including camera based detectors.

[0039] “Diffraction limit” of a light beam means the minimum separation of two optical sources that can be distinguished by a detector. The Abbe diffraction limit d for a microscope having a numerical aperture (NA) and operating at a wavelength is defined as d = / (2 • NA). Physical restraints on the numerical aperture of a microscope prohibit very large numerical apertures, and therefore the diffraction limit of a microscope depends strongly upon the operating wavelength used for detection, with large wavelengths corresponding to relatively poor resolution and high wavelengths corresponding to increased precision.

[0040] “Illuminate,” “illuminating,” and “illumination” mean to direct radiation at an object, for example a surface of a sample. Illumination may include radiation in the infrared wavelength range, visible, and other wavelengths from ultraviolet to a millimeter or more. Illumination may include any arbitrary configuration of radiation sources, reflecting elements, focusing elements and any other beam steering or conditioning elements.

[0041] An illumination source can be one of a large number of sources, including a gas laser, a laser diode, a superluminescent diode (SLD), or any other laser having either narrowband or broadband illumination output at any of a number of frequencies. Other illumination sources usable at higher wavelengths can include thermal or Globar sources, supercontinuum laser sources, frequency combs, difference frequency generators, sum frequency generators, harmonic generators, optical parametric oscillators (OPOs), optical parametric generators (OPGs), quantum cascade lasers (QCLs), interband cavity lasers (ICLs), synchrotron infrared radiation sources, nanosecond, picosecond, femtosecond and attosecond laser systems, CO2 lasers, microscopic heaters, electrically or chemically generated sparks, and / or any other source that produces emission of radiation. Broadband sources can be made narrow band with filters, monochromators and other devices. A “narrowband light source” a light source with a narrow bandwidth or linewidth, for example a light of linewidth smaller than 8 cm-1, but in general it can be a light source with a linewidth narrow enough that the linewidth does not cover a spectral range of interest of the sample. An illumination source can create a light beam, also referred to herein as an optical signal, that can be directed through a variety of optical components such as mirrors, lenses, objectives, and the like.

[0042] “Modulating” or “modulation” when referring to radiation incident on a sample refers to changing the intensity at a location periodically, and “modulator” is a device for accomplishing this. Modulating the light beam intensity can be achieved by means of mechanical chopping of the beam, controlled laser pulsing, and / or deflecting the laser beam, for example by a tilting mirror that is driven electrostatically, electromagnetically, with piezo actuators or other means to tilt or deform the mirror, or high-speed rotating mirror devices. Modulation can also be accomplished with devices that provide time varying transmission like acousto-optic modulators, electro-optic modulators, photoelastic modulators, pockel cells, and the like. Modulation can also be accomplished with diffraction effects, for example by diffractive MEMS-based modulators, or by high-speed shutters, attenuators, or other mechanisms that change the intensity, angle, and / or phase of the laser intensity incident on the sample.

[0043] “Optical property” refers to a characteristic of a sample, including but not limited to index of refraction, absorption coefficient, reflectivity, absorptivity, real and / or imaginary components of the index refraction, real and / or imaginary components of the sample dielectric function and / or any property that is mathematically derivable from one or more of these optical properties.

[0044] “Optical response” refers to the result of interaction of radiation with a sample. The optical response is related to one or more optical properties defined above. The optical response can be an absorption of radiation, a temperature increase, a thermal expansion, a photo-induced force, the reflection and / or scattering of light or other response of a material due to the interaction with illuminating radiation.

[0045] “Signal indicative of’ refers to a signal that is mathematically related to a property of interest. The signal may be an analog signal, a digital signal, and / or one or more numbers stored in a computer or other digital electronics. The signal may be a voltage, a current, or any other signal that may be readily transduced and recorded. The signal may be mathematically identical to the property being measured, for example explicitly an absolute phase signal or an absorption coefficient. It may also be a signal that is mathematically related to one or more properties of interest, for example including linear or other scaling, offsets, inversion, or even complex mathematical manipulations.

[0046] A “signal processor” is an analog and / or digital device that is used to process one or more signal, for example performing filtering, level shifting, squaring, summing, root- mean-square summing and / or any other computation or transformation of an analog and / or digital signal. A signal processor may be an analog circuit where all filtering / shifting / computations are performed with discrete analog components, or one of more digital processors, for example a CPU, computer, field programmable gate array, digital signal processor, or any other suitable digital computation means. The signal processor may be a hybrid of both analog and digital components.

[0047] “Spectrum” refers to a measurement of one or more properties of a sample as a function of wavelength or equivalently (and more commonly) as a function of wavenumber.

[0048] The terms “about” or “approximate” and the like are synonymous and are used to indicate that the value modified by the term has an understood range associated with it, where the range can be ±20%, ±15%, ±10%, ±5%, or ±1%.

[0049] The term “substantially” is used to indicate that a result (e.g., measurement value) is close to a targeted value, where close can mean, for example, the result is within 80% of the value, within 90% of the value, within 95% of the value, or within 99% of the value.

[0050] Various examples of the disclosure include one or more adjustment mechanisms in an Oblique Plane Microscopy (OPM) multi-modal microscope, such as, e.g., remote refocus, illumination pupil position, and illumination steering in order to account for sample-induced optical responses causing imaging errors at progressive depths into 3D specimens. An apparatus for performing the above-discussed adjustments may include a motorized lens configured to adjust a remote focus intermediate image position in an OPM microscope. Adjusting the image position in the 0PM microscope can include adjusting which oblique plane of the specimen volume is imaged onto the detector. A motorized actuator can similarly adjust the lateral position of a beam steering mirror, which translates the beam orthogonally to the optical axis to adjust the off-axis position of the illumination beam on the pupil of the primary objective, thereby changing the angle of the oblique illumination sheet in the sample, and a motorized actuator configured to adjust the tilt of a beam steering mirror in order to control the position of the light sheet illumination focal point with respect to the optical axis of the primary objective.

[0051] In various examples, operation of examples of the disclosure may include interrogating the sample, e.g., identifying a region of interest, before imaging the sample, in order to determine a desired focus, desired pupil position, or desired steering position. To that end, the sample may be divided into layers along a vertical direction such as, e.g., a “Z” direction, move the light incident on the sample to the center of each layer and adjust the focus, pupil position, and / or steering to determine desired settings corresponding to that layer. For examples, the layers may have thicknesses in a range of 0-50 pm, 50-100 pm, 100-150 pm, and the like. For each adjustment, a number of images through a range of positions may be collected. Desired settings may increase or maximize, e.g., contrast, resolution, and field of view. A software automated focus score or other average and regional contrast metrics may be used to determine desired settings, which may be saved for each layer to be recalled during later imaging acquisition steps. A focus score can be determined by detecting image crispness algorithmically (see, e.g., Rajevenceltha and Gaidhane, “A novel approach for image focus measure,” 15 Signal, Image and Video Processing 547-555 (2021)).

[0052] A workflow of examples of the disclosure may include scanning the sample in the Z direction by moving the primary objective along the optical axis while adjusting to a pre-determined light sheet remote focus position, pupil positions, and / or steering for optimal performance. The scanning may be performed in a stepwise fashion, or a continuous scan, and similarly the light sheet adjustments may be controlled to move at each step or continuously synchronized to interpolated positions based on pre-determined settings. Another workflow may include scanning the sample in a layer-wise fashion in a direction perpendicular to the Z direction in a stepwise fashion or as a continuous scan by utilizing the sample stage, and at each layer, pre-determined adjustments may be performed to remote focus, pupil position and / or steering to achieve increased or optimal performance. In examples, images with variable adjustment settings in focus, pupil position, and steering, may be collected and combined together to form a resultant image stack using known tomographic techniques to include weighted averaging, cropping, and tiling. Individual settings can be adjusted to improve contrast and resolution.

