Patent Grant
11506877
There is an increasing need for methods and devices that permit rapid high-resolution imaging of the structure and function of large three-dimensional objects, for example broad slabs of substantial thickness, in particular, fully intact biological samples such as large slices of biological tissues. An example of this is brain tissues the importance of which, to neuroscience research is underscored by the announcement of high profile initiative including White House sponsored BRAIN Initiative for building Neurotechnologies.
In the known technique of light sheet microscopy (LSM), a thin sheet of light illuminates a sample from the side and an orthogonally arranged objective detects the emitted signal from the illuminated plane.
The configuration of light sheet microscopy provides two key advantages: minimum possible energy load on the sample (therefore minimum photobleaching and phototoxicity) and very high imaging speeds. The imaging depth (i.e., the sample dimension along the imaging axis) is generally determined by the working distance of the detection objective, which puts a physical limit, and the optical scattering properties of biological tissue being imaged. The overall intact sample size that can be imaged is also restricted in one of the two other sample dimensions (i.e., the dimension along the light sheet propagation direction indicated in
A commonly used configuration (
Presently described are embodiments of a microscope which may be identified as Light Sheet Theta Microscopy (LSTM), also referred to herein as LSTM. The LSTM microscope provides the ability to resolve images in substantial depth as in a light sheet microscopy while providing the ability to scan samples having a large area. Variations also provide faster scanning by imaging on opposite sides of the sample.
Objects and advantages of embodiments of the disclosed subject matter will become apparent from the following description when considered in conjunction with the accompanying drawings.
Embodiments will hereinafter be described in detail below with reference to the accompanying drawings, wherein like reference numerals represent like elements. The accompanying drawings have not necessarily been drawn to scale. Where applicable, some features may not be illustrated to assist in the description of underlying features.
Presently described are embodiments of a microscope which may be identified as Light Sheet Theta (LS-θ) Microscopy, also referred to herein as LSTM. LSTM achieves planar imaging by employing obliquely arranged illumination light sheets from the same side of the sample as the detection objective. This configuration alleviates limitations on the lateral dimensions of the sample, while providing similar or better imaging depth, quality, low photo-bleaching and high imaging speed for larger samples. LSTM allows imaging of larger samples with high uniform resolution across the entire sample. In addition, LSTM achieves uniform illumination across the entire imaging field-of-view, in contrast to the non-uniform Gaussian illumination commonly used in LSM, thus enabling quantitative imaging across the entire sample. LSTM allows imaging of large samples including thick slabs of cleared and labelled post-mortem healthy and diseased human brains as well as imaging of large animal intact brains, including rat and primate brains. Moreover, LSTM facilitates in situ detection of thousands of transcripts in expanded tissue samples.
An embodiment is illustrated figuratively in
To provide the ability to scan large samples as in large sample imaging capabilities without affecting the imaging depth or the image quality. The optical layout is summarized in
Referring momentarily to
By scanning this illuminated linear volume 219, the entire front focal plane may be illuminated and captured. The scanning on the illumination side may be performed, as later explained, by one or more mirrors, electrically tunable lenses, or alternatives thereto (e.g., rotating prisms, diffraction optics, MEMs pico projectors, electrically controllable spatial light modulators, tunable Acoustic lens (e.g. TAG Lens) and others commended by the state of the art for this purpose. This can also be achieved by synchronously scanning the illumination objective along the light sheet propagation direction alternatively or in conjunction with other scanning mechanisms.). The scanning of the light sheet in combination with the selective sampling of lines on the sCMOS camera imaging plane together may capture a “tile” portion of a larger sample to be imaged in its entirety. By syncing the illuminated linear volume 219 with the line-by-line detection of the sCMOS camera 110, high quality images of the plane are acquired.
According to embodiments, three different modes of detection may be provided with respective embodiments or through a single embodiment with multiple user-selectable modes. Referring to
A second mode is as that illustrated in
Other variant scanning modes are possible for each of the foregoing modes. For example, there are two scanning modes for scanning the illuminated linear volume 219 for planar illumination. Referring to
Referring to
To characterize and compare these modes of illumination and detection, micron scale fluorescent beads and CLARITY-cleared human brain tissue stained with nuclear stain DAPI were imaged. The resulting image volumes revealed that 2-AS allows for much more uniform axial resolution across the entire field-of-view, whereas both 1-AS and conventional LSM imaging produce reduced image quality on the periphery of the field-of-view. Simultaneous two-sided illumination from two symmetrically arranged illumination arms (as seen in
The three-dimensional volumes are acquired by either moving the sample across the illuminated sheet in a stepwise manner (or continuously at slow speed, while imaging), or by synchronously moving the effective illumination light-sheet and the exposed pixel line resolved by the detection arm. Imaging speed can be further increased by simultaneously imaging the sample from opposed detection arms on respective opposite faces of the sample. In addition, a continuous motion of the sample may be used to traverse laterally instead of moving the exposed pixel line. The continuous traversal is faster than step-wise motion as linear stages may take significant amount of time for starting and stopping.
