Multi-modal imaging systems and methods

Multi-modality imaging systems and methods for enabling controllable and/or automated switching between different imaging systems or modes. An imaging system includes a base plate having a first exposed region and a second region, a sample stage configured to hold a sample platform, and a first translation mechanism configured to translate the sample stage on the base plate along a first axis between a first position and a second position. In the first position the sample stage is positioned proximal to the first exposed region, and in the second position, the sample stage is positioned proximal to the second region. An illumination device is configured to illuminate a portion of the first exposed region, and a second translation mechanism is configured to translate the illumination device along a second axis substantially perpendicular to the first axis.

SUMMARY

The present disclosure provides multi-feature imaging systems and methods.

Various embodiments advantageously provide systems and methods for imaging a sample with one or multiple imaging modalities using the same imaging system layout. Various embodiments advantageously combine optical imaging modalities such as a line-scanning imaging system with an area imaging system on the same system platform layout. In certain embodiments, the systems and methods advantageously enable controllable and/or automated switching between imaging modalities and capturing images of a sample using the various imaging modalities.

The various embodiments advantageously enable optimized quantitative measurements of a sample plane, e.g., one or more targets of interest at or on the sample plane, which may be applicable for various scientific applications, such as but not limited to, e.g., electromagnetic spectrum imaging, fluorescence imaging, chemiluminescence imaging and the like.

According to an embodiment, an imaging system is provided that includes a base plate having a first exposed region and a second region, a sample stage, configured to hold a sample platform, and a first translation mechanism, coupled with the base plate and the sample stage and configured to translate the sample stage on the base plate along a first axis between a first position and a second position, wherein when in the first position a first portion of the sample stage is positioned proximal to the first exposed region in the base plate, and when in the second position, the first portion of the sample stage is positioned proximal to the second region of the base plate. The system may also include an illumination device located proximal to the base plate, and configured to illuminate a portion of the first exposed region in the base plate, and a second translation mechanism, coupled with the base plate and the illumination device and configured to translate the illumination device along a second axis, the second axis being substantially perpendicular to the first axis. The system may also include a first imager located proximal to the base plate on a side opposite the sample stage and configured to image the portion of the first exposed region in the base plate, and a third translation mechanism, coupled with the base plate and the first imager and configured to translate the first imager along a third axis, the third axis being parallel to the second axis.

In certain aspects, the system further comprising a fourth translation mechanism coupled with the third translation mechanism and configured to translate the first imager along a fourth axis, the fourth axis being substantially perpendicular to both the third axis and the first axis. In certain aspects, the second region is a second exposed region in the base plate, and the system further comprising a second imager located proximal to the base plate and configured to image a region proximal to the second exposed region in the base plate. In certain aspects, the second imager is located proximal the base plate on the side opposite the sample stage. In certain aspects, the second imager is located proximal the base plate on the same side as the sample stage.

In certain aspects, the second translation mechanism and the third translation mechanism are configured to move together so as to translate the illumination device along the second axis simultaneously or in step with moving the first imager along the third axis. In certain aspects, the second region of the base plate is a loading region that provides access to load the sample platform on the sample stage. In certain aspects, the second region is a second exposed region in the base plate, wherein the first translation mechanism is further configured to translate the sample stage on the base plate along the first axis to a third position proximal to a loading region of the base plate, wherein when in the third position the first portion of the sample stage is positioned proximal the loading region and provides access to load the sample platform on the sample stage.

In certain aspects, the system further includes a control system comprising one or more processors, the control system configured to control operation of at least the first translation mechanism, the second translation mechanism and the third translation mechanism. In certain aspects, the control system is further configured to control operation of the first imager and the illumination device. In certain aspects, the control system is further configured to control operation of the fourth translation mechanism.

In certain aspects, the base plate includes grid lines thereon to enable accurate positioning of the sample on the sample platform when the sample platform is coupled with the sample stage. In certain aspects, the illumination device is located proximal to the base plate on an opposite side of the base plate as the sample stage, and configured to illuminate a portion of the first exposed region in the base plate. In certain aspects, the illumination device includes one or multiple illumination sources. In certain aspects, the one or multiple illumination sources are located proximal to the base plate on an opposite side and/or same side of the base plate as the sample stage, and configured to illuminate a portion of the first exposed region in the base plate. In certain aspects, each of the one or multiple illumination sources emit light at different wavelengths.