[0053] FIG. 1 is a schematic view of a multimode imaging system, according to examples of this disclosure. The multimode imaging system 100 includes an optical arrangement that may be operated in one or more imaging modes including a SPIM imaging mode. FIG. 1 shows an illumination source 110 such as, e.g., a laser 110, configured to generate an illumination light or incident light “IL,” a fiber selector 120 configured to direct the illumination light IL along a given or desired light path, filters 130 and 135, an ocular lens 190, and an image acquisition device 195 such as, e.g., a camera. The fiber selector 120 may include a selectable first optical fiber 122 and a selectable second optical fiber 124, each selectable optical fiber 122 or 124 being configured to emit the illumination light IL therethrough towards a given signal path. In examples, either or both of the first optical fiber 122 and the second optical fiber 124 may be or include a fiber bundle, the bundle including a plurality of fibers.

[0054] FIG. 1 also describes a first lens 140 and a second lens 150, a mirror 160, and an objective lens 170. It should be understood that any number of additional or alternative focusing optics and other elements may be used in various embodiments for light handling in various examples. The objective lens 170 may face a sample 180 arranged on a movable stage 185, and the sample 180 is movable via the movable stage 185 in a planar or XY direction that is parallel to the objective lens 170. The controller 102 is configured to switch between various imaging modes including the SPIM mode and, e.g., select the optical fibers 122 and 124 in view of a desired imaging mode. For example, the optical fiber 122 may be selected in the SPIM mode. Operation of at least one of the illumination source 110 or the fiber selector 120 is controlled via the controller 102 to transmit the illumination light IL in a SPIM light path, the illumination light IL being referred to in this case as the illumination signal.

[0055] The selectable first optical fiber 122 of the fiber selector 120 is configured to emit the illumination signal therethrough towards the light sheet light path, and the selectable second optical fiber 124 is configured to emit another light source signal therethrough towards, e.g., a widefield or confocal light path (not shown). The controller 102 may be coupled to or include a computing system such as, e.g., the computer system 600 discussed below with respect to FIG. 6.

[0056] In operation during the SPIM mode, also referred to herein as light sheet mode, the illumination source 110 generates the illumination light IL that is transmitted through fiber selector 120 to the dichroic mirror 125. The illumination light received at the dichroic mirror 125 is reflected onto a mirror 160 and onto objective lens 170. The path of the illumination light IL is radially off-centered with respect to the objective lens 170 or other focusing optic, such that the illumination light IL reaches the sample 180 at a non-perpendicular angle with respect to the surface of the sample 180. In an example, the light path between the illumination source 110 and the sample 180 is referred to as an optical subassembly. One example of such an optical subassembly can include fiber selector 120, dichroic mirror 125, mirror 160, and objective 170, though in alternative embodiments there may be more, fewer or different elements as part of the optical subassembly that accomplish the same effect of directing the illumination light IL to the sample at an angle.

[0057] The illumination light IL is received at the objective lens 170 in an off-centered configuration and is transmitted to the sample 180 at an incident angle a, as illustrated in FIG. 1. FIG. 1 also depicts an optical light (optical signal), which may be an emission light or a so-called returned light “RL,” which is used herein for clarity.

[0058] For example, the returned light RL may be a fluorescent emission. Fluorescent emissions are the emissions of light from a sample at one wavelength or a range of wavelengths due to excitation at another wavelength. Fluorescent excitation and emission processes are a form of inelastic scattering of incident light and can be used to characterize a sample by providing information about the types of fluorescent emissions (number of photons emitted, and wavelength of emitted photons) based on a particular intensity and spectrum of incident light. “Autofluorescence” refers to fluorescence that occurs naturally upon exposure of a sample to an excitation source, while fluorescence more broadly can refer either to autofluorescence or to exogenous fluorescence via the application / integration of external fhiorophores like fluorescent dyes, fluorescent proteins, and fluorescent nanoparticles or other fluorescence treatments.

[0059] Confocal fluorescence microscopy is a laser-based technique where radiation of one wavelength excites a fluorescent response in a sample that is detected at second wavelength or range of wavelengths. Extensive libraries of fluorescent dyes have been developed to target different functional and structural elements of biological materials, for example cells, tissues, and organisms. Fluorescence microscopy enables researchers and clinicians to create, visualize and analyze micrographs of a sample where each color represents the distribution of specific target structures within the biological material. Various fluorescence microscopy techniques are described, for example in Renz, “Fluorescence Microscopy — A historical and Technical Perspective,” Cytometry Part A, Vol 83, pp. 767-779 (2013) and Sanderson et al., “Fluorescence Microscopy,” Cold Spring Harb Protoc. 2014(10): pdb.top071795. doi:10.1101 / pdb.top071795.

[0060] Emitted or returned light emanates from the sample 180 at an oblique angle that is substantially equal and opposite to the incident angle a, and travels through the objective lens 170 in an off-centered configuration in the opposite direction of the illumination light IL and at an oblique angle to a central axis of the objective lens 170. In another example, the optical light may be a fluorescent light or signal that follows the same path as the returned light RL and is characterized by the emission of light by a sample that has absorbed light or other electromagnetic radiation. The returned light RL travels through an off-centered radial portion of the objective lens 170, is reflected from an off-centered radial portion of the mirror 160, and travels through an off-centered radial portion of the dichroic mirror 125 and of the second lens 150. Upon leaving the second lens 150, the returned light RL continues to travel through an off-centered radial portion of the first lens 140, through filters 130 and 135, and onto a focusing module 175. The focusing module 175 may be movable and may be configured to receive the off-centered returned light RL and convert the returned light RL from an off-centered beam to a parallel beam 178 onto ocular lens 190. The ocular lens 190 is configured to focus the parallel beam returned light RL onto the image acquisition device 195.

[0061] In the light sheet imaging system 100, the movable focusing module 175 may be movable in the Z direction, (relative, e.g., to the XY movement of the stage 185) as illustrated in FIG. 1 , and may be configured to be placed on the path of the returned light RL in order to convert the returned light RL from an off-centered beam to the parallel beam 178 that then projects onto ocular lens 190. For example, when the fiber selector 120 directs the illumination light IL to the dichroic mirror 125 in a light sheet mode, the movable focusing module 175 is moved along the Z direction to be placed on the light path of the returned light RL. On the other hand, when the fiber selector directs the illumination light IL to the filter 130 and through a path corresponding to the light path of another imaging mode such as, e.g., a widefield, brightfield, or confocal mode (not shown), the movable focusing module 175 may be moved off the light path of the returned light RL. In another example, the light path described above between the sample 180, the objective lens 170, the mirror 160, the dichroic mirror 125, the second lens 150, the first lens 140, the filters 130 and 135, the focusing module 175 and the ocular lens 190 may be referred to herein, or included within, as a transmission subassembly. In an example, the second lens 150 may be movable along its optical axis via a moving mechanism 152 so as to adjust a distance between the second lens 150 and the first lens 140 along the optical axis. Alternatively, any one of the first lens 140 and the second lens 150 may be movable with respect to each other along the optical axis in order to adjust the distance therebetween. Adjusting the distance between the first lens 140 and the second lens 150 may improve the quality, e.g., the focus, contrast and resolution, of the image formed on the image acquisition device.

[0062] FIG. 2A is a schematic view of a movable SPIM focusing module in a multimode imaging system, according to various examples of this disclosure. FIG. 2A depicts a movable focusing module 200, similar to the movable focusing module 175 illustrated in FIG. 1, that converts the transmitted returned light RL from an off-centered beam of returned light RL to a parallel beam 288. With reference to FIG. 1, the parallel beam 288 is handled in a similar manner to the way that beam 178 is modified in the embodiment of FIG. 1 before it exits the movable focusing module 175. In FIG. 2A, the returned light RL is received at a mirror 281 that redirects the returned light RL with respect to the initial light path of the returned light RL onto a first lens 282, also referred to herein as first relay lens 282. The returned light RL is received in a radially off-centered portion of the relay lens 282 and onto another relay lens 283, also in a radially off-centered portion thereof. The lens 283 then directs the returned light RL to a radially off-centered portion of a mirror 284 which redirects the returned light RL at an angle with respect to the incident returned light RL and towards a first relay objective 286. Other optical arrangements are possible, but importantly the returned light RL is transmitted such that it is received off-center at the first relay objective 286. The mapping of the pupils of first and second relay objectives 286 and 287 results in spatially-resolved data that can be used as described herein. Movement of the upper stage 204 affects the spatial position of the incoming returned light RL beam and these lenses map that incoming position to a 1 : 1 spatially-resolved position on the objectives 286, 287, either centered or offset depending upon the incoming returned light RL.