The various LSTM system embodiments enable imaging experiments of large sample size with extremely high resolution similar that of light sheet microscopy. In LSTM microscopy the detection objective remains normal to the sample surface, and the illumination objectives are placed at an angle (significantly different from 90 degrees, as close to 90 degrees as allowed by the geometry), thus removing the limitations demonstrated in
Some application examples include:
Imaging of thick slabs of human brain, or other organs, that are cleared by using any of the tissue clearing procedures including CLARITY, iDISCO, uDISCO, SeeDB, ClearT and Scale. These procedures employ a cocktail of chemicals to render the tissue transparent while preserving structural and molecular content. These tissues can come from normal or diseased states including, but not limited to, various types of cancers, neurological disorders and other diseased biopsies.
Imaging of large animal intact brains, including rat and primate brains. The current state-of-the-art technology has been limited to imaging samples of mouse brain size.
Live imaging applications in imaging mouse brain for functional activity.
Live imaging of 3D cell cultures.
Referring to
Note that instead of subsystems 300A and 300B being positioned to scan different layers of the sample, they may image the same layer simultaneously, permitting the captured image data to be combined to increase resolution. Variations may also be provided such as illumination arm or arms on one side and detection arm on the opposite side of the sample. Also, the block 140 may be eliminated and also made movable so that it can be adjusted from a range of 0% blockage to a major fraction of the aperture of the illumination objective. Any of the claimed embodiments may be modified to provide additional embodiments with zero or adjustable blockage.
It will be clear that any of the mirror or other deflectors such as optical scanning device 320 may be substituted by equivalent light redirectors. Such may include refracting as well as reflecting components or other types of light manipulation devices. Lenses may be of any suitable type. Wave-guide-based devices including fiber optics may also be substituted for beam reflectors. Devices other than ETLs may be employed to manipulate the axial position of the front focal plane of the illumination arm, including but not limited to, mounting of illumination objective on fast piezo motors for axial motion profile. The camera 330 may also be replaced, for example by a scannable line sensor. Instead of a sheet beam, a scanned pencil beam may be projected over a target illuminated linear volume 219 although such alternatives may have characteristics that are adverse to the functions of speed provided by the illustrated and described embodiments.
A sample 336 may be mounted for translation and rotation movement, for example, mounted on an XYZ-theta motorized stage. This may permit the sample 336 to be scanned over multiple tiles. The illumination arm of
Referring to
The LSTM system may have a controller to perform a function that automatically performs a calibration by sparsely automatically sampling of the imaging planes at multiple positions in the sample. According to an embodiment, the LSTM system may be controlled to perform a sparse traversal of a large sample in order to extract parameters that affect the positions of the illuminated linear volumes 219 such as surface variations and variations in index of refraction. Thus, a sparse matrix of samples can be captured and analyzed for these variations and stored in order to create a fine traversal itinerary. This ultimately may speed and improve the imaging quality of the acquisition of optical data in a large sample. The traversal itinerary may employ adaptive correction of the detected parameters and interpolation of the parameters from the sparse intervals. The adaptive optics may correct, for example, light sheet displacement relative to the detection focal plane.
Note that although the drawings of
In some embodiments, the illumination arms 500 are mounted at an approximately 60° angle relative to the detection arm 550 fixed on an optic table. In alternative embodiments, this angle may vary within the parameters discussed in connection with
In this embodiment, two thin light sheets are generated by using two illumination optical arms 500. When the illumination objectives are air objectives, a 3D printed cap with a coverslip (e.g., a 1 inch diameter quartz coverslip) may be used to seal the objective for oil immersion use.
The detection arm 500 in this embodiment includes a detection objective 552 (e.g., Olympus 10×/0.6 NA/8 mmWD or 25×/1.0 NA/8 mmWD), a tube lens 556, and an sCMOS camera 558 (e.g., a Hamamatsu Orca Flash 4.0 V3). In some preferred embodiments, an emission filter (not shown) is disposed between the detection objective 552 and the tube lens 556.
A list of suitable parts and their sources that may be used in this embodiment is provided in Table 1.
Because LSTM involves scanning of a line illumination-detection profile generated by the intersection of the light sheet and the detection plane, some preferred embodiments use static sheets (generated by the use of a cylindrical lens and associated optics), instead of a dynamic sheet (generated by rapid scanning of a pencil beam) to maximize imaging speeds. But some alternative embodiments can use dynamic sheets.
A galvo scanner 514 is used to achieve rapid translation of light sheets perpendicular to their propagation direction. For the 2-AS mode, rapid translation of the thinnest part of the sheet along the propagation direction is required. This may be accomplished using a variety of approaches including but not limited to using fast piezo motors to translate the illumination objectives, using holographic spatial light modulators or an electrically tunable lens (ETL) 508 to induce divergence and convergence of a collimated beam. The use of piezo motors for rapid scanning of objective often results in vibrations and require additional settling time, and the spatial light modulators are limited in modulation speed because of slower refresh rates. ETLs, on the other hand, can achieve high frequency modulation of focal plane position without the need for moving optics of significant mass. Accordingly, some preferred embodiments use ETLs 508. In practice, an ETL based approach has proven to be highly effective for achieving uniform simultaneous 2-axes scanning.