In certain aspects, the first imager comprises a fluorescence imaging system and the second imager includes a chemiluminescence imaging system. In certain aspects, the fluorescence imaging system includes a bi-telecentric imaging system. In certain aspects, the first imager includes an imaging device and one or multiple light sources positioned such that either 1) light from the one or multiple light sources incident on the sample stage proximal the first exposed region reflects toward the imaging device, or 2) light incident on the sample stage is coaxial with an imaging axis of the imaging device.

According to another embodiment, a method of imaging a sample using any of the systems described herein is provided. The method includes receiving a sample on the sample platform on the sample stage, wherein the sample is positioned on the sample platform proximal to the first portion of the sample stage, and imaging the sample using the first imager when the sample stage is in the first position, wherein the sample is proximal to the first exposed region in the base plate when the sample stage is in the first position.

In certain aspects, the imaging the sample using the first imager includes acquiring a first image of the sample, translating, using the third translation mechanism, the first imager along the third axis, and acquiring a second image of the sample, wherein the second image of the sample and the first image cover different portions of the sample. In certain aspects, the acquiring an image includes line-scanning a portion of the sample. In certain aspects, the acquiring an image includes illuminating the sample using the illumination device, the method further including translating the illumination device along the second axis, so that the illumination device is positioned to illuminate at least a portion of the sample being imaged by the first imaging device.

In certain aspects, the imaging the sample using the first imager includes acquiring a first image of the sample, translating, using the fourth translation mechanism, the first imager along the fourth axis, and acquiring a second image of the sample, wherein the second image is taken such that a focal plane of the first imager is located at a different position in the sample when compared with the first image.

In certain aspects, the receiving the sample platform on the sample stage includes translating the sample stage, using the first translation mechanism, to the loading position on the base plate, receiving a sample on the sample platform, receiving the sample platform on the sample stage, and thereafter translating the sample stage, using the first translation mechanism, to the first position.

In certain aspects, the method further includes translating, using the first translation mechanism, the sample stage along the first axis to the second position, and imaging the sample using the second imager when the sample stage is in the second position, wherein the sample is proximal to the second exposed region in the base plate when the sample stage is in the second position.

According to another embodiment, a method of imaging a sample using any of the systems described herein is provided. The method includes receiving the sample platform on the sample stage, wherein the sample is positioned on the sample platform proximal to the first portion of the sample stage, and imaging the sample using the second imager when the sample stage is in the second position, wherein the sample is proximal to the second region in the base plate when the sample stage is in the first position, wherein the second region is an exposed region in the base plate.

In certain aspects, the receiving the sample platform on the sample stage includes translating the sample stage, using the first translation mechanism, to the loading position on the base plate, receiving a sample on the sample platform, receiving the sample platform on the sample stage, and thereafter translating the sample stage, using the first translation mechanism, to the second position.

In certain aspects, the method further includes translating, using the first translation mechanism, the sample stage along the first axis to the first position, and imaging the sample using the first imager when the sample stage is in the first position, wherein the sample is proximal to the first exposed region in the base plate when the sample stage is in the first position.

According to another embodiment, a method of imaging a sample using any of the systems described herein is provided. The method includes receiving the sample platform on the sample stage, wherein the sample is positioned on the sample platform proximal to the first portion of the sample stage, acquiring one or more first images of the sample using the first imager when the sample stage is in the first position, wherein the sample is proximal to the first exposed region in the base plate when the sample stage is in the first position, and acquiring one or more second images of the sample using the second imager when the sample stage is in the second position, wherein the sample is proximal to the second region of the base plate when the sample stage is in the second position.

In certain aspects, the method further includes combining the one or more first images with the one or more second images to form one or more combined images of the sample. In certain aspects, the one or more first images are line-scan images, each first image taken at a different position relative to a prior first image, and wherein the method further includes stitching the one or more first images together to form a combined image of the sample.

In certain aspects, the systems described herein may further include a control system module including at least one processor, wherein the control system module is communicably coupled with and adapted to control operation of the system components, such as activating illumination sources (e.g., LED(s), lasers, etc.), activating or reading detectors, controlling various adjustable optical components and adjustable mechanical components, including mechanical actuators for adjusting or translating physical positions of various components such as illumination devices and imaging devices, and a stage or platform that holds a sample, which may include one or more targets of interest at or on the sample plane, etc.

In certain aspects, a sample may include one or more targets of interest, and the multi-modality imaging system is configured to capture or acquire images of the one or more targets of interest using one or multiple imaging systems such as a line-scanning system and an area imaging or wide-field imaging system. Specific examples include an electromagnetic spectrum imaging system, a fluorescence imaging system, a chemiluminescence imaging system, or like imaging systems. In certain aspects, for fluorescence imaging as an example, the one or more targets of interest (e.g., within or on a sample, which may be located on a sample platform) may comprise a fluorescent material, and the illumination device emits light at one or more wavelengths in the absorption band(s) of the fluorescent material(s) comprised within the one or more targets of interest.

