Systems and methods for multimode content screening

Abstract

An optical arrangement includes a light source, an objective lens in proximity to a sample, an ocular lens in proximity to an image detecting 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 transmits a first fluorescent signal from the sample to the image detecting 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 is movable with respect to an optical axis of the ocular lens and can transmit a second fluorescent signal at an oblique angle to the objective lens from the sample to the image detecting device.

Description

[0001] SYSTEMS AND METHODS FOR MULTIMODE CONTENT SCREENING

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 510,015, filed June 23, 2023, the disclosure of which is hereby incorporated herein by reference in its entirety.

[0004] INTRODUCTION

[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 there are several 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 arrangement including a light source, an objective lens in proximity to a sample, an ocular lens in proximity to an image detecting device, a first optical subassembly 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 returned light from the sample to the image detecting 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 modified returned light at an oblique angle to the objective lens from the sample to the image detecting device.

[0008] In another example of the above aspect, the first light source signal is transmitted to the sample through a central axis of the objective lens. In an example, each of the returned light and the modified returned light includes one of a fluorescent signal and a transilluminated signal. In another example, the optical arrangement further includes a controller configured to control the light source to transmit at least one of the first light source and the second light source. In a further examples, the optical arrangement further includes a moving mechanism, wherein the controller is further configured to control the moving mechanism to shift the movable portion of the second optical subassembly at least one of onto the optical axis and off the optical axis in a direction substantially perpendicular to the optical axis. In examples, when the movable portion is on the optical axis, the modified returned light is received at the second optical subassembly as an oblique plane signal and is transmitted to the image detecting device as a signal central to an axis of the image detecting device. In yet another example, the objective lens, the first optical subassembly, the second optical subassembly, and the ocular lens are arranged on a single system. In a further example, the moving mechanism is configured to shift the movable portion with respect to the optical axis on the single system.

[0009] In another example of the above aspect, the second optical subassembly further includes a dichroic mirror on a path of the second light source signal. In a further example, the first optical subassembly includes one of a wide field microscope, a confocal microscope, and a brightfield transillumination microscope, and the second optical subassembly includes a light sheet microscope. For example, the optical arrangement further includes a movable stage configured to move the sample in one or more directions on a plane that is central to the objective lens. In another example, the optical arrangement further includes a light source signal selector configured to select one of the first light source signal and the second light source signal. For example, the light source signal selector includes at least a first optical fiber configured to emit the first light source signal therethrough and a second optical fiber configured to emit the second light source signal therethrough. In an example, at least one of the first optical fiber and the second optical fiber include a fiber bundle, the fiber bundle including a plurality of fibers.

[0010] In another aspect, the technology relates to a method of performing multimode imaging, the method including generating a first light source signal at a light source, transmitting, via a first optical subassembly, the first light source signal to a sample through an objective lens, transmitting, via the first optical subassembly, a returned light from an area of the sample to an image detecting device, generating a second light source signal at the light source, transmitting, via a second optical subassembly including a fixed portion and a movable portion, the second light source signal through an off-centered portion of the objective lens to the sample in an oblique illumination, and transmitting, via the movable portion of the second optical subassembly, a modified returned light from a portion of the area of the sample to the image detecting device at an oblique angle to the objective lens.

[0011] In an example of the above aspect, at least one of transmitting the returned light from the area of the sample includes forming a first image of the area of the sample at the image detecting device based on the transmitted returned light, and transmitting the modified returned light from the portion of the area of the sample includes forming a second image of the portion of the area of the sample at the image detecting device based on the transmitted modified returned light. In another example, transmitting the first light source signal includes transmitting the first light source signal to the sample through a central axis of the objective lens. In a further example, when the second light source signal is transmitted to the sample: the movable portion of the second optical subassembly is positioned on an optical axis of the image detecting device, and transmitting the modified returned light includes transmitting a fluorescent oblique plane signal from the sample to the image detecting device. In yet another example, when the first light source signal is transmitted to the sample, the movable portion of the second optical subassembly is off the optical axis of the image detecting device. In a further example, generating the first light source signal includes performing one of widefield microscopy and confocal microscopy, and generating the second light source signal includes performing light sheet microscopy.

[0012] In other examples of the above aspect, the method further includes switching between the one of widefield microscopy and confocal microscopy and the light sheet microscopy based on a selection. In a further example, the method further includes arranging a second sample in place of the sample, performing one of widefield microscopy and confocal microscopy on an area of the second sample, and performing light sheet microscopy on a portion of the area of the second sample. In an additional example, the method further includes transmitting, via the movable portion of the second optical subassembly, a further modified returned light from a different portion of the area of the sample to the image detecting device at an oblique angle to the objective lens. In yet another example, transmitting the returned light includes transmitted a first fluorescent signal, and transmitting the modified returned light includes transmitted a second fluorescent signal.

[0013] In another aspect, the technology relates to an optical arrangement including a light source on a first side of a sample, an objective lens on opposite side of the sample, an ocular lens in proximity to an image detecting device, a first optical subassembly configured to transmit a light source signal from the light source to the sample, the first optical subassembly being further configured to transmit a returned light from the sample to the image detecting device, a second optical subassembly including a fixed portion and a movable portion and configured to receive the returned light through an off-centered portion of the objective lens 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 the returned light to the image detecting device.