One way to optically align the LSTM assembly is to place a prism mirror (e.g., with fine scratches in the center) in the focal plane of detection optics, to visualize the location and cross- section of the light sheet relative to the detection focal plane. The light sheet positioning parameters can then optimized such that the thinnest part was in alignment with the center of the field-of-view of the detection plane. Next, the mirror can be replaced with a high concentration (>2%) agarose gel containing fluorescent beads (Note. The high concentration of agarose may be used to ensure that only the surface plane of the agarose gel is visible during the alignment optimization.). By examining the extent and quality of the illuminated beads located on the surface, optimal galvo scanner and ETL parameters for achieving uniform planar illumination across the entire field-of-view can be identified.
A series of calculations was used to assess and compare various properties of the
where W1 and W2 are the working distances of the detection and illumination objectives respectively, D1 and D2 are the diameters of the detection and illumination objectives respectively, and θi and θf are the boundary angular positions. When the objectives are designed to be used in air, Snell's law may be used to calculate the approximate effective working distance (e.g., in a refractive index liquid of 1.454, as shown in
12 depicts the influence of angular separation of the illumination arms 500 and the detection arms 550 in the
The graph in
The graph in
The imaging performances of LSTM and LSM was compared using a thick (˜5 mm deep and ˜2 centimeters wide) cleared rat brain section stained for vasculature, with a highly transparent sample with bright uniformly distributed signal. LSTM enabled imaging of the entire sample with uniform high-resolution, whereas the lack of penetration of the illumination light sheets in LSM resulted in progressively poorer image quality towards the center of the tissue. These and other experiments help demonstrate the suitability of LSTM for quantitative high-resolution imaging of large samples, without any limits on lateral sizes.
The use of lower NA illumination in LSTM 1-AS configuration in a large sample was tested in a CLARITY-cleared thick coronal section of Thyl-eYFP transgenic mouse brain. Low numerical aperture illumination was used to generate a large field-of-view (and hence thicker light sheets). While, the LSTM 1AS mode allowed for high-quality imaging of the section, image quality was reduced for peripheral portions of the field-of-view. This result was similar to the imaging performance of a LSM system employing Gaussian beams for illumination.
Next, the imaging performance of LSTM in simultaneous 2-AS mode was assessed using a thick (˜9.6 mm×13.5 mm×5.34 mm) coronal section of CLARITY-cleared Thyl-eYFP transgenic mouse brain, with 10×/0.6 NA/8 mmWD and 25×/1.0 NA/8 mm objectives. A larger input beam diameter was used to employ full NA (0.28) of the illumination objective. With this configuration, LSTM provided rapid high-resolution quantitative imaging of these large samples without any reduction in the image quality across the sample dimensions.
As a follow up to these experiments, an entire intact cleared mouse brain (with connected spinal cord) of Thyl-eYFP transgenic mouse was successfully imaged. A detailed high-resolution 3D rendering was performed and it demonstrated uniform, high quality imaging. One can therefore conclude that LSTM allows high-resolution quantitative imaging of large intact biological systems with no limitations on the lateral dimensions. Sample thickness that can be imaged remains limited by the working distance of the detection objective and also by level of tissue transparency and penetration of labelling reagents.
Due to the non-orthogonal incidence of the light sheet on the detection plane, the effective light sheet thickness can be approximated as the projection of the original thickness on to the detection direction, resulting in b/sin(θ), where b is the original light sheet thickness at the most focused position, and θ is the angle of incidence relative the detection axis.
Throughout the specification, reference to embodiments includes those defined by the claims. Thus, any proposed modifications to embodiments contemplates corresponding variations of the claims and such variations are considered to be disclosed in the present application.
Many of the disclosed embodiments contemplate the use of one or more movable stages carrying specimens, optical components, or both. The details of such devices and technology which are sufficient to implement the disclosed embodiments and variations thereof are in the public domain and are therefore not disclosed.
Motion stage-driven microtome may also be combined with the disclosed LSTM microscope embodiments. The microtome may be controlled automatically, in synchrony with a tile-wise scan of a sample, to remove a layer of the sample after an outer layer has been scanned into memory. Once a layer is removed, the traversing system can move the excitation and detection arms to scan the exposed deeper layer automatically. A microtome may be provided for opposite surfaces of a sample, for example, separate microtomes may be provided for the opposing sample faces of the embodiment of
In any of the embodiments, further processing such as compression, may be performed on acquired data in parallel with scanning. For example, as the optical data are acquired for a tile, the data from a previous tile may be further processed to reduce the data volume, for example, by algorithms such as JPEG2000 2D or JPEG2000 3D. The further processing may also include other image processing tasks such as image segmentation, feature recognition or other data reduction procedures.
It will be understood that although a camera is shown for each detection arm, in alternative embodiments, a single camera can receive light, with a suitable optical arrangement, from both sides of a slab to be imaged.