[0063] The first relay objective 286 transmits that redirected returned light RL to a second relay objective 287, the second relay objective 287 converts the radially off- centered returned light RL to a parallel beam onto a mirror 289. The mirror 289 then redirects the received parallel beam of the returned light RL out of the movable focusing module 200 as a parallel beam 288. As a result, the returned light RL, initially entering the movable focusing module 200 as a radially off-centered light signal or light source signal, is converted to a centered parallel beam 288 that is usable to an image acquisition device such as, e.g., the camera 195 illustrated in FIG. 1. The relay objectives 286 and 287 may be aligned and fixed in space on, e.g., a platform, to preserve the alignment therebetween.

[0064] The movable focusing module 200 may include a first portion 204 and a second portion 208. The first portion 204, also referred to herein as movable portion 204, includes the mirrors 281 and 289. The second portion 208, also referred to herein as fixed portion, includes a plurality of relay lenses such as, e.g., relay lenses 282, 283, and a plurality of relay objectives such as, e.g., relay objectives 286 and 287, and the mirror 284. The first portion 204 is configured to receive the optical signal of the returned light RL along a first axis, e.g., the axis of the returned light RL. Moving mechanism 279 is mechanically coupled to first portion 204 to cause translation thereof relative to second portion 208. Movement mechanism 279 can be controlled by, e.g., the controller 102 illustrated in FIG. 1. In other examples, both the portions 204 and 208 may be movable via, e.g., the moving mechanism 279. In alternative examples, the portion 204 is fixed and the portion 208 is movable via, e.g., the moving mechanism 279. Although only two relay lenses 282 and 283 are depicted and discussed herein, more than two relay lenses (or other optical handling components) may be part of the movable focusing module 200, or part of the second portion thereof 208. 204 and 208 are independent but optically aligned to each other. First portion 204 can select whether light is transmitted through second portion 208 or not, and returns the output back into the widefield path, creating a multimodal system for use in OPM.

[0065] Accordingly, in operation, when the SPIM or light sheet mode is activated, the first portion 204 is placed on the light path of the optical signal or returned light RL on the optical axis of the ocular lens 190, and directs the returned light RL from the first portion 204 into the second portion 208 before returning to the first portion 204 and out of mirror 289 as parallel beam 288. In particular, the second portion 208 receives, from the first portion 204 along an axis of the Z direction, the optical signal / returned light RL at the first relay lens 282. The second portion 208 transmits the returned light RL from the relay lens 282 to the second relay lens 283 and to a first relay objective 286, then from the first relay objective 286 to the second relay objective 287, and then transmits the returned light RL to the first portion 204 from the second relay objective 287. The first portion 204 then transmits the returned light RL received from the second relay objective 287 to an image acquisition device such as, e.g., the camera 195 illustrated in FIG. 1. The second portion 208 may include a moving mechanism 280 that is configured to adjust, typically along the Z direction or axis, a position of at least one of the first relay lens 282 and the second relay lens 283 based upon an instruction from the controller 102. As shown in FIG. 2A, a sufficiently large face of the mirror 281 can receive returned light RL at a variety of Z positions, and translation of first portion 204 in the Y axis can also reflect incoming returned light RL to different spatially-resolved positions within second portion 208. Whether in the Z direction or some other direction, a controller (e.g., 102) can be used to implement relative movement between first portion 204 and second portion 208. Although two relay lenses 282 and 283 are illustrated in FIG. 2A, other relay lenses may be present in the second portion 208 to relay the returned light RL from the first relay lens 282 to the first relay objective 286.

[0066] In order to improve the quality, e.g., the focus, contrast or resolution, of the image formed at the image acquisition device, either the relay objectives 286 and 287 may be moved with respect to each other, or the relay lenses 282 and 283 may be moved with respect to each other. Moving the relay objectives 286 and 287 is typically not desirable because it is advantageous to preserve the alignment between the relay objectives 286 and 287 and fix the relay objectives 286 and 287 relative to one another in a fixed coordinate system on, e.g., a platform that supports the movable focusing module 200 and more broadly that supports the multimode imaging system 100. Accordingly, the quality of the image may be improved by adjusting the distance between the relay lenses 282 and 283 along the optical axis thereof, or the Z direction illustrated in FIG. 2A. As such, the relay lens 282 may be movable with respect to the relay lens 283 along the Z direction. Alternatively, the relay lens 283 may be movable with respect to relay lens 282 along the Z direction, or both relay lenses 282 and 283 may be movable with respect to each other along the Z direction. As a result of the adjustment of the relative movement of the relay lenses 282 and 283 with respect to each other, the distance therebetween may be adjusted, and the quality of the image formed at the image acquisition device, e.g., the focus, contrast or resolution, can be improved.

[0067] As discussed above, the distance between the relay lenses 282 and 283 is adjustable via moving mechanism 280. In addition, the distance between the transmission lenses 140 and 150 may also be adjustable via moving mechanism 152. Accordingly, it may also be possible to improve the quality of the image formed on the image capturing device by contemporaneously, or simultaneously, adjusting the distances between the relay lenses 282 and 283 and between the transmission lenses 140 and 150 in order to improve the focus, contrast or resolution of the resulting image formed on the image capturing device.

[0068] FIG. 2B is a schematic view of a movable SPIM focusing module in a multimode imaging system, according to various examples of this disclosure. FIG. 2B depicts a movable focusing module 205, similar to the movable focusing module 175 illustrated in FIG. 1, that converts the redirected beam from an incoming off-centered beam RL into a parallel beam 288. With reference to FIG. 1, the parallel beam 288 is equivalent to the beam 178 that exits the movable focusing module 175. In FIG. 2B, unlike in FIG. 2A, the off-centered incident returned light RL is received directly at the relay objective 286. The first relay objective 286 transmits the incident returned light RL to a second lens 287, and the second relay objective 287 converts the radially off-centered returned light RL to parallel beam onto a mirror 289. As a result, the returned light RL, initially entering the movable focusing module 200 as a radially off-centered light signal or light source signal, is converted to a centered parallel beam 288 that is usable to an image acquisition device such as, e.g., the camera 195 illustrated in FIG. 1. The relay objectives 286 and 287 may be aligned and fixed in space on, e.g., a platform, to preserve the alignment therebetween.

[0069] The movable focusing module 205 may include a moving mechanism 285 that is configured to adjust, along a direction that is the same as, or parallel to, the optical axis of the returned light RL axis incoming to the module at the first relay objective 286, a position of at least one of the first relay objective 286 and the second relay objective 287. Although two relay objectives 286 and 287 are illustrated in FIG. 2B, other relay objective or lenses may be present in the movable focusing module 205 to relay the returned light RL from the first relay objective 286 to the second relay objective 287. The second portion 208 may further include a tunable lens configured to focus the returned light RL transmitted from the second relay objective to the mirror 289. For example, the tunable lens may be or include a liquid tunable lens.

[0070] FIGS. 3 A-3F illustrate images formed via confocal imaging and via light sheet imaging at a plurality of magnifications. In particular, FIGS. 3 A and 3B are comparative images showing the difference between a confocal image in FIG. 3 A and a light sheet or SPIM image in FIG. 3B. It is apparent that the quality of the image 320 illustrated in FIG. 3B and illustrative of SPIM imaging is better than the quality of the image 310 illustrated in FIG. 3 A and illustrative of confocal imaging, thus highlighting the advantage of using light sheet or SPIM imaging over confocal imaging. However, further observation of light sheet images in FIGS. 3C-3F illustrate some unwanted features of SPIM imaging. For example, FIGS. 3C-3F illustrate a same area being imaged at increasing magnifications by a SPIM imaging device similar to the device illustrated in FIGS. 1, 2 A, and 2B, but where the relay lenses 282 and 283 and transmission lenses 140 and 150 are at fixed distances from each other. FIG. 3C has a portion 332 that has a good image quality, and a portion 334 that has a lesser image quality than the portion 332 because focus, contrast and resolution are lost in portion 334. Similarly, FIG. 3D, which illustrates a slightly greater magnification of the image illustrated in FIG. 3C, also has a good quality area 342 and a poor quality area 344. The difference in image quality between areas 334 and 344 and areas 332 and 342, respectively, may be due to the fact that the relay lenses, such as relay lenses 282 and 283 illustrated in FIG. 2A, are fixed in distance with respect to each other. In other examples, the difference in image quality between areas 334 and 344 and areas 332 and 342, respectively, may also be due to the fact that the transmission lenses, such as the transmission lenses 140 and 150 illustrated in FIG. 1, are fixed in distance with respect to each other.