[0014] In another example, the optical arrangement further includes a controller configured to control the light source to transmit the light source to the sample. In an example, the optical arrangement further includes a moving mechanism, wherein the controller is further configured to control the moving mechanism to shift the movable portion of the second optical subassembly at least one of onto the optical axis and off the optical axis in a direction substantially perpendicular to the optical axis. For example, when the movable portion is on the optical axis, the returned light is received at the second optical subassembly as an oblique plane signal and is transmitted to the image detecting device as a signal central to an axis of the image detecting device. In yet another example, the objective lens, the first optical subassembly, the second optical subassembly, and the ocular lens are arranged on a single system. In a further example, the moving mechanism is configured to shift the movable portion with respect to the optical axis on the single system. In a further example, the second optical subassembly further includes a dichroic mirror on a path of the transmitted signal. In an additional example, the first optical subassembly includes a brightfield microscope, and the second optical subassembly includes a light sheet microscope. In another example, the returned light includes a transilluminated signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGS. 1A-1E are schematic views of a multimode imaging system and components thereof, configured in various modes, according to examples of this disclosure.

[0016] FIG. 2 is a schematic view of a movable focusing module, according to various examples of this disclosure.

[0017] FIGS. 3A-3D are partial perspective views of a multimode imaging system, according to various examples of this disclosure.

[0018] FIGS. 4A-4B depict a method of performing multimode imaging, in accordance with various examples of the disclosure.

[0019] FIG. 5 depicts a block diagram of a computing device.

[0020] DETAILED DESCRIPTION

[0021] Conventional optical microscopy provides high resolution (about 200 nm) images and has a wide range of applications, from inspection of electronic devices to cell biology. In many cases, it is desirable to obtain optically sectioned images, e.g., images of axially thin slices through the sample. The conventional method for obtaining high quality optically sectioned images is confocal microscopy, which includes scanning a point of illumination and detecting the reflected or fluorescent light back to a confocal point detector, and typically requires scanning the point source and detection region over the sample in two or three dimensions, depending on whether a 2D or 3D image is required or desired. Such scanning can limit the data acquisition rate or, if rapid scanning is employed, may increase the peak power at the sample, which can lead to increased photodamage and phototoxicity of biological samples. Other conventional optical microscopy techniques include spinning disc confocal microscopy, which has, rather than a single pinhole, a spinning disc confocal microscope that includes a large number of pinholes arranged in spirals on an opaque disk which rotates at high speed. When spun, the pinholes scan across the sample in rows, building up an image. Using a spinning disc typically improves the speed of image acquisition compared to a conventional optical microscope, but suffers from the same shortcomings as discussed above with respect to conventional optical microscopes. Another technique for obtaining optically sectioned images is that of SPIM (Selective Plane Illumination Microscopy), also referred to as light sheet microscopy, which uses two objective lenses, separated by an angle of 90° 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. One such 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 may be required to be used. Other drawbacks to overcome for SPIM in high content screening include difficulty in sample preparation.

[0022] SPIM is described, for example, in US Patent Application Serial No. 63 / 614 / 743, which is hereby incorporated by reference in its entirety.

[0023] Other challenges may be encountered when using SPIM including, e.g., that consumable compatibility with an imaging platform may be limited in a multimodal SPIM platform. Other challenges include the fact that finding objects of interest and collecting the images via SPIM may be slow and may require a large amount of data storage while smaller field of view (FOV) of SPIM reduces throughput of acquisition and makes imaging the volume of interest more difficult. In addition, image stitching for larger FOV is necessary but challenging to do in SPIM, presenting data to quickly orient the user to the sample in real world is often cumbersome or inaccurate due to aberrations and errors in the imaging system, and the sample index of refraction may limit 3D image reconstruction and image quality of SPIM images.

[0024] There is therefore a need to be able to use a technique similar to SPIM, but using a single objective lens at the sample while avoiding or reducing the aberration effects. In order to address the above drawbacks, examples of the disclosure provide a multi-mode 3D imaging platform that allows an array of microscopy techniques including widefield, confocal, brightfield and SPIM technologies with automated control and integrated workflows. An advantage of such a system is the use of a single microscope having a common optical axis with the ability to share some of the same objectives, lenses, filters, and cameras. Another advantage is the ability to automatically, or manually, choose the mode (confocal, widefield, brightfleld, or SPIM) by redirecting the optical path without needing to perform a difficult realignment or switch between multiple microscope systems. This enables a wide range of sample consumable formats, such as microscope slides and microplates, to use a common environmental control. Additionally, it enables fast switching between imaging modes for improved performance, simplifies setup and verification method between imaging modes and 3D reconstruction, and reduces the chance of moving the sample within the consumable.

[0025] Other advantages of the disclosure include having an integrated arrangement which uses both widefield and SPIM to survey a wide sample area in widefield mode, and to locate regions of interest to be 3D scanned in SPIM mode with 3D stitching of small FOV SPIM images, solving the FOV problem. Additional advantages include image software reconstruction that may allow a user to view widefield, brightfleld, confocal, and SPIM images with similar and compatible visualization of the same volume in the sample. Consumables can be added or removed within the ecosystem, such as an incubator, plate reader, or other components, without introducing or removing those components to or from the environmentally controlled area. Further advantages include workflow with independent adjustment of the illumination and imaging paths for SPIM which may reduce some of the impact with the sample index of refraction and consumable compatibility. Other advantages may include continuous axial series imaging in SPIM that extends the range of sample depth as well as increase the acquisition speed, measuring and compensating optical aberrations by using widefield illumination and / or imaging to identify optical distortions and image plane tilt for corrections -such as those used for measuring optical distortion and image plane tilt- in SPIM acquisition, image reconstruction, stitching, and analysis.