Note that in any of the embodiments employing an electrically tunable lens, any other suitable mechanism configured to move the sheet beam waist along an axis of the illumination objective may instead, or additionally—as feasible may be employed. These include, but are not limited to, alone or in combination, acousto-optics tunable lenses, spatial light modulators, digital micromirror devices or mounting of illumination objective on a piezo motor, gradient-index (GRIN) lenses, micro electromechanical machines (MEMS) carrying fibers, beam redirectors, for example a GRIN lens at the end of a fiber driven by a MEMS motor. See, for example, “Multimode fibre: Light-sheet microscopy at the tip of a needle by Ploschner at al, (http://dx.doi.org/10.1038/srep18050). Those of skill in the art will recognize that other equivalent or similar mechanisms may also be used.
Suitable mechanisms such as stage on tracks, linkages, or flexible composite structures can be used to maintain the orientation of the GRIN lens throughout the sweep of the scan. Note that other sweep paths may be implemented other than those shown in
In any embodiment described as including an sCMOS camera, other suitable imaging devices may be substituted, including, but not limited to CCD or line detectors.
As used in the current specification and in the claims, the term “objective” may refer to any lens or set of lens capable of being used as an objective or a part thereof. So, for example, the excitation arm is described, in embodiments, as using an objective to form a sheet beam. In this role it is not used for forming an image but the optical element itself may have features of an objective including characterizing features such as a numerical aperture.
In any of the embodiments, provisions may be made to alter the depth of field of the microscope and to select a number of depths to scan for a given tile in order to increase the speed of throughput for live imaging of tissue samples, for example. The depth of field may be selected by any suitable mechanism such as an objective turret selector or replaceable objectives.
According to first embodiments, an optical imaging apparatus includes a first illumination arm having optical components arranged for forming a first sheet beam. A first detection arm has a front focal plane and having optical components for imaging a linear volume where the sheet beam intersects the front focal plane. The first illumination arm and first detection arm optical components each include a respective illumination objective and detection objective, the angle of the first sheet beam is oblique to the optical axis of the illumination objective, and the first detection arm front focal plane is perpendicular to the detection objective.
The first embodiments include variants in which the first illumination arm optical components include scanning components that cause a beam waist of the sheet beam to traverse the first detection arm front focal plane. The first embodiments include variants that include a controller connected to a first camera to read image data therefrom, wherein the first detection arm includes a two-dimensional image detector of the camera, the controller reading and storing selected pixels of the image detector responsively to positions of the sheet beam waist, thereby causing the selected pixels to follow the traversal of the beam waist. The first embodiments include variants in which the controller controls the scanning components. The first embodiments include variants in which the scanning components include an electrically tunable lens configured to move the sheet beam waist along an axis of the illumination objective. The first embodiments include variants that include a traversing system, the controller configured to control the scanning components and to form an image slice from successive samples of the linear volume. The first embodiments include variants that include a second illumination arm identical to the first and configured to form a second sheet beam that intersects the first sheet beam. The first embodiments include variants in which the first and second illumination arm illumination objective optical axes each forms an angle to the surface of the first detection arm front focal plane. The first embodiments include variants in which each of the illumination arms includes a partial beam block which causes the sheet beam to be formed using a side portion of the illumination objective. The first embodiments include variants in which the angles of the sheet beams are responsive to the position of the beam block. The first embodiments include variants that include a controller connected to a traversal system, the controller is programmed to optically scan portions of a degree of freedom of the traversing system such that the traversing system can move in steps along an axis corresponding to the degree of freedom whose size is responsive to a dimension of the optical scan, to permit the capture of a sample larger than the dimension of the optical scan. The first embodiments include variants that include a second illumination arm and a second detection arm, both identical, respectively, to the first illumination and detection arms, the second illumination and detection arms is positioned on opposite sides of a movable stage. The first embodiments include variants that include cameras on each of the first and second illumination arms, a controller connected to first and second image detectors to read image data therefrom, wherein the first detection arms each includes a two-dimensional image detector of the camera, the controller reading and storing selected pixels of the image detector responsively to positions of the sheet beam waist, thereby causing the selected pixels to follow the traversal of the beam waist. The first embodiments include variants in which the controller is configured to control the sheet beams of the first and second illumination arms to maintain a distance between the intersections of the sheet beams and the front focal planes of the detection arms. The first embodiments include variants in which the controller is configured to control the sheet beams of the first and second illumination arms to maintain a distance between the intersections of the sheet beams and the front focal planes of the detection arms by maintain a phase difference between the scan cycling of the two sheet beams.
According to second embodiments, the disclosed subject matter includes a method of imaging an elongate volume within a sample. The method includes forming a sheet beam by passing a beam of light through a side of a first objective such that the beam is blocked from passing through a portion to a side of an optical axis of the first objective. The method includes detecting light from a linear volume illuminated by the sheet where the sheet intersects the focal plane of the first objective or a second objective. The forming includes scanning the illumination beam such that the linear volume traverses an axis that is perpendicular to an axis of the linear volume.