[0071] FIGS. 3E and 3F illustrate images formed via SPIM imaging at greater magnifications still. FIG. 3E shows an area 352 that has a relatively good image quality, e.g., image focus, image contrast and image resolution, and an area 354 has poor image quality. However, FIG. 3F, which is an even greater magnification than FIG. 3E, illustrates an image formed via SPIM imaging that is fairly uniformly of poor quality. Such poor quality may be attributed, as discussed above, to the fact that the relay lenses 282 and 283 illustrated in FIG. 2A are fixed in distance with respect to each other, or to the fact that the transmission lenses 140 and 150 illustrated in FIG. 1 are fixed in distance with respect to each other and do not allow further magnification of the image formed. Accordingly, as the magnification increases the resolution of the formed images 320-360 decreases.

[0072] FIGS. 4A-4B illustrate images formed via SPIM imaging in various focus positions, in accordance with examples of the disclosure. A focus position or configuration is a given configuration of the illumination light that is irradiated on the sample in relation to the optical light that is emitted by the sample as the result of the illumination thereof. FIGS. 4 A and 4B illustrate images of the same sample in two different focus positions or configurations. FIG. 4A illustrates an image 410 formed at the focus position illustrated by 412, and FIG. 4B illustrates an image 420 formed at the focus position illustrated by 422. Image 410 shows a portion 416 that exhibits a good focus, contrast or resolution, and a portion 414 that exhibits poor focus, contrast and resolution. Image 420 shows a portion 428 that exhibits a good focus, contrast and resolution, and portions 424 and 426 that exhibit poor focus, contrast and resolution. The images 410 and 420 have portions thereof exhibiting a good quality, e.g., good focus, contrast and resolution, and portions thereof exhibiting a poor quality because each image has different overlaps such as, e.g., overlap 436 illustrated in FIG. 4D, between the angle or the volume of the optical light path and the angle or the volume of the illumination light path, as further discussed below with respect to FIGS. 4C and 4D.

[0073] FIGS. 4C and 4D illustrate an adjustable focus configuration 430, in accordance with various examples of the disclosure. FIG. 4C illustrates the position 432 of optical light or radiation of a sample, which is the light or radiation emitted by the sample. FIGS. 4C and 4D also show the position 434 of illumination light or radiation, which is portion of the sample that is illuminated. In other words, the optical light portion 432 corresponds to light that is transmitted, diffracted, or otherwise emanating from the sample as a result of the sample being illuminated at the illumination light portion 434. The optical light 432 may be diffracted, or bent by the sample after being received at illumination light portion 434, which causes the focus of the image to be in a different location than the location of illumination.

[0074] Increasing the focus, contrast and resolution of the resulting image by, e.g., adjusting the distance between the relay lenses 282 and 283, can be achieved by moving the position of the optical light portion 432 with respect to the illumination light portion 434, and vice-versa. The optical light portion 432 and the illumination light portion 434 may be moved with respect to each other until an overlap 436 is formed, as illustrated in FIG. 4D. When the overlap 436 between the angle or volume of a path of the optical light 432 and the angle or volume of a path of the illumination light 434 is sufficiently large, the resulting image has good quality at the portion of the sample that corresponds to the overlap 436.

[0075] Conversely, in regions of the sample where there is little or no overlap between a path of the optical light 432 and a path of the illumination light 434, the quality of the resulting image is poor. In examples, varying the distance between the relay lenses 282 and 283 illustrated in FIG. 2A results in a lateral movement of the optical light 432 and / or the illumination light 434. In other examples, increased overlap 436 between the path of the optical light 432 and the path of the illumination light 434 may be achieved by tilting the illumination light 434 or tilting the optical light 432, or tilting and shifting the illumination light 434 and the optical light 432 with respect to each other. Tilting the illumination light 434 and tilting the optical light 432 may be achieved by varying the orientation of the dichroic mirror 125 illustrated in FIG. 1. Shifting the illumination light 434 with respect to the optical light 432, as illustrated in the passage from the FIG. 4C to FIG. 4D, may be achieved by adjusting the distance between the relay lenses 282 and 283, and alternatively or in combination by adjusting the distance between the transmission lenses 140 and 150. Generally, increasing the overlap 436 between the path of the illumination light 434 and the path of the optical light 432 results in increased image quality with better focus, contrast and resolution.

[0076] FIG. 5 depicts an imaging method 500 that includes operation 510 which includes receiving, at a first portion of an optical subassembly, an optical signal along a first axis, the optical subassembly further comprising a second portion that comprises a plurality of relay lenses and a plurality of relay objectives. With reference to FIG. 2 A, the first portion and the second portion of the optical subassembly may correspond to the first portion 204 and the second portion 208 of the optical subassembly 200, and the first axis may correspond to the axis of the returned light RL.

[0077] In some examples, prior to operation 510, the method 500 includes generating a light source signal at a light source, transmitting, via at least one objective lens and via a transmission subassembly that includes a plurality of transmission lenses, the light source signal to a sample. Prior to operation 510, the method 500 may also include transmitting, via the at least one objective and the plurality of transmission lenses, the optical signal generated by the sample to the optical subassembly. Prior to operation 510, the method 500 may also include adjusting a position of at least one of a first transmission lens and a second transmission lens along the first axis so as to adjust the quality of the image formed at the image acquisition device, and thus the quality, e.g., focus, contrast or resolution, of the image formed. With reference to FIGS. 1 and 2, prior to operation 510, the method 500 includes generating the illumination light IL at the laser 110, transmitting the illumination light IL to sample 180 via the objective lens 170, transmitting the optical signal of the returned light RL from the sample 180 to the focusing unit 175 or 200, and adjusting a position of the transmission lenses 140 or 150 along the axis of the illumination light IL and a position of the relay lenses 282 and 283 along the Z direction.

[0078] Operation 520 includes transmitting the received optical signal from the first portion along a second axis to a first relay lens of the plurality of relay lenses in the second portion, the optical signal being transmitted through the plurality of relay lenses and to a first relay objective of the plurality of relay objectives. With reference to FIG. 2 A, the second axis may correspond to the Z direction, the first relay objective corresponds to the relay objective 286 and the second relay objective corresponds to the relay objective 287. Accordingly, during operation 520, the returned light RL is transmitted from the first portion 204 to the second portion 208, and in particular to an off-center portion of the first relay lens 282. The returned light RL is then transferred to an off-center portion of the second relay lens 283, and then to relay objectives 286 and 287.

[0079] Operation 530 includes transmitting the optical signal to the first portion from the second relay objective. With reference to FIG. 2A, the returned light RL is transmitted from the second potion 208 back to the first portion 204, and in particular is transmitted from the second relay objective 287 to the first portion 204.

[0080] Operation 540 includes transmitting the optical signal received from the second relay objective to an image acquisition device to form an image. With reference to FIGS. 1 and 2, the returned light RL that is transmitted from the second relay objective 287 to the first portion 204 is transmitted to the image acquisition device 195.

[0081] Operation 550 includes adjusting, along the second axis, a position of at least one of the first relay lens and a second relay lens to increase a quality of the image formed the image acquisition device. With reference to FIG. 2A, the position of the relay lens 282 may be adjustable with respect to the position of the relay lens 283 along the Z direction, via the moving mechanism 280, and adjusting the position of the first relay lens 282 and the second relay lens 283 may result in improving a focus, contrast or resolution of the image formed at the image acquisition device. The method 500 may also include, with reference to FIGS. 1 and 2, contemporaneously adjusting the position of at least one of the first relay lens 282 and the second relay lens 283 and the position of at least one of the first transmission lens 140 and the second transmission lens 150 so as to improve the quality of the image formed at the image acquisition device 195. With respect to FIGS. 4C and 4D, adjusting the distance between the first relay lens 282 and the second relay lens 283, and alternatively or in combination adjusting the distance between the first transmission lens 140 and the second transmission lens 150, may result in the illumination light 434 to shift laterally with respect to the optical light 432.