[0026] Examples of the disclosure include novel solutions in hardware and software to support current and future high content / high throughput screening needs with a single microscope comprising a multimode imaging system. Examples of the disclosure increase assay and sample feasibility and help unlock the potential to screen complex 3D structures (including organoids and other models) at high speed, contrast, and resolution while maintaining compatibility with existing analysis and visualization tools from common widefield, brightfleld and confocal modalities. Fluorescent emissions are the emissions of light from a sample at one wavelength 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 fluorophores like fluorescent dyes, fluorescent proteins, and fluorescent nanoparticles or other fluorescence treatments.

[0027] 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.

[0028] FIGS. 1A-1D are schematic views of a multimode imaging system 100, according to various examples of this disclosure. The multimode imaging system 100 includes an optical arrangement that may be operated in one or more of a widefield imaging mode 100A (depicted in FIG. 1A), a confocal imaging mode 100B (depicted in FIG. IB), a brightfield imaging mode 100C (depicted in FIG. 1C), and a SPIM imaging mode 100D (depicted in FIG. ID). FIGS. 1A-1D are described concurrently and not every component described is depicted in every figure. FIGS. 1A-1D describe a laser 110 or other light source that is configured to generate an incident light “IL,” a fiber selector 120 configured to direct the IL to a given or desired light path, filters 130 and 135, an ocular lens 190, and an image detecting device such as, e.g., at the camera 195. In other embodiments, laser 110 could be replaced with an alternative light source, such as a light emitting diode (LED). LEDs can provide advantages in providing noncoherent light sources, highly defined wavelengths of light output, and lower power use per lumen created, as well as being lower cost and having longer life than a laser. In still further embodiments, laser 110 could be replaced with a light source that produces multiple wavelengths and a filter or other optical componentry to transmit only desired wavelengths.

[0029] 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 IL therethrough. In examples, the selectable first optical fiber 122 or the selectable second optical fiber 124 may be or include a fiber bundle, the bundle including a plurality of fibers. In one aspect of the invention, the fiber selector 120 comprises a motorized holder including a demultiplexer 196 (FIG. IE) configured to allow the selectable optical fibers 122 and 124 to be proximity coupled with greater than 85 percent efficiency. The proximity coupled demultiplexer 196 allows lens free selection of the desired optical path with low loss. In another aspect of the invention, the demultiplexer 196 may be combined with a multiplexer in a single unit referred to as a multiplexer 197. The multiplexer 197 may be used to allow multiple light sources, for example 110a and 110b to be used on either of the selectable optical fibers 122, 124 for use in a desired imaging modes. This multiplexer / demultiplexer enables flexibility to use multiple sources and optical modes in as single microscope without tedious realignment. Multiple demultiplexer / multiplexers may be connected in series to increase options, for example either mixing or not mixing sources for the same optical mode, in addition to just switching between optical modes or optical sources.

[0030] In the multimode imaging system 100, the spatial irradiance on the distal end of the selectable optical fibers 122, 124 is important, as well as the need to improve radiant distribution. Bending fibers can improve radiant distribution. The spatial radiance can be improved by using the same or slightly different core size / shaped fibers, or mixing rods, for example, using 1mm core fiber coupled to 1.5mm core fiber, using a square core fiber or mixing rod in-line with the final system fiber. Additionally, using Anti-Reflection (AR) coated fibers and rods, the losses between interfaces can be minimal and can be fully aligned with lower mechanical tolerances when changing fiber or mixing rod shapes.

[0031] By adjusting the lateral and / or axial spacing between the selectable optical fibers 122 and 124, the coupling efficiency will decrease. This decreased light exposure to the sample 180 can be lower than the light source dynamic range and guarantee protection of the sample 180 from excessive light.

[0032] In this manner, the uniformity can be improved using fiber alone, without the need for additional lenses or optics. Additionally, it is possible to change the intensity to less than 10% of the maximum by decentering the fibers alone without adding any optical elements or parts in the path. This cuts cost and complexity while improving reliability and functionality. Furthermore, this configuration enables the incorporation of additional light sources on different fibers to be switched to without the need for tedious optical alignment or concern for lateral and axial color aberrations.

[0033] FIG. 1A-1D also depict a first lens 140 and a second lens 150, a mirror 160, an objective lens 170. 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 central to the objective lens 170. Details regarding the above components are described further below, as relevant to the various modes (widefield 100A, confocal 100B, and SPIM 100D). Controller is configured to drive a motor (not shown) connected to the mechanical stages, which move the optics with respect to the optical axis. Through its configuration, controller 102 can switch between the various modes illustrated in FIGS. 1A-1D and, e.g., select the optical fibers 122 and 124 in view of a desired imaging mode. Operation of at least one of the laser 110 or the fiber selector 120 is controlled via controller 102 to transmit the IL in a light sheet light path, the IL being referred to in this case as a first light source signal, or in a widefield or confocal light path, the IL being referred to in this case as a second light source signal, though the terminology “first” and “second” here are interchangeable.

[0034] FIG. 1A illustrates a schematic view of the multimode imaging system 100 in a widefield imaging mode 100A, in accordance with examples of the disclosure. In FIG. 1A, the IL is generated by the laser 110 and transmitted through the fiber selector 120 to the filter 130 which may be or include, e.g., a dichroic mirror, and is transmitted to first and second lenses 140 and 150. The IL emitted from the laser 110 thus travels through the center of each lens 140 and 150, intersects the center of the mirror 160, passes through the central axis of the objective lens 170, and reaches the sample 180. The objective lens 170 thus illuminates the sample 180 in a perpendicular direction with respect to a plane of the objective lens 170 and allows an observation of the sample 180. The sample 180 may be disposed within a multi-well plate or microplate that is movable via a movable stage 185 in a planar or XY direction parallel to the objective lens 170, so as to remain perpendicular to the angle of incidence of the IL passing through the center portion of the objective lens 170.