The second embodiments include variants in which the detecting includes sampling a line of pixels of a two-dimensional image plane of a camera, the line of pixels coinciding with an image of the linear volume. The second embodiments include variants in which the first objective or a second objective is the first objective. The second embodiments include variants in which the first objective or a second objective is a second objective. The second embodiments include variants in which the scanning includes traversing an axial position of the linear volume along an axis of the first objective. The second embodiments include variants in which the traversing an axial position includes actuating an electrically tunable lens. The second embodiments include variants that include repeating the forming and detecting on an opposite side of a single sample. The second embodiments include variants that include mechanically traversing a sample after each of the steps of scanning and repeating the forming and detecting at each position of the mechanically traversing and compositing the images corresponding to each scan to form a three-dimensional image of a sample.
According to third embodiments, the disclosed subject matter includes a microscope. A detection objective has optical axis is at a normal to a predefined plane defined by two axes of a traversing system. One or more illumination objectives are positioned and oriented to generate one or more light sheets at an angle oblique to the normal such that the light sheets enter the sample from the same side of the predefined plane as the detection objective.
The third embodiments include variants that include a detection arm that conveys light from the detection objective to a two-dimensional image sensor. The third embodiments include variants in which the image sensor is a sCMOS type sensor. The third embodiments include variants that include illumination arms that form beams that pass through the illumination objectives. The third embodiments include variants in which the illumination arms are configured to pass light asymmetrically through the illumination objectives such that the light sheets form an angle relative to the illumination objective optical axes. The third embodiments include variants in which the illumination arms each block a beam applied to a respective one of the illumination objectives. The third embodiments include variants that include a controller and scanning optics controlled by the controller in the illumination arms to cause an intersection between the light sheets and a front focal plane of the detection objective to traverse the front focal plane. The third embodiments include variants that include the controller captures a line of pixels of an imaging device corresponding to an image of an illuminated region located at the intersection.
According to embodiments, the disclosed subject matter includes a microscope. An imaging component has an imaging objective. A first light sheet projector is located on a same side of a sample region as the imaging component. The imaging component itself can include all the part of a conventional microscope, for example. The imaging objective and first light sheet projector have respective optical axes that intersect in the sample region. This occurs because of the way they are positioned and oriented in a common mounting with the microscope. A sample support is positioned and oriented with respect to the imaging objective such that, when a sample is placed on the sample support, the imaging objective optical axis is perpendicular to a surface of the sample.
In variations of the above microscope, the sample support is a generally flat element has a major plane and shaped to hold a slab-shaped sample, the imaging objective optical axis is perpendicular to the major plane. In further variations, which may be combined with the preceding variations, a second light sheet projector is also provided. The second light sheet projector has an optical axis forming an angle with the imaging objective optical axis equal to an angle formed by the first sheet projector optical axis with the imaging objective optical axis.
According to further embodiments, the disclosed subject matter includes a microscope with an imaging component, has an imaging objective, and a first light sheet projector located on a same side of a sample region. The imaging objective and first light sheet projector have respective axes that intersect in the sample region. A sample support is positioned to hold a sample in the sample region. The light sheet projector has light scanning and focusing optics to permit the selection of an axial position of a light sheet beam waist and the selection of a position of the light sheet along an axis perpendicular to the light sheet propagation direction. A controller is connected to control the light scanning and focusing optics to translate the beam waste along a linear path. The imaging objective optical axis are perpendicular to the linear path.
In variations of the foregoing microscope, the focusing optics include an electrically tunable lens (ETL). In further variations, the sample support is a generally flat element shaped to hold a slab-shaped sample. In further variations, a second light sheet projector has an optical axis forming an angle with the imaging objective optical axis equal to an angle formed by the first sheet projector optical axis with the imaging objective optical axis.
In still further variations, one or more further light sheet projectors and an imaging component are arranged on an opposite side of the sample region such that two opposite faces of a sample can be scanned. In still further variations, a translating stage is connected to move the sample support to permit the sequential scanning of portions of a sample.
According to embodiments, the disclosed subject matter includes a microscope with an imaging objective and a first light sheet projector located on a same side of a sample region. The imaging objective and first light sheet projector have respective optical axes that intersect in the sample region. A sample support is provided on a translating stage that translates the sample support along at least one linear axis. The translating stage is positioned and oriented with respect to the imaging objective such that the imaging objective optical axis is perpendicular to the at least one linear axis.
The sample support may be a generally flat element has a major plane and shaped to hold a slab-shaped sample, the imaging objective optical axis is perpendicular to the major plane. A second light sheet projector may be provided having an optical axis forming an angle with the imaging objective optical axis equal to an angle formed by the first sheet projector optical axis with the imaging objective optical axis.
Any of the microscope embodiments may include one or more further light sheet projectors and an imaging component arranged on an opposite side of the sample region such that two opposite faces of a sample can be scanned and/or a translating stage connected to move the sample support to permit the sequential scanning of portions of a sample.