[0082] Image quality can be determined as a focus score or other metric. Focus score can include one or more determinations of contrast, resolution, field of view, or some combination of these factors. A focus score or image quality score can be determined for multiple images obtained for a given layer of a 3D image, and the images of the layers can be ranked based on the focus score associated with that image. The instrument settings associated with the highest-ranked images can be compared and combined to determine which settings provide the best results on each layer, or across all layers. Operation 560 includes determining whether the quality of the image formed at the image acquisition device meets or exceeds a threshold value. For example, operation 560 can include determining whether the focus, contrast and / or resolution of the image formed at the image acquisition device exceed a predetermined minimum threshold value. When the quality of the image formed at the image acquisition device is determined not to be sufficient (i.e., at least one of the threshold values is not met), the method 500 continues to operation 550, during which the relative positions of the relay lenses, or the relative positions of the transmission lenses, are further adjusted. With reference to FIG. 2A, when the quality of the image formed at the image acquisition device is not determined to be sufficient, the relative positions of the relay lenses 282 and 283, or the relative positions of the transmission lenses 140 and 150, are further adjusted.

[0083] FIG. 6 depicts a block diagram of a computing device 600 similar configured to control the multimode imaging device 100 discussed above with respect to FIG. 1. In the illustrated example, the computing device 600 may include a bus 602 or other communication mechanism of similar function for communicating information, and at least one processing element 102 (collectively referred to as processing element 102) coupled with bus 602 for processing information. Processing element 102 can be used as shown in FIG. 1 as a controller for a fiber selector or laser in embodiments described herein. Processing element 102 and / or communication device 620 can drive automated or manual movement of components, such as the relative movement between first portion 204 and second portion 208 as shown in FIG. 2A, the relative movement between lenses 282 and 283 in FIG. 2A, or the relative movement between objectives 286 and 287 as shown and described with respect to FIG. 2B.

[0084] As will be appreciated by those skilled in the art, the processing element 102 may include a plurality of processing elements or cores, which may be packaged as a single processor or in a distributed arrangement. Furthermore, a plurality of virtual processing elements 102 may be included in the computing device 600 to provide the control or management operations for, e.g., the multimode imaging device 100 illustrated above.

[0085] The computing device 600 may also include one or more volatile memory(ies) 606, which can for example include random access memory(ies) (RAM) or other dynamic memory component(s), coupled to one or more busses 602 for use by the at least one processing element 102. Computing device 600 may further include static, nonvolatile memory(ies) 608, such as read only memory (ROM) or other static memory components, coupled to busses 602 for storing information and instructions for use by the at least one processing element 102. A storage component 610, such as a storage disk or storage memory, may be provided for storing information and instructions for use by the at least one processing element 102. As will be appreciated, the computing device 600 may include a distributed storage component 612, such as a networked disk or other storage resource available to the computing device 600.

[0086] The computing device 600 may be coupled to one or more displays 614 for displaying information to a user. Optional user input device(s) 616, such as a keyboard and / or touchscreen, may be coupled to Bus 602 for communicating information and command selections to the at least one processing element 102. An optional cursor control or graphical input device 618, such as a mouse, a trackball or cursor direction keys for communicating graphical user interface information and command selections to the at least one processing element. The computing device 600 may further include an input / output (I / O) component, such as a serial connection, digital connection, network connection, or other input / output component for allowing intercommunication with other computing components and the various components of, e.g., the multimode imaging device 100 discussed above.

[0087] In various examples, computing device 600 can be connected to one or more other computer systems via a network to form a networked system. Such networks can for example include one or more private networks or public networks, such as the Internet. In the networked system, one or more computer systems can store and serve the data to other computer systems. The one or more computer systems that store and serve the data can be referred to as servers or the cloud in a cloud computing scenario. The one or more computer systems can include one or more web servers, for example. The other computer systems that send and receive data to and from the servers or the cloud can be referred to as client or cloud devices, for example. Various operations of, e.g., multimode imaging device 100 may be supported by operation of the distributed computing systems.

[0088] The computing device 600 may be operative to control operation of the components of the multimode imaging device 100 through a communication device such as, e.g., communication device 620, and to handle data generated by components of the multimode imaging device 100 through the processing element 102. In some examples, feedback in the form of an image is provided by the computing device 600 in response to the processing element 102 executing instructions contained in memory 606 or 608 and performing operations on data received from the multimode imaging device 100 such as, e.g., receiving and transmitting optical signals and adjusting the positions of relay lenses within a focusing module. Execution of instructions contained in memory 606 and / or 608 by the at least one processing element 102 can render, e.g., the multimode imaging device 100 to perform methods described herein.

[0089] The term “computer-readable medium” as used herein refers to any media that participates in providing instructions to the processing element 102 for execution. Such a medium may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media includes, for example, optical or magnetic disks, such as disk storage 610. Volatile media includes dynamic memory, such as memory 606. Transmission media includes coaxial cables, copper wire, and fiber optics, including the wires that include bus 602. Various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to the processing element 102 for execution. For example, the instructions may initially be carried on the magnetic disk of a remote computer. The remote computer can load the instructions into its dynamic memory and send the instructions over a telephone line using a modem. A modem local to computing device 600 can receive the data on the telephone line and use an infra-red transmitter to convert the data to an infra-red signal. An infra-red detector coupled to bus 602 can receive the data carried in the infra-red signal and place the data on bus 602. Bus 602 carries the data to memory 606, from which the processing element 102 retrieves and executes the instructions. The instructions received by memory 606 and / or memory 608 may optionally be stored on storage device 610 either before or after execution by the processing element 102.

[0090] In accordance with various examples, instructions operative to be executed by a processing element to perform a method are stored on a computer-readable medium. The computer-readable medium can be a device that stores digital information. For example, a computer-readable medium includes a compact disc read-only memory (CD-ROM) as is known in the art for storing software. The computer-readable medium is accessed by a processor suitable for executing instructions configured to be executed.

[0091] This disclosure described some examples of the present technology with reference to the accompanying drawings, in which only some of the possible examples were shown. Other aspects can, however, be embodied in many different forms and should not be construed as limited to the examples set forth herein. Rather, these examples were provided so that this disclosure was thorough and complete and fully conveyed the scope of the possible examples to those skilled in the art.

[0092] Common forms of computer-readable media or computer program products include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, a CD-ROM, digital video disc (DVD), a Blu-ray Disc, any other optical medium, a thumb drive, a memory card, a RAM, PROM, and EPROM, a FLASH- EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read. FIGS. 7A-7D are schematic views of a multimode imaging system including SPIM, according to examples of this disclosure. The multimode imaging system illustrated in FIGS. 7A-7D is substantially similar to the multimode imaging system illustrated in FIG. 1 and discussed above, with the following modifications. In FIG. 7A, the multimode imaging system 100 further includes beam shaping optics or a beam shaping device 123 and an adjustable mirror 127. The beam shaping optics or beam shaping device 123 may achieve a high aspect ratio while providing extended depth of field, and may be configured to generate a light sheet beam such as, e.g., a Gaussian light sheet beam. The adjustable mirror 127 may also be referred to herein as beam steering mirror 127, or pupil position adjustment mirror 127, and is configured to reflect the incident light “IL” received form the fiber selector 120 onto the dichroic mirror 125. The multimode imaging system 100 may also include a rotation and translation mechanism 129, also referred to as motorized actuator, coupled to the adjustable mirror 127 and configured to rotate the adjustable mirror 127 by an angle around an axis thereof. As such, the adjustable mirror 127 is configured to translate the adjustable mirror 127 along a direction “X” of the incident light IL, or a direction “X” that is parallel to the direction of the incident light IL. Although the rotation and translation mechanism 129 is discussed above and illustrated in the drawings as a single mechanism, the rotation and translation mechanism 129 may be or include an individual rotation mechanism or an individual translation mechanism separate from the rotation mechanism. FIGS. 7B and 7C illustrate the above-discussed configurations in greater detail. In various examples, the beam shaping device 123 may replace the adjustable mirror 127.