[0035] When the IL is received at the objective lens 170 and transmitted to the sample 180, a sample emitted signal “SES” (also referred to as Returned Light RL, as defined elsewhere herein) is emitted off the sample 180 and travels through the objective lens 170 in the generally opposite direction of the IL. The SES may be a fluorescent signal or light, and may be a transilluminated signal or light, e.g., a transmitted illuminated signal or light. For example, the SES travels through an off-centered radial portion of the objective lens 170, is reflected off of the mirror 160, and travels through the second lens 150 and the first lens 140 in an opposite direction of the IL.

[0036] The SES is at a different wavelength than the IL. Accordingly, where filter 130 is a dichroic mirror or the like, instead of reflecting back to the fiber selector 120 the SES continues to travel through filters 130 and 135 before reaching an ocular lens 190 that focuses the SES onto an image detecting device 195 such as, e.g., a camera. In an example, the SES, which may be a fluorescent signal or a transmitted illuminated signal, is reflected or emitted from the sample in the off-centered radial portion of the objective lens 170 but remains substantially symmetrical with respect to the central axis thereof. In addition, any portion of the SES that is reflected or emitted off of the sample in a region corresponding to the center of the objective lens 170 may be reflected or emitted along the central axis thereof. In the widefield imaging mode 100A the controller 102 is configured to, inter alia, control operation of the laser 110 and of the fiber selector 120.

[0037] FIG. IB illustrates a schematic view of the multimode imaging system 100 in a confocal imaging mode 100B, in accordance with examples of the disclosure. In FIG. IB, similar to the multimode imaging system 100, in the widefield imaging mode 100 A, illustrated in FIG. 1A, the IL is generated by the laser 110 and transmitted through the fiber selector 120 to the filter 130, and is then transmitted to first and second lenses 140 and 150. In the case of the multimode imaging system 100 in the confocal imaging mode 100B, the system further includes a confocal spinning disk 155 between the first lens 140 and the second lens 150. The rest of the path of the IL is similar to the path described above with respect to FIG. 1 A. With respect to the SES, when reflected or emitted off the sample 180, the SES passes through the objective lens 170 in a radially off-centered position, is reflected by the mirror 160 and passes through the second lens 150 before passing through the confocal spinning disk 155 and then reaching the first lens 140. The rest of the path of the SES is similar to the path described above with respect to FIG. 1A.

[0038] The optical assembly that includes the laser 110, the fiber selector 120, the filter 135, the first lens 140, the second lens 150, optionally the confocal spinning disk 155, the mirror 160, the objective lens 170, and the ocular lens 190 may be referred to herein as a first optical subassembly. The first optical subassembly may also include other elements as described with respect to FIGS. 1A-1D. Similar to the multimode imaging system 100 in the widefield imaging mode 100 A, the multimode imaging system 100 in the confocal imaging mode 100B further includes a controller 102 configured to control operation of the laser 110 and of the fiber selector 120, as well as other components of the system, and the multimode imaging system 100 in the brightfield mode 100C also includes controller 102 to control operation of the brightfield illumination source 112, as well as other components of the system. Accordingly, in the case of the confocal 100B and widefield 100A modes, the first optical subassembly is configured to transmit the light source signal, also referred to as the IL, from the laser 110 through a central axis of the objective lens 170 to the sample 180, and to transmit the fluorescent or emitted signal, also referred to as the SES or RL, from the sample 180 through an off- centered or radial portion of the objective lens 170 to the image detecting device 195.

[0039] In the case of the brightfield imaging mode 100C, the first optical subassembly is configured to transmit the light source signal, also referred to as the IL, from the brightfield illumination source 112 to the sample 180, and to transmit the attenuated signal, also referred to as the SES, from the sample 180 through an off-centered or radial portion of the objective lens 170 to the image detecting device 195. In operation during the SPIM mode, also referred to herein as light sheet mode, the laser 110 generates the IL that is transmitted through fiber selector 120 to the filter 130 (e.g., a dichroic mirror). The illumination light received at the filter 130 that is reflected onto mirror 160 passes to objective lens 170. The path of the IL is radially off- centered with respect to the objective lens 170 or other focusing optic, such that the 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 110 is referred to as an optical subassembly. One example of such an optical subassembly can include fiber selector 120, filter 130, 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 IL to the sample at an angle.

[0040] FIG. 1C also depicts an optical light, which may be an emission light or a so- called returned light “RL,” which is used herein for clarity. Emitted or returned light RL can be emanate from the sample 180 at any of a range of oblique angles that affect how the RL travels through the objective lens 170 in an off-centered configuration in the opposite direction of the 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 RL and is characterized by the emission of light by a sample that has absorbed light or other electromagnetic radiation. The 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 RL continues to travel through an off-centered radial portion of the first lens 140, through filters 130 and 135, and onto a movable selection module 175. The movable selection module 175 may be movable and may be configured to receive the off-centered RL and convert the 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 RL onto the image acquisition device 195 such as, e.g., camera 195.