According to embodiments, the disclosed subject matter includes an imaging instrument including a microscope with an objective has an optical axis. A first light beam projector has imaging optics separate from the microscope positioned to direct a beam of light that crosses the optical axis. The first light beam projector forms a sheet has a beam waist in the form of a line of maximum intensity. The first light beam projector has a scanning mechanism that scans the line of maximum intensity through a range of positions along a scan direction perpendicular to the line of maximum intensity. The microscope objective optical axis is perpendicular to the scan direction.
In the imaging instrument, the objective and the light beam projector are located on a same side of a sample volume such that an extended slab-shaped sample can be scanned by moving the sample relative to the objective and the light beam projector. In the imaging instrument and any variations thereof, a traversing system may be connected to one or both of the microscope and a sample support to move them such that a slab-shaped sample can be scanned. In the imaging instrument and any variations thereof, the traversing system is an X-Y traversing system. In the imaging instrument and any variations thereof, the traversing system is an X-Y traversing system, where X and Y axes are perpendicular to the optical axis. In the imaging instrument and any variations thereof, a controller may be connected to the traversing system and scanning mechanism to scan each of multiple tile segments of a sample using the scanning mechanism and to shift the sample support relative to the microscope to scan additional tile segment. In the imaging instrument and any variations thereof, the scanning mechanism includes electrically tunable lens (ETL). In the imaging instrument and any variations thereof, the ETL may change a position of the line of maximum intensity along a length of the sheet in the direction of propagation of the sheet.
In the imaging instrument and any variations thereof, the scanning mechanism includes a light redirector that scans the sheet such that the line of maximum intensity traverses an axis perpendicular to a direction of propagation of the sheet. In the imaging instrument and any variations thereof, the scanning mechanism may include a light redirector that scans the sheet such that the line of maximum intensity traverses an axis perpendicular to a direction of propagation of the sheet. In the imaging instrument and any variations thereof, the scanning mechanism may include a light redirector that scans the sheet such that the line of maximum intensity traverses an axis perpendicular to a direction of propagation of the sheet.
In the imaging instrument and any variations thereof, the light redirector may include a galvo-motor. In the imaging instrument and any variations thereof, the light redirector may include a galvo-mirror. In the imaging instrument and any variations thereof, the scanning mechanism may scan in two axes, one to changes a position of the line of maximum intensity along a length of the sheet in the direction of propagation of the sheet and one to change the position of the line of maximum intensity along an axis perpendicular to the direction of propagation of the sheet. In the imaging instrument and any variations thereof, the scanning mechanism may scan in two axes, one to changes a position of the line of maximum intensity along a length of the sheet in the direction of propagation of the sheet and one to change the position of the line of maximum intensity along an axis perpendicular to the direction of propagation of the sheet. In the imaging instrument and any variations thereof, the scanning mechanism may include an electrically tunable lens (ETL) to change the position of the line of maximum intensity along a length of the sheet in the direction of propagation of the sheet.
According to embodiments, the disclosed subject matter includes an imaging instrument that includes a microscope with an objective. A first light beam projector has imaging optics separate from the microscope positioned to direct a beam of light that crosses the optical axis. The first light beam projector forms a sheet that has a beam waist in the form of a line of maximum intensity. The first light beam projector has a scanning mechanism that scans the line of maximum intensity through a range of positions along a scan direction perpendicular to the line of maximum intensity.
In variations, the objective and the light beam projector are located on a same side of a sample volume such that an extended slab-shaped sample can be scanned by moving the sample relative to the objective and the light beam projector. According to embodiments, the disclosed subject matter includes the microscopes include a traversing system connected to one or both of the microscope and a sample support to move them such that a slab-shaped sample can be scanned. In variations, the traversing system is an X-Y traversing system. In variations, the traversing system is an X-Y-Z traversing system, where X and Y axes are parallel to a major plane of the sample volume and the Z axis is perpendicular thereto. In variations, a controller is connected to the traversing system and scanning mechanism to scan each of multiple tile segments of a sample using the scanning mechanism and to shift the sample support relative to the microscope to scan additional tile segment. In variations, the scanning mechanism includes electrically tunable lens (ETL). In variations, the ETL changes a position of the line of maximum intensity along a length of the sheet in the direction of propagation of the sheet. In variations, the scanning mechanism includes a light redirector that scans the sheet such that the line of maximum intensity traverses an axis perpendicular to a direction of propagation of the sheet. In variations, the scanning mechanism includes a light redirector that scans the sheet such that the line of maximum intensity traverses an axis perpendicular to a direction of propagation of the sheet. In variations, the scanning mechanism includes a light redirector that scans the sheet such that the line of maximum intensity traverses an axis perpendicular to a direction of propagation of the sheet. In variations, the light redirector includes a galvo-motor. In variations, the light redirector includes a galvo-mirror. In variations, the scanning mechanism scans in two axes, one to changes a position of the line of maximum intensity along a length of the sheet in the direction of propagation of the sheet and one to change the position of the line of maximum intensity along an axis perpendicular to the direction of propagation of the sheet. In variations, the scanning mechanism scans in two axes, one to changes a position of the line of maximum intensity along a length of the sheet in the direction of propagation of the sheet and one to change the position of the line of maximum intensity along an axis perpendicular to the direction of propagation of the sheet. In variations, wherein the scanning mechanism includes electrically tunable lens (ETL) to change the position of the line of maximum intensity along a length of the sheet in the direction of propagation of the sheet.