[0093] FIG. 7B is a schematic view of a multimode imaging system including SPIM, according to examples of this disclosure. In FIG. 7B, the adjustable mirror 127, proximal to the illumination source 110 or the fiber selector 120, is configured to reflect light received from the illumination source 110 onto the objective lens 170 and to be rotatable around an axis thereof so as to reflect the light signal received from the fiber selector 120 onto the dichroic mirror 125 at varying angles such as, e.g., angles al and a2. For example, a rotation of the adjustable mirror at a first angle reflects the light received from the light source at a corresponding first location of the objective lens, and a rotation of the adjustable mirror at a second angle different from the first angle reflects the light received from the light source at a corresponding second location of the objective lens, the second location being different from the first location. The axis of rotation of the adjustable mirror 127 may be along an axis or direction that is angled with respect to both the Z direction and the X direction. As the angle of reflection ai varies, the location of incidence of the illumination light IL on the sample 180 varies. As a result, the angle of the returned light RL also varies, and the overlap 436 between the path of the optical light and the angle or the path of the illumination light discussed above with respect to FIG. 4D varies. Accordingly, it is possible to increase or optimize the amount of overlap between the path of the optical light and the path of the illumination light by varying the angle ai of incidence of the light received from the fiber selector 120 and transmitted to the dichroic mirror 125. As the amount of overlap between the path of the optical light and the path of the illumination light is increased, the quality of the image eventually formed at the image acquisition device or camera 195 is also increased or optimized.

[0094] In examples, the distance between the relay lenses 282 and 283 may be varied together with the angle ai so as to increase the amount of overlap between the path of the optical light and the path of the illumination light. Accordingly, both variations, e.g., variation of the distance between the relay lenses 282 and 283 and the variation in the angle of the incident light, may be performed in a coordinated matter so as to increase or optimize the amount of overlap between the path of the optical light and the path of the illumination light, and as a result increase or optimize the quality of the image formed at the image acquisition device 195.

[0095] FIG. 7C is a multimode imaging system including SPIM, according to examples of this disclosure. In FIG. 7C, the mirror adjustment mechanism 129 is configured to translate the adjustable mirror 127 along a longitudinal direction X that is parallel to the direction of the light received from the illumination source 110 or fiber selector 120, which varies a position of the pupil of the adjustable mirror 127. For example, a longitudinal movement of the adjustable mirror 127 at a first distance along the longitudinal direction X reflects the light received from the light source 110 at a corresponding first location of the objective lens 170, and a longitudinal movement of the adjustable mirror 127 at a second distance along the longitudinal direction X different from the first distance reflects the light received from the illumination source 110 at a corresponding second location of the objective lens 170, the second location being different from the first location. Accordingly, the adjustable mirror 127 is configured to be translatable along a direction “X” that is the direction of the incident light IL, or a direction parallel to the direction of the illumination light IL so as to reflect the light signal received from the fiber selector 120 onto the dichroic mirror 125 at varying locations xO, xl and x2. As the location xi of incidence of illumination light IL on the dichroic mirror 125 varies, the location of incidence of the incident illumination light IL on the sample 180 varies. As a result, the angle of the returned light RL also varies, and the overlap 436 between the path of the optical light and the path of the illumination light discussed above with respect to FIG. 4D varies. Accordingly, it is possible to increase or optimize the amount of overlap between the path of the optical light and the path of the illumination light by varying the location xi of incidence of the light received from the fiber selector 120 and transmitted to the dichroic mirror 125. As the amount of overlap between the path of the optical light and the path of the illumination light is increased, the quality of the image eventually formed at the image acquisition device or camera 195 is also increased or optimized.

[0096] In examples, the distance between the relay lenses 282 and 283 may be varied together with the angle ai and the location xi of incidence of the illumination light IL so as to increase the amount of overlap between the path of the optical light and the path of the illumination light. Accordingly, the three types of variations, e.g., variation of the distance between the relay lenses 282 and 283, variation of the angle ai of the incident light IL, and variation of the location of incidence xi of the IL, may be performed in a coordinated matter so as to increase or optimize the amount of overlap between the path of the optical light and the angle or the volume of the illumination light. As a result, the quality of the image formed at the image acquisition device 195 may be increased or optimized.

[0097] FIG. 7D is a multimode imaging system including SPIM, according to examples of this disclosure. The multimode imaging system illustrated in FIG. 7D is similar to the ones illustrates in FIGS. 7B and 7C, but instead of an adjustable mirror 127, the multimode imaging system illustrated in FIG. 7D includes a spatial light modulator 128 configured to translate the incident illumination light IL as illustrated in FIG. 7B, or to rotate the incident illumination light IL as illustrated in FIG. 7C.

[0098] In one example, instrument positions are determined for an oblique plane microscope as described with respect to FIGS. 1 and 7A.

[0099] First, a sample is modeled as a set of layers. As described above, a series of layers each extending in a plane (referred to above as the corresponding X-Y plane) are stacked one above another in the Z direction, also referred to herein as the height of the layers. These layers can have the same thickness as one another, and the thickness of the layers in the Z direction can vary based upon the desired resolution of the image and upon the wavelengths of the light source to be used to illuminate the layers.

[0100] For each of the layers, an oblique plane image can be formed, such as with a camera or other detector. Each image is taken of an illuminated layer, and is associated with a remote focus position, a pupil position or a steering position at one or more different orientations to the obliquely illuminated plane that makes up that layer.

[0101] As used herein, the remote focus position, pupil position, or steering position refer to the various settings and distances between the components described above with respect to FIGS. 2A, 2B, 7B, 7C, and 7D. For example, a focus position can refer to the focal length of a variable objective or lens described herein. The description of FIG. 7 A refers specifically to pupil positions, though it should be understood that in any use of the objectives described throughout the application a pupil position could be adjusted to form an image of a desired layer. “Steering position,” as used herein refers to the changeable angles or distances between components that are described throughout the application, such as the angle of the reflector 127 in FIG. 7B. Other settings that can be adjusted, falling within the realm of steering positions, can include a distance between a pair of objectives, or a distance between a pair of lenses, a tilt, and / or associated pupil position of an objective.

[0102] For each of the images, a focus score can be determined. As described above, a focus score can be based on contrast, resolution, field of view, or some combination of these factors. A focus score is a data type that is related to the purpose of the imaging process — for example, sufficient contrast to ensure signal-to-noise that sufficient resolution to make out a feature of interest, or sufficient field of view to capture an entire feature or field of interest.

[0103] Where there are multiple images obtained for a given layer, the images can be ranked for each layer based on the focus score associated with that image. The instrument settings associated with the highest-ranked images can be compared and combined to determine which settings provide the best results on each layer, or across all layers. These instrument settings can be stored for each layer as candidate instrument positions, and can be specific to the highest-ranking positions for remote focus position, the pupil position and the steering position, for example.

[0104] Once these instrument settings are stored, one or more final images can be acquired for each of the layers based on at least one candidate instrument position associated with each layer.

[0105] The layer information can include the height, as well as one or more sample features of interest. The sample features can be input by a user at a user interface or obtained automatically using imaging techniques using the plurality of oblique plane images. As the images are taken, they can be prepared at a high-contrast location within each of the layers, and they can be directed to the sample features (if any) that are input as being of interest.

[0106] The images for each layer can be multiple captured or detected datasets at the detector or image forming device, which are then averaged. The averaging can be a straight average, a weighted average, or any other combination of the two captured data sets of two or more images taken at a corresponding two or more locations within each of the one or more layers.