[0041] FIG. 1C illustrates a schematic view of the multimode imaging system 100 in a brightfield imaging mode 100C, in accordance with examples of the disclosure. In brightfield imaging, sample illumination is transmitted from one side thereof and observed at an opposite side thereof. The sample illumination is typically performed via a white bright light, and the contrast observed on the other side of the sample is typically cause by attenuation of the transmitted light in dense areas of the sample. Brightfield imaging typically allows for label free imaging to find or study samples without, e.g., bleaching the sample. In FIG. 1C, the RL is generated by a brightfield illumination source 112 directly onto the sample 180 located at the movable stage 185. The sample 180 may be disposed within a multi-well plate or microplate that is movable via movable stage 185 in a planar or XY direction parallel to the objective lens 170, so as to remain perpendicular to the angle of incidence of the RL. The RL emitted from the brightfield illumination source 112 thus travels through the sample 180 and reaches the objective lens 170. The objective lens 170 thus receives the RL from the sample 180 and transmits the sample illuminated signal SES, to the objective lens 170. For example, the SES travels through a symmetric and on-axis portion of the objective lens 170, is reflected off of the mirror 160, and travels through the second lens 150 and the first lens 140, continues to travel through filter 135 before reaching the ocular lens 190 that focuses the SES onto image detecting device 195 such as, e.g., a camera 195.

[0042] As discussed above, the SES, which may be a fluorescent signal or a transilluminated signal, is reflected from the sample in the off-centered radial portion of the objective lens 170 but remains substantially symmetrical with respect to the central axis thereof. As also discussed above, any portion of the SES that is reflected off of the sample in a region corresponding to the center of the objective lens 170 may be reflected along the central axis thereof. In the brightfield imaging mode 100C the controller 102 is configured to, inter alia, control operation of the brightfield illumination source 112.

[0043] FIG. ID illustrates a schematic view of the multimode imaging system 100 in a SPIM imaging mode 100D, in accordance with examples of the disclosure. In FIG. ID, the laser 110 generates the IL that is transmitted through fiber selector 120 to the dichroic mirror 125. The fiber selector 120 directs the IL emitted by the laser 110 to the dichroic mirror 125 instead of the to the filter 130 as is the case in the widefield 100A and confocal 100B modes illustrated in FIGS. 1A and IB. Accordingly, in the SPIM imaging mode 100D, also referred to herein as light sheet mode, the fiber selector 120 directs the IL to a different light path than the light paths described above with respect to the widefield 100A and confocal 100B modes. The light received at the dichroic mirror 125 is reflected onto a mirror 160 and onto objective lens 170. The path of the IL that is received at the dichroic mirror 125 is radially off-centered with respect to a center of the dichroic mirror 125. Similarly, as the IL is transmitted to the mirror 160 and to the objective lens 170, the IL is radially off-centered with respect to a center of the mirror 160 and with respect to a center of the objective lens 170. As a result, the IL reaches the sample 180 at a non-perpendicular angle with respect to the surface of the sample 180.

[0044] In FIG. ID, when the 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. ID, the SES is also reflected from the sample 180 at an oblique angle that is 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 IL and at an oblique angle to a central axis of the objective lens 170. For example, the SES 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 SES continues to travel through an off-centered radial portion of the first lens 140, through filters 130 and 135, and onto a movable selection module 175. In examples, the movable selection module 175 is configured to receive the off-centered SES and converts the SES to beam 178 onto ocular lens 190, the beam 178 being along the optical axis of the ocular lens 190. The ocular lens 190 is configured to focus the beam SES onto the image detecting device 195 such as, e.g., camera 195.

[0045] In the multimode imaging system 100 in a light sheet imaging mode or SPIM imaging mode 100D, the movable selection module 175 may be movable in the Z direction, (relative, e.g., to the XY movement of the movable stage 185) as illustrated in FIG. ID, and may be configured to be placed on the path of the SES in order to convert the SES from an off-centered beam to the beam 178 that then projects onto ocular lens 190, the beam 178 being along the optical axis of the ocular lens 190. For example, when the fiber selector 120 directs the IL to the dichroic mirror 125 of FIG. ID in a light sheet mode, also referred to as SPIM imaging mode 100D, then the movable selection module 175 is moved along the Z direction to be placed into the light path of the SES. On the other hand, when the fiber selector 120 directs the IL to the filter 130 and through a path corresponding to the light path of a widefield or confocal mode as illustrated in FIGS. 1A-1B, then the movable selection modulel75 may be moved in a direction out of the light path of the SES. In the illustration of FIG. ID, the movable selection module 175 may be moved downwards so as to be off the light path of the SES. In other examples, the movable selection module 175 may be movable in other directions.

[0046] Coordinating the selection of the light path by the fiber selector 120 to be in a SPIM configuration, with movement of the movable selection module 175 in the path of the SES, may be performed via the controller 102 which may be similar to, e.g., the computing device 500 discussed below with respect to FIG. 5. Similarly, coordinating the selection of the light path by the fiber selector 120 in a widefield or confocal configuration, with movement of the movable selection module 175 out of the path of the SES, may also be performed via the computing device500. Operation of at least one of the laser 110 or the fiber selector 120 is controlled via controller 102 to transmit the IL in a light sheet light path, the IL being referred to in this case as a first light source signal, or in a widefield or confocal light path, the IL being referred to in this case as a second light source signal. The selectable first optical fiber 122 of the fiber selector 120 is configured to emit the first light source signal therethrough towards the light sheet light path, and the selectable second optical fiber 124 is configured to emit the second light source signal therethrough towards the widefield or confocal light path. The controller 102 may be coupled to or include a computing system such as, e.g., the computing device500 discussed below with respect to FIG. 5.

[0047] The optical assembly that includes the laser 110, the fiber selector 120, the dichroic mirror 125, the mirror 160, the objective lens 170, the filters 130 and 135, the movable selection module 175, and the ocular lens 190 may be referred to herein as a second optical subassembly. Accordingly, the second optical subassembly is configured to transmit the light source signal from the laser 110 through an off-centered portion of the objective lens 170 to the sample 180 in an oblique illumination, and to transmit the fluorescent signal from the sample 180 at an oblique angle to the objective lens 170 to be imaged at the image detecting device 195. The objective lens 170, the first optical subassembly, the second optical subassembly, and the ocular lens 190 may be arranged on a single system.