According to embodiments, the disclosed subject matter includes a method of acquiring a three-dimensional image of a sample. The method includes projecting a beam of light into a sample, the beam has a line of maximum intensity perpendicular to a direction of propagation of the beam and scanning the beam to scan the line of maximum intensity along a path oblique to both the direction of propagation and perpendicular to it. The method may include capturing images of the line on respective portions of an imaging transducer. The method may be such that, additionally or alternatively, such that the imaging transducer includes a camera sensor. The method may be such that, additionally or alternatively, such that the capturing includes imaging light from the line using a microscope. The method may be such that, additionally or alternatively, such that the capturing includes receiving and imaging secondary light induced by the line of maximum intensity. The method may be such that, additionally or alternatively, such that the capturing includes receiving and imaging a region of fluorescence induced by the line of maximum intensity. The method may be such that, additionally or alternatively, such that the scanning includes mechanically moving the end of a wave guide. The method may include, additionally or alternatively, moving the sample relative to the microscope and the beam of light and repeating the scanning and capturing for a different region of the sample. The method may include, additionally or alternatively, creating two-dimensional images from the capturing and stitching them together to form larger composite images by matching features of the edges of the images. The method may be such that, additionally or alternatively, such that the moving includes moving in one or all of three orthogonal directions. The method may be such that, additionally or alternatively, such that the moving includes moving in all of three orthogonal directions to traverse a three-dimensional volume of positions.
The method may include, additionally or alternatively, changing the focus of an imaging device to optically discriminate multiple depths of the sample after each scan of the scanning. The method may be such that, additionally or alternatively, such that the light sheet projector includes a GRIN lens as an objective.
According to embodiments, the disclosed subject matter includes a microscopy method that includes positioning a detection objective oriented with its optical axis perpendicular to a facing surface of a sample slab. The method further includes positioning an excitation objective oriented to form an acute angle with the detection objective optical axis. The positioning a detection objective and the positioning an excitation objective is such that the detection and excitation objectives is on a same side of the sample slab. The method further includes applying light off-center to the back aperture of the excitation objective such that a beam emerges at an angle with respect to an optical axis of the excitation objective and with a major axis parallel to its direction of propagation that forms a larger angle than the acute angle.
The method may be such that, additionally or alternatively, such that the applying includes partially optically blocking the back aperture. The method may be such that, additionally or alternatively, such that the major axis forms a more acute angle with the facing surface than the detection objective optical axis.
According to embodiments, the disclosed subject matter includes a microscopy method that includes arranging a detection objective of a microscope with its optical axis normal to a surface of a sample slab and injecting light along directions forming equal and opposite angles with the detection objective optical axis. The method includes imaging light resulting from the injecting using the microscope. The method may be such that, additionally or alternatively, such that the light resulting from the injecting includes fluorescence. The method may be such that, additionally or alternatively, such that the injecting results in a single line-shaped region of maximum light intensity in the sample. The method may include, additionally or alternatively, scanning the region perpendicular to a major axis thereof to trace out a planar segment of the sample while sampling the light resulting from the injecting using a light sensor.
The method may be such that, additionally or alternatively, such that the sensor includes a camera. The method may be such that, additionally or alternatively, such that the imaging includes receiving the resulting slight through a microscope. The method may be such that, additionally or alternatively, such that the detection objective is an objective of a microscope.
The method may, additionally or alternatively, include scanning the region perpendicular to a major axis thereof to trace out a planar segment of the sample that is parallel to the sample surface while sampling the light resulting from the injecting using a light sensor.
According to embodiments, the disclosed subject matter includes a microscope with at least two excitation optical elements arranged to form and direct respective sheets of light such that they intersect to form an illuminated line of intersection in a sample region, the sheets has major planes that form angles of at least 75 degrees. A detection optical element is arranged with respect to the excitation optical elements with an optical axis forming equal angles with the major planes of the sheets.
In variations of the microscope, at least two excitation optical elements include objective lenses. In further variations of the microscope, at least two excitation optical elements include objective lenses each has an adjustable partial beam block to cause the sheet beam to be emerge from a side portion of the objective. In further variations of the microscope at least two excitation optical elements include GRIN lenses. In further variations of the microscope the two excitation optical elements include scanning optical elements arranged to cause the illuminated line to traverse the sample region. In further variations of the microscope the two excitation optical elements include scanning optical elements arranged to cause the illuminated line to traverse the sample region along a focal plane of the detection optical element. In further variations of the microscope a traversing system is provided and the controller is configured to control scanning components and to form an image slice from successive detections of the sample region.
According to embodiments, the disclosed subject matter includes a microscope. First and second imaging components, each have an imaging objective, and first and second light sheet projectors each located on a same side of a sample region as a respective on of the first and second imaging components. Each imaging objective and each light sheet projector has respective axes that intersect in the sample region. A sample support is positioned to hold a sample in the sample region. Each light sheet projector has light scanning and focusing optics to permit the selection of an axial position of a light sheet beam waist and the selection of a position of the light sheet along an axis perpendicular to the light sheet propagation direction. A controller is connected to control the light scanning and focusing optics to translate the beam waste of each light sheet projector independently along a respective linear path.