[0107] The varied candidate instrument positions (e.g., remote focus position, a pupil orientation, or a steering position) can be modified in a stepwise fashion or a continuous scan to determine the set point that will result in the highest-ranked image. However, checking every possible setting would be difficult if not impossible. The individual settings can be synchronized to interpolated positions from predetermined positions in some example. That is, if an expected best value is at setpoint A, and test data is taken at A, B, and C, the final position can be set at some fourth value D that is a best estimate of an ideal or suitable position to obtain the desired overall focus score or image quality. The focus score may be determined by a user selecting from among the plurality of oblique plane images a number of images which have a higher contrast, resolution or field of view. These images could be used to set the instrument settings as well, to correspond to the images that were preferred by the user. The focus score itself can be determined automatically by a processor based on contrast, resolution, or field of view metrics. After the user selection(s), the at least one candidate instrument position associated with each layer used to acquire the one or more final images can be selected either automatically based on those selections, or can be set directly by the user from the stored candidate instrument positions.

[0108] The candidate instrument position associated with each layer used to acquire the one or more final images can selected from between at least two candidate instrument positions by selecting the two instrument positions having substantially similar scores, averaging the associated instrument settings, acquiring the one or more final images for each of the at least one or more layers comprises using the same at least one candidate instrument position to acquire each of the multiple images within the layer, and stitching the final images for each of the at least one or more layers together. The multiple images within the layer can differ from one another based on the associated instrument settings for at least one of the one or more layers, such as a position in an X-Y plane associated with a steering position or an initial calibration position. The initial calibration position can be based on a known standard and the associated instrument settings include boundaries in the X-Y plane based on the known standard.

[0109] Although specific examples were described herein, the scope of the technology is not limited to those specific examples. One skilled in the art will recognize other examples or improvements that are within the scope of the present technology. Therefore, the specific structure, acts, or media are disclosed only as illustrative examples. Examples according to the technology may also combine elements or components of those that are disclosed in general but not expressly exemplified in combination, unless otherwise stated herein. The scope of the technology is defined by the following claims and any equivalents therein.

Claims

CLAIMSWhat is claimed is:

1. An optical subassembly comprising: a first portion configured to receive an optical signal along a first axis; a second portion comprising a plurality of relay lenses and a plurality of relay objectives, wherein the second portion is configured to receive the optical signal from the first portion along a second axis, wherein the second portion transmits the optical signal from the plurality of relay lenses to a first relay objective of the plurality of relay objectives and transmits the optical signal to the first portion from a second relay objective of the plurality of relay objectives, and wherein the first portion transmits the optical signal received from the second relay objective to an image acquisition device; and a first moving mechanism configured to adjust, along the second axis, a position of at least one of a first relay lens and a second relay lens of the plurality of relay lenses; wherein moving the at least one of the first relay lens and the second relay lens adjusts a quality of the image formed at the image acquisition device.

2. The optical subassembly of claim 1, wherein the position of the at least one of the first relay lens and the second relay lens adjusts a resolution of the image formed at the image acquisition device.

3. The optical subassembly of either of claims 1 or 2, wherein the optical signal comprises a fluorescent signal.

4. The optical subassembly of any preceding claim, wherein the first portion is a fixed portion and the second portion is a movable portion, and the optical subassembly further comprises a second moving mechanism configured to move the movable portion with respect to the fixed portion along a direction perpendicular to the first axis.

5. The optical subassembly of any preceding claim, wherein the second axis is perpendicular to the first axis.

6. The optical subassembly of any preceding claim, wherein the plurality of relay lenses comprise two relay lenses.

7. The optical subassembly of any preceding claim, wherein the plurality of relay objectives comprise two relay objectives.

8. An optical arrangement, comprising: the optical subassembly of any of claims 1-7; a light source; an objective lens in proximity to a sample; a transmission subassembly comprising a plurality of optical components configured to transmit a light source signal from the sample to an image acquisition device; and a third moving mechanism configured to adjust a position of at least one of a first transmission lens of the plurality of optical components and a second transmission lens of the plurality of optical components along the first axis.

9. The optical arrangement of claim 8, wherein the first moving mechanism of the optical subassembly is configured to adjust the position of at least one of the first relay lens and the second relay lens so as to increase an overlap between an optical light path and an illumination light path.

10. The optical arrangement of claim 8 or claim 9, wherein moving the at least one of the first transmission lens and the second transmission lens adjusts the quality of the image formed at the image acquisition device by increasing the overlap between the optical light path and the illumination light path.

11. The optical arrangement of any of claims 8-10, wherein the position of at least one of the first relay lens and the second relay lens and the position of at least one of the first transmission lens and the second transmission lens are contemporaneously adjusted via the first moving mechanism and the third moving mechanism, respectively, so as to improve the quality of the image formed at the image acquisition device.

12. The optical arrangement of any of claims 8-11, wherein the position of at least one of the first relay lens and the second relay lens and the position of at least one of the first transmission lens and the second transmission lens are contemporaneously adjusted via the first moving mechanism and the third moving mechanism, respectively, so as to improve a resolution of the image formed at the image acquisition device.

13. An imaging method comprising: receiving, at a first portion of an optical subassembly, an optical signal along a first axis, the optical subassembly further comprising a second portion that comprises a plurality of relay lenses and a plurality of relay objectives; transmitting the received optical signal from the first portion along a second axis to a first relay lens of the plurality of relay lenses in the second portion, the optical signal being transmitted through the plurality of relay lenses and to a first relay objective of the plurality of relay objectives; transmitting the optical signal to the first portion from a second relay objective of the plurality of relay objectives; transmitting the optical signal received from the second relay objective to an image acquisition device to form an image; and adjusting, along the second axis, a position of at least one of the first relay lens and a second relay lens of the plurality of relay lenses to increase a quality of the image formed at the image acquisition device.

14. The method of claim 13, wherein adjusting the position of at least one of the first relay lens and a second relay lens comprises adjusting a resolution of the image formed at the image acquisition device.

15. The method of claim 13 or claim 14, wherein prior to receiving the optical signal, the method comprises: generating a light source signal at a light source; transmitting, via at least one objective lens and via a transmission subassembly that comprises a plurality of transmission lenses, the light source signal to a sample; transmitting, via the at least one objective and the plurality of transmission lenses, the optical signal generated by the sample to the optical subassembly; and adjusting a position of at least one of a first transmission lens of the plurality of transmission lenses and a second transmission lens of the plurality of transmission lenses along the first axis so as to increase a quality of the image formed at the image acquisition device.

16. The method of claim 15, wherein adjusting the position of at least one of the first transmission lens and the second transmission lens comprises adjusting a resolution of the image formed at the image acquisition device.

17. The method of claim 15 or claim 16, further comprising contemporaneously adjusting the position of at least one of the first relay lens and the second relay lens and the position of at least one of the first transmission lens and the second transmission lens so as to improve the quality of the image formed at the image acquisition device.

18. An optical arrangement comprising: a first portion configured to receive an optical signal along a first axis; a second portion comprising a plurality of relay lenses and a plurality of relay objectives, wherein the second portion receives the optical signal from the first portion, along a second axis, and wherein the second portion transmits the optical signal from the plurality of relay lenses to a first relay objective of the plurality of relay objectives and transmits the optical signal to the first portion from a second relay objective of the plurality of relay objectives, and wherein the first portion transmits the optical signal received from the second relay objective to an image acquisition device;a light source; an objective lens in proximity to a sample stage configured to hold a sample; a transmission subassembly between the light source and the objective lens, the transmission subassembly comprising an adjustable mirror coupled to a mirror adjustment mechanism and a plurality of adjustable transmission lenses, the transmission subassembly being configured to transmit a light source signal from the light source to the sample and to transmit the optical signal emitted from the sample to the optical arrangement via the plurality of adjustable transmission lenses; wherein the adjustable mirror is configured to reflect light received from the light source onto the objective lens.

19. The optical arrangement of claim 18, wherein the transmission subassembly further comprises a beam shaping device between the light source and the adjustable mirror.

20. The optical arrangement of claim 18 or claim 19, wherein the mirror adjustment mechanism is configured to rotate the adjustable mirror around an axis of rotation thereof.

21. The optical arrangement of any of claims 18-20, wherein: a rotation of the adjustable mirror at a first angle reflects the light received from the light source at a corresponding first location of the objective lens; and a rotation of the adjustable mirror at a second angle different from the first angle reflects the light received from the light source at a corresponding second location of the objective lens, the second location being different from the first location.