[0048] FIG. 2 shows the RL (i.e., the SES) received at a mirror 281 that redirects the optical light RL with respect to the initial light path of the RL onto a first lens 282, also referred to herein as first relay lens 282. The 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 RL to a radially off-centered portion of a mirror 284 which redirects the RL at an angle with respect to the incident RL and towards a first relay objective 286. Other optical arrangements are possible, but importantly the RL is directed 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 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 RL.

[0049] The first relay objective 286 transmits that redirected RL to a second relay objective 287, the second relay objective 287 converts the radially off-centered RL to a parallel beam onto a mirror 289. The mirror 289 then redirects the received parallel beam of the RL out of the movable focusing module 200 as a parallel beam 288. As a result, the 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 FIGS. 1A-1C. The relay objectives 286 and 287 may be aligned and fixed in space on, e.g., a platform, to preserve the alignment therebetween.

[0050] 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 RL along a first axis, e.g., the axis of the 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 FIGS. 1A-1E. 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.

[0051] 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 RL on the optical axis of the ocular lens 190, and directs the 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 RL at the first relay lens 282. The second portion 208 transmits the 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 RL to the first portion 204 from the second relay objective 287. The first portion 204 then transmits the RL received from the second relay objective 287 to an image capture device such as, e.g., the camera 195 illustrated in FIGS. 1A-1C. 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. 2, a sufficiently large face of the mirror 281 can receive RL at a variety of Z positions, and translation of first portion 204 in the Y axis can also reflect incoming 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. 2, other relay lenses may be present in the second portion 208 to relay the RL from the first relay lens 282 to the first relay objective 286. It should be understood that mode selection module 204 is not limited to use in SPIM, and could be used for SPIM or any number of optical modules that could be assembled on an instrument.

[0052] Movable selection module 175 converts from an off-centered beam SES to a central beam 288 that may be emitted along the optical axis of, e.g., an ocular lens. As a result, the SES, initially entering the movable focusing module 200 as a radially off- centered light signal or light source signal, is converted to a central beam 288 that is usable to an image detecting device such as, e.g., the camera 195 illustrated in FIG. ID.

[0053] FIGS. 3A-3D are top views of a multimode imaging system 300, according to various examples of this disclosure. FIGS. 3A-3D are described concurrently and not every component described is depicted in every figure. FIGS. 3A-3D illustrate a multimode imaging system 300 where, as illustrated in FIG. 3A which is a side view thereof, the multimode imaging system 300 has a first side 310 and a second side 320 on each side of a platform 322. In examples, the first side 310 includes a multimode imaging system 300 such as the imaging system illustrated in FIGS. 1C as well as the moving mechanism 379, which corresponds to the moving mechanism 279 illustrated in FIG. 2. With reference to FIG. 2, the movable portion 204 may be located on the first side 310, and the fixed portion 208 may be on the second side 320. FIG. 3B illustrates the multimode imaging system 300 with a fixed portion 308 similar to the fixed portion 208 illustrated in FIG. 2, and a movable focusing module 304, similar to the movable portion 204 in FIG. 2, that can be placed in the path of the SES when in the SPIM mode 100D, where the SES is transmitted by the objective lens 370 and reflected from the mirror 360 towards the image detecting or image capture device 395. The objective lens 370 may be similar to the objective lens 170 discussed above. In FIG. 3B, the movable focusing module 304 is off of the path of the SES, which indicates that the imaging mode is either a confocal 100B, a widefield 100A, or a brightfield mode 100C. In FIG. 3C, the movable selection module 304 (which is a version of the movable selection module 175 described above) is on the path of the SES on the way to the image capture device 395. In this case, the imaging mode is a light sheet or SPIM imaging mode 100D, the operation of which is discussed with respect to FIGS. ID and 2 above. In FIG. 3D, the multimode imaging system 300 includes the movable focusing module 304 and the fixed portion 308. The movable focusing module 304 and the fixed portion 308 respectively correspond to the movable portion 204 and the fixed portion 208 described above with respect to FIG. 2. FIG. 3D illustrates operation of the light sheet or SPIM imaging mode 100D, where the movable focusing module 304 is placed on the path of the SES that is reflected from the mirror 360 and through the filter 330 towards the image capture device 395. The fixed portion 308 is configured to complete the light path of the SES traveling through the movable focusing module 304 before reaching the image detecting or capture device 395. A more detailed light path within the fixed portion 308 is described with respect to the fixed portion 208 in FIG. 2.

[0054] FIGS. 4A-4B depict a method 400 of performing multimode imaging, in accordance with various examples of the disclosure. Operation 410 of the method 400 includes generating a first light source signal at a light source. The light source may be, e.g., the laser 110 discussed above with respect to FIG. 1A. In an example, generating the first light source signal during operation 410 includes performing one of widefield microscopy, confocal microscopy, and brightfield microscopy, and, e.g., selecting an area of a sample to evaluate. Operation 420 includes transmitting, via a first optical subassembly, the first light source signal to a sample through an objective lens, e.g., via a central axis of the objective lens 170. With reference to FIGS. 1 A-1B, the first optical subassembly may include, e.g., the laser 110, the fiber selector 120, the filter 135, the first lens 140, the second lens 150, optionally the confocal spinning disk 155, the mirror 160, the objective lens 170, and the ocular lens 190 illustrated in FIGS. 1A-1B. The first optical subassembly may also include the image detecting or image capture device 195.