In variations, the imaging objective optical axes are perpendicular to a respective one of the linear paths. In variations, the imaging objective optical axes are perpendicular to a both of the linear paths. In variations, the focusing optics include an electrically tunable lens (ETL). In variations, the sample support is a generally flat element shaped to hold a slab-shaped sample. It will be appreciated that the modules, processes, systems, and sections described above can be implemented in hardware, hardware programmed by software, software instruction stored on a non-transitory computer readable medium or a combination of the above. For example, a method for capturing three dimensional optical images can be implemented, for example, using a processor configured to execute a sequence of programmed instructions stored on a non-transitory computer readable medium. For example, the processor can include, but not be limited to, a personal computer or workstation or other such computing system that includes a processor, microprocessor, microcontroller device, or is comprised of control logic including integrated circuits such as, for example, an Application Specific Integrated Circuit (ASIC). The instructions can be compiled from source code instructions provided in accordance with a programming language such as Java, C++, C#.net or the like. The instructions can also comprise code and data objects provided in accordance with, for example, the Visual Basic™ language, LabVIEW, or another structured or object-oriented programming language. The sequence of programmed instructions and data associated therewith can be stored in a non-transitory computer-readable medium such as a computer memory or storage device which may be any suitable memory apparatus, such as, but not limited to read-only memory (ROM), programmable read-only memory (PROM), electrically erasable programmable read-only memory (EEPROM), random-access memory (RAM), flash memory, disk drive and the like.
Furthermore, the modules, processes, systems, and sections can be implemented as a single processor or as a distributed processor. Further, it should be appreciated that the steps mentioned above may be performed on a single or distributed processor (single and/or multi-core). Also, the processes, modules, and sub-modules described in the various figures of and for embodiments above may be distributed across multiple computers or systems or may be co-located in a single processor or system. Exemplary structural embodiment alternatives suitable for implementing the modules, sections, systems, means, or processes described herein are provided below.
The modules, processors or systems described above can be implemented as a programmed general purpose computer, an electronic device programmed with microcode, a hard-wired analog logic circuit, software stored on a computer-readable medium or signal, an optical computing device, a networked system of electronic and/or optical devices, a special purpose computing device, an integrated circuit device, a semiconductor chip, and a software module or object stored on a computer-readable medium or signal, for example.
Embodiments of the method and system (or their sub-components or modules), may be implemented on a general-purpose computer, a special-purpose computer, a programmed microprocessor or microcontroller and peripheral integrated circuit element, an ASIC or other integrated circuit, a digital signal processor, a hardwired electronic or logic circuit such as a discrete element circuit, a programmed logic circuit such as a programmable logic device (PLD), programmable logic array (PLA), field-programmable gate array (FPGA), programmable array logic (PAL) device, or the like. In general, any process capable of implementing the functions or steps described herein can be used to implement embodiments of the method, system, or a computer program product (software program stored on a non-transitory computer readable medium).
Furthermore, embodiments of the disclosed method, system, and computer program product may be readily implemented, fully or partially, in software using, for example, object or object-oriented software development environments that provide portable source code that can be used on a variety of computer platforms. Alternatively, embodiments of the disclosed method, system, and computer program product can be implemented partially or fully in hardware using, for example, standard logic circuits or a very-large-scale integration (VLSI) design. Other hardware or software can be used to implement embodiments depending on the speed and/or efficiency requirements of the systems, the particular function, and/or particular software or hardware system, microprocessor, or microcomputer being utilized. Embodiments of the method, system, and computer program product can be implemented in hardware and/or software using any known or later developed systems or structures, devices and/or software by those of ordinary skill in the applicable art from the function description provided herein and with a general basic knowledge of control systems, kinematic design, optics, microscopy and/or computer programming arts.
Moreover, embodiments of the disclosed method, system, and computer program product can be implemented in software executed on a programmed general purpose computer, a special purpose computer, a microprocessor, or the like.
It is, thus, apparent that there is provided, in accordance with the present disclosure, optical imaging systems, devices, and methods. Many alternatives, modifications, and variations are enabled by the present disclosure. Features of the disclosed embodiments can be combined, rearranged, omitted, etc., within the scope of the invention to produce additional embodiments. Furthermore, certain features may sometimes be used to advantage without a corresponding use of other features. Accordingly, Applicants intend to embrace all such alternatives, modifications, equivalents, and variations that are within the spirit and scope of the present invention.
This Application is a US national stage filing under 35 U.S.C. § 371 of International Application No. PCT/US2017/061165, filed Nov. 10, 2017, which claims the benefit of U.S. Provisional Application 62/420,349 filed Nov. 10, 2016, and U.S. Provisional Application 62/473,944, filed Mar. 20, 2017, each of which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/061165 | 11/10/2017 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/089839 | 5/17/2018 | WO | A |
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