22. The optical arrangement of any of claims 18-21, wherein a rotation of the adjustable mirror at a non-perpendicular angle to reflect the light received from the light source at a corresponding location of the objective lens varies an overlap between an optical light path emitted by the sample and an illumination light path transmitted to the sample from the light source compared to a light that is reflected at a perpendicular angle.

23. The optical arrangement of claim 22, wherein an increase in the overlap between the optical light path and the illumination light path increases a quality of the image formed at the image acquisition device.

24. The optical arrangement of any of claims 18-23, wherein the mirror adjustment mechanism is configured to translate the adjustable mirror along a longitudinal direction that is parallel to a direction of the light received from the light source.

25. The optical arrangement of claim 24, wherein: a longitudinal movement of the adjustable mirror at a first distance along the longitudinal direction reflects the light received from the light source at a corresponding first location of the objective lens; and a longitudinal movement of the adjustable mirror at a second distance along the longitudinal direction different from the first distance reflects the light received from the light source at a corresponding second location of the objective lens, the second location being different from the first location.

26. The optical arrangement of claim 24 or claim 25, wherein a longitudinal movement of the adjustable mirror along the longitudinal direction to reflect the light received from the light source at a corresponding location of the objective lens varies an overlap between a path of an optical light emitted by the sample and a path of an illumination light transmitted to the sample from the light source.

27. The optical arrangement of claim 26, wherein an increase in the overlap between the optical light path and the illumination light path increases a quality of the image formed at the image acquisition device.

28. An imaging method comprising: generating a light source signal at a light source;directing the illumination light from the light source signal to a sample via at least one objective lens and via a transmission subassembly that comprises a plurality of transmission lenses and an adjustable mirror proximal to the light source; directing, via the at least one objective and the plurality of transmission lenses, the returned optical signal from the sample to an optical subassembly; receiving, at a first portion of the optical subassembly, the returned optical signal along a first axis, the optical subassembly further comprising a second portion that comprises a plurality of relay lenses and a plurality of relay objectives; transmitting the returned optical signal from the first portion along a second axis to a first relay lens of the plurality of relay lenses in the second portion, the returned optical signal being transmitted through the plurality of relay lenses and to a first relay objective of the plurality of relay objectives; transmitting the returned optical signal to the first portion from a second relay objective of the plurality of relay objectives; transmitting the returned optical signal received from the second relay objective to an image acquisition device to form an image; and adjusting a configuration of the adjustable mirror so as to increase a quality of the image formed at the image acquisition device.

29. The method of claim 28, wherein the transmission subassembly further comprises a beam shaping device between the light source and the adjustable mirror.

30. The method of claim 28 or claim 29, wherein adjusting the configuration of the adjustable mirror comprises adjusting one of a resolution and a contrast of the image formed at the image acquisition device.

31. The method of any of claims 28-30, wherein adjusting the configuration of the adjustable mirror comprises rotating the adjustable mirror around an axis of rotation so as to improve the quality of the image formed at the image acquisition device.

32. The method of claim any of claims 28-31, wherein rotating the adjustable mirror around the axis of rotation comprises varying an overlap between a path of the returned optical signal from the sample and a path of the illumination light transmitted to the sample from the light source.

33. The method of claim 32, wherein increasing the overlap between the returned optical signal path and the illumination light path comprises increasing a quality of the image formed at the image acquisition device.

34. The method of any of claims 28-33, wherein adjusting the configuration of the adjustable mirror comprises translating the adjustable mirror along a longitudinal direction parallel to a direction of incidence of the light received from the light source so as to improve the quality of the image formed at the image acquisition device.

35. The method of claim 34, wherein translating the adjustable mirror along the longitudinal direction comprises varying an overlap between a path of the optical light emitted by the sample and a path of the illumination light transmitted to the sample from the light source.

36. The method of claim 35, wherein increasing the overlap between the optical light path and the illumination light path comprises increasing a quality of the image formed at the image acquisition device.

37. An optical subassembly comprising: a focusing module configured to receive an optical signal along an optical axis and comprising a plurality of relay objectives, wherein the focusing module receives the optical signal at a first relay objective of the plurality of relay objectives along the optical axis, and transmits the optical signal from the first relay objective to a second relay objective of the plurality of relay objectives along the optical axis, and then to an image acquisition device; anda moving mechanism configured to adjust, along the axis, a position of at least one of the first relay objective and a second relay objective of the plurality of relay objectives along the optical axis; wherein moving the at least one of the first relay objective and the second relay objective adjusts a quality of the image formed at the image acquisition device.

38. The optical subassembly of claim 37, wherein the position of the at least one of the first relay objective and the second relay objective adjusts a resolution of the image formed at the image acquisition device.

39. The optical subassembly of claim 37 or claim 38, wherein the plurality of relay objectives comprise two relay objectives.

40. A method of determining instrument positions for an oblique plane microscope, the method comprising: receiving layer information from the division of a three-dimensional sample into one or more layers; for each of the one or more layers, receiving a plurality of oblique plane images of the three-dimensional sample, each image associated with a remote focus position, a pupil position or a steering position at one or more different orientations to an obliquely illuminated plane of the three-dimensional sample; determining, for each of the plurality of images, a focus score that is related to at least one of contrast, resolution, and field of view; ranking the images received for each layer based on the focus score associated with that image; storing instrument settings associated with a number of highest ranking images for each layer as candidate instrument positions, the instrument settings comprising one or more of the remote focus position, the pupil position and the steering position; andacquiring one or more final images for each of the at least one or more layers based on at least one candidate instrument position associated with each layer.

41. The method of claim 40, wherein the one of more layers comprises at least two layers.

42. The method of claim 40 or claim 41 , wherein the layer information comprises a height of the layer within the sample.

43. The method of any of claims 40-42, wherein the layer information comprises one or more sample features, and wherein the sample features can be input by a user at a user interface or obtained automatically using imaging techniques using the plurality of oblique plane images.

44. The method of any of claims 40-43, wherein the plurality of oblique plane images are taken at a high-contrast location within each of the one or more layers.

45. The method of any of claims 40-44, wherein the plurality of oblique plane images are each an average of two or more images taken at a corresponding two or more locations within each of the one or more layers.

46. The method of any of claims 40-45, wherein a remote focus position, a pupil orientation, or a steering position can be determined in a stepwise fashion or a continuous scan, or can be synchronized to interpolated positions from predetermined positions.

47. The method of any of claims 40-46, wherein the focus score is determined by a user selecting from among the plurality of oblique plane images a number of images which have a higher contrast, resolution or field of view.

48. The method of any of claims 40-46, wherein the focus score is determined automatically by a processor based on contrast, resolution, or field of view metrics.

49. The method of any of claims 40-48, wherein the at least one candidate instrument position associated with each layer used to acquire the one or more final images is selected by the user from the stored candidate instrument settings.

50. The method of any of claims 40-49, wherein the at least one candidate instrument position associated with each layer used to acquire the one or more final images is selected from between at least two candidate instrument positions by: selecting the two instrument positions having substantially similar scores; averaging the associated instrument settings; acquiring the one or more final images for each of the at least one or more layers comprises using the same at least one candidate instrument position to acquire each of the multiple images within the layer; and stitching the final images for each of the at least one or more layers together, wherein the multiple images within the layer differ from one another are based on the associated instrument settings for at least one of the one or more layers.

51. The method of any of claims 40-50, wherein the focus score is based upon contrast.

52. The method of any of claims 40-50, wherein the focus score is based upon field of view.

53. The method of any of claims 40-52, wherein the associated instrument settings include a distance between a pair of objectives, or a distance between a pair of lenses.

54. The method of any of claims 40-52, wherein the associated instrument settings include a tilt and associated pupil position of an objective.

55. The method of any of claims 40-52, wherein the associated instrument settings include a position in an X-Y plane associated with a steering position.

56. The method of any of claims 40-55, wherein the associated instrument settings are based upon an initial calibration position.

57. The method of claim 56, wherein the initial calibration position is based on a known standard and the associated instrument settings include boundaries in an X-Y plane based on the known standard.

Images