[0055] Operation 430 includes transmitting, via the first optical subassembly, a first fluorescent signal from an area of the sample that received the transmitted first light source signal, to the image detecting device. For example, the area of the sample is the area that received the first light source signal, and the fluorescent signal is a result of the excitation of the sample at the area by the received first light source signal. Transmitting the first light source signal may include transmitting the first light source signal to the sample through a central axis of the objective lens, e.g., a central axis of objective lens 170 illustrated in FIGS. 1A-1B. When the first light source signal is transmited to the sample, the movable portion (e.g., 204 of FIG. 2) of the second optical subassembly, illustrated above with respect to FIGS. ID-2, is shifted out of the optical axis of the image detecting device so as not to interfere with the transmited first fluorescent signal. Operation 440 may be part of the method 400 and includes forming a first image of the area of the sample at the image detecting device, e.g., at the camera 195 illustrated in FIGS. 1A-1B, based on the transmitted first fluorescent signal.

[0056] In various examples of method 400, operation 450 includes generating a second light source signal at the light source. The generated second light source signal may be a laser generated by, e.g., the laser 110 illustrated in FIG. ID. In an example, generating the second light signal during operation 450 includes performing light sheet microscopy. Once the second light source signal is generated during operation 450, operation 460 includes transmiting, via a second optical subassembly, the second light source signal through an off-centered portion of the objective lens to the sample in an oblique illumination. For example, the second light source signal is transmited to the area of the sample that received the first light source signal. Operation 460 includes transmiting the second light source to the area of the sample as a result of receiving a selection by, e.g., a user. The second optical subassembly may include e.g., the optical assembly that includes the laser 110, the fiber selector 120, the dichroic mirror 125, the mirror 160, the objective lens 170, the filters 130 and 135, movable selection module 175, and the ocular lens 190.

[0057] As a result of the excitation of the second light source signal received by the sample, the sample emits a second fluorescent signal, and operation 470 includes transmiting the emited second fluorescent signal from the portion of the area of the sample that has been excited by the second light source signal to the image detecting device at an oblique angle to the objective lens. When the second fluorescent signal is transmited from the portion of the area of the sample during operation 470, the movable portion of the second optical subassembly, e.g., the movable portion 204 of movable selection module 175 illustrated in FIGS. ID-2, may be positioned on an optical axis of the image detecting device so that the fluorescent oblique plane signal SES received from the sample can be transmited to the image detecting device as a central beam. Prior to operation 450, operation may be switched via, e.g., the fiber selector 120 illustrated in FIG. ID, from transmiting the first light source signal to transmitting the second light source signal. For example, operation may be switched between widefield / confocal microscopy to light sheet microscopy, and vice-versa, based on the received selection. Operation 480 includes forming a second image of the portion of the area of the sample at the image detecting device, e.g., at the camera 195, based on the transmitted second fluorescent signal emitted by the sample when the portion of the area of the sample is excited by the second light source signal. Alternatively, operation 480 may also include forming a third image of a different portion of the area of the sample based on a transmitted third fluorescent signal from the different portion of the area of the sample.

[0058] In various examples, method 400 can include switching between the widefield / confocal microscopy and the light sheet / SPIM microscopy modes based on a selection received from, e.g., a user. The method 400 can include operating first in widefield, confocal, or brightfield mode to interrogate the entire portion of the sample. Then, a decision can be made by user instruction or analysis metrics (such as size, shape, intensity, or other sample features) whether or not to proceed with SPIM imaging. The method 400 can then include switching to selective plane illumination for SPIM imaging. The method 400 can include switching to or from either of these modes, starting from either SPIM or widefield / confocal / brightfield, and can involve multiple switches between modes as needed to accomplish a desired imaging task.

[0059] FIG. 5 depicts a block diagram of a computing device configured to control the multimode imaging system 100 discussed above with respect to FIGS. 1A-1D. In the illustrated example, the computing device 500 may include a bus 502 or other communication mechanism of similar function for communicating information, and at least one processing element 504 (collectively referred to as processing element 504) coupled with bus 502 for processing information. As will be appreciated by those skilled in the art, the processing element 504 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 504 may be included in the computing device 500 to provide the control or management operations for, e.g., the multimode imaging system 100 illustrated above.

[0060] The computing device 500 may also include one or more volatile memory(ies) 506, which can for example include random access memory(ies) (RAM) or other dynamic memory component(s), coupled to one or more busses 502 for use by the at least one processing element 504. Computing device 500 may further include static, non-volatile memory(ies) 508, such as read only memory (ROM) or other static memory components, coupled to busses 502 for storing information and instructions for use by the at least one processing element 504. A storage component 510, 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 504. As will be appreciated, the computing device 500 may include a distributed storage component 512, such as a networked disk or other storage resource available to the computing device 500.

[0061] The computing device 500 may be coupled to one or more displays 514 for displaying information to a user. Optional user input device(s) 516, such as a keyboard and / or touchscreen, may be coupled to Bus 502 for communicating information and command selections to the at least one processing element 504. An optional cursor control or graphical input device 518, 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 500 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 system 100 discussed above.

[0062] In various examples, computing device 500 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 system 100 may be supported by operation of the distributed computing systems. The computing device 500 may be operative to control operation of the components of the multimode imaging system 100 through a communication device such as, e.g., communication device 520, and to handle data generated by components of the multimode imaging system 100 through the processing element 504. In some examples, analysis results are provided by the computing device 500 in response to the processing element 504 executing instructions contained in memory 506 or 508 and performing operations on data received from the multimode imaging system 100. Execution of instructions contained in memory 506 and / or 508 by the at least one processing element 504 can render, e.g., the multimode imaging system 100 to perform methods described herein.

[0063] The term “computer-readable medium” as used herein refers to any media that participates in providing instructions to the processing element 504 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 510. Volatile media includes dynamic memory, such as memory 506. Transmission media includes coaxial cables, copper wire, and fiber optics, including the wires that include bus 502.

[0064] 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.

[0065] Various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to the processing element 504 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 500 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 502 can receive the data carried in the infra-red signal and place the data on bus 502. Bus 502 carries the data to memory 506, from which the processing element 504 retrieves and executes the instructions. The instructions received by memory 506 and / or memory 508 may optionally be stored on storage device 510 either before or after execution by the processing element 504.

[0066] 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.

[0067] 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.

[0068] 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

CLAIMS1. An optical arrangement, comprising: a light source; an objective lens in proximity to a sample stage configured to hold a sample; an ocular lens in proximity to an image detecting device; a first optical subassembly 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 returned light from the sample to the image detecting device; and a second optical subassembly comprising 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 modified returned light at an oblique angle to the objective lens from the sample to the image detecting device.

2. The optical arrangement of claim 1, wherein the first light source signal is transmitted to the sample through a central axis of the objective lens.

3. The optical arrangement of claim 1 or claim 2, wherein each of the returned light and the modified returned light comprises one of a fluorescent signal and a transilluminated signal.

4. The optical arrangement of any one of claims 1-3, further comprising a controller configured to control the light source to transmit at least one of the first light source signal and the second light source signal.

5. The optical arrangement of claim 4, further comprising a moving mechanism, wherein the controller is further configured to control the moving mechanism to shift the movable portion of the second optical subassembly at least one of onto the optical axis and off the optical axis in a direction substantially perpendicular to the optical axis.

6. The optical arrangement of claim 5, wherein when the movable portion is on the optical axis, the modified returned light is received at the second optical subassembly as an oblique plane signal and is transmitted to the image detecting device as a signal central to an axis of the image detecting device.

7. The optical arrangement of any one of claims 1-6, wherein the objective lens, the first optical subassembly, the second optical subassembly, and the ocular lens are arranged on a single system.

8. The optical arrangement of claim 7, wherein the moving mechanism is configured to shift the movable portion with respect to the optical axis.

9. The optical arrangement of any one of claims 1-8, wherein the second optical subassembly further comprises a dichroic mirror on a path of the second light source signal.

10. The optical arrangement of any one of claims 1-9, wherein the first optical subassembly and the second optical subassembly are configured to be operated together in any of a widefield, brightfield, confocal, or SPIM mode.

11. The optical arrangement of any one of claims 1-10, further comprising a movable stage configured to move the sample in one or more directions on a plane that is parallel to the objective lens.

12. The optical arrangement of any one of claims 1-11, further comprising a multiplexor configured to select one of the first light source signal and the second light source signal.

13. The optical arrangement of claim 12, wherein the light source signal selector comprises at least a first optical fiber configured to emit the first light source signal therethrough and a second optical fiber configured to emit the second light source signal therethrough.

14. The optical arrangement of claim 13, wherein at least one of the first optical fiber and the second optical fiber comprises a fiber bundle, the fiber bundle comprising a plurality of fibers.

15. A method of performing multimode imaging, the method comprising: generating a first light source signal at a light source; transmitting, via a first optical subassembly, the first light source signal to a sample through an objective lens; transmitting, via the first optical subassembly, a returned light from an area of the sample to an image detecting device; generating a second light source signal at the light source; transmitting, via a second optical subassembly comprising a fixed portion and a movable portion, the second light source signal through an off-centered portion of the objective lens to the sample in an oblique illumination; and transmitting, via the movable portion of the second optical subassembly, a modified returned light from a portion of the area of the sample to the image detecting device at an oblique angle to the objective lens.

16. The method of claim 15, wherein at least one of: transmitting the returned light from the area of the sample comprises forming a first image of the area of the sample at the image detecting device based on the transmitted returned light; and transmitting the modified returned light from the portion of the area of the sample comprises forming a second image of the portion of the area of the sample at the image detecting device based on the transmitted modified returned light.

17. The method of claim 15 or claim 16, wherein transmitting the first light source signal comprises transmitting the first light source signal to the sample through a central axis of the objective lens.

18. The method of any one of claims 15-17, wherein when the second light source signal is transmitted to the sample: the movable portion of the second optical subassembly is positioned on an optical axis of the image detecting device; and transmitting the modified returned light comprises transmitting an oblique plane signal from the sample to the image detecting device.

19. The method of claim 18, wherein when the first light source signal is transmitted to the sample, the movable portion of the second optical subassembly is off the optical axis of the image detecting device.

20. The method of any one of claims 15-19, wherein: generating the first light source signal comprises performing one of widefield microscopy and confocal microscopy; and generating the second light source signal comprises performing light sheet microscopy.

21. The method of claim 20, further comprising switching between the one of widefield microscopy and confocal microscopy and the light sheet microscopy based on a selection.

22. The method of any one of claims 15-21, further comprising: transmitting, via the movable portion of the second optical subassembly, a modified returned light from a different portion of the area of the sample to the image detecting device at an oblique angle to the objective lens.

23. The method of any one of claims 15-22, wherein: transmitting the returned light comprises transmitted a first fluorescent signal; and transmitting the modified returned light comprises transmitted a second fluorescent signal.

Images