In a further embodiment, a non-transitory computer readable medium is provided that stores instructions, which when executed by at least one processor, causes the at least one processor to control operation of the system components and to implement any method as described herein. Examples of computer readable media include RAM, ROM, CDs, DVDs, ASICs, FPGAs or other circuit elements including memory elements.

DETAILED DESCRIPTION

Various system and method embodiments are provided for enabling automatic switching between multiple imaging modalities for imaging a sample and/or one or more targets of interest on or within the sample. In certain embodiments, the systems and methods advantageously enable imaging of a sample and/or one or more targets of interest on or within the sample using one or multiple different imaging modalities associated with the same system platform. Such information may be useful for research scientists who would like to know more detail, such as where in a sample a certain target of interest, biomarker, probe, tracer, contrast agent or dye molecule and the like is located in addition to spatial and temporal information, the physiological information, phenotypical information, efficacy and potency information that may be derived from imaging a sample, for example.

FIG.1Ashows an isometric view of a multi-modality imaging system100, according to an embodiment. System100includes a base plate12, a sample stage14and a first translation mechanism10coupled with the base plate12and the sample stage14. Base plate12functions as a central supportive element or structure. Sample stage14functions as a supportive element or structure for receiving and holding sample platform16(position of platform16shown inFIG.1A,FIG.1B, andFIG.2A; physical representation shown inFIG.3.) The first translation mechanism10is configured to translate the sample stage14along a first axis relative to the base plate12. For example, with the arbitrary X-Y-Z axis shown inFIG.1A, the first translation mechanism10is configured to translate the sample stage14along the Y-axis relative to the base plate12.FIGS.2A-2E, which will be discussed in more detail below, illustrate examples of the sample stage14in different positions along the Y-axis relative to the base plate12.

Sample stage14is configured to hold a sample platform16, which is adapted to receive and hold a sample thereon; sample platform16, in an embodiment, provides a surface that removably interconnects with the sample stage14to hold and/or position one or more samples. The sample platform16may be removed, e.g., for cleaning, replacing or repairing and may have interchangeable inserts used for holding and imaging different samples or sample types.

Embodiments of the present invention with optical imaging systems address to imaging targets of interest contained within or on a sample. A “sample” includes and may refer to any liquid, solid, or other type of material that may be comprised of or as, in or on a cell or cells (e.g. in whole or lysed); a slurry or an extraction of cellular components; a tissue or tissues; an organ, organs, organoid or other organ-like materials; organisms such as but not limited to invertebrate or vertebrate organisms (i.e. in whole or in part); substrates such as but not limited to western blots, membranes, woven networks of fibers, gels, plastic media, glass media or other media in the form of a plate, dish, tube, slide, etc.; or any combination thereof. It should also be appreciated that “imaging system” and “imaging device” and “imager” may be used interchangeably herein, and that each includes one or more detector elements for detecting light from the sample and one or more optical elements for directing and/or focusing light onto the one or more detector elements. One skilled in the art will understand that many types of useful sensors or detectors and arrays of detectors may be used, including but not limited to CCD and CMOS sensors. Other useful detectors or sensors might include sCMOS sensors, photodiodes, avalanche photodiodes, silicon photomultiplier devices, an array of photomultiplier tubes, a focal plane array, etc.

Accordingly, when the first translation mechanism10operates to move the sample stage14along the Y-axis relative to the base plate12, an attached sample platform16(and any sample that may be on sample platform16) concomitantly moves relative to base plate12. Sample platform16may comprise an optically transparent glass or plastic material or may comprise an optically opaque plastic or metal or glass material, depending on the application and system configuration.

FIG.1Bshows a top view of the multi-modality imaging system100ofFIG.1A, according to an embodiment. As shown, a first region21and a second region22in base plate12are identified. In an embodiment, each of first region21and second region22may include an exposed region of base plate12, e.g., devoid of material, or including an optically transparent material or window. In some embodiments, one or both of first region21and second region22may be a solid region of base plate12. As shown inFIG.1A, sample stage14is located in a loading position, away from first region21and second region22. When in the loading position, sample stage14may partially overlap with first region21. Controlled movement of sample stage14between various positions will be discussed below with reference toFIGS.2A-2E.

In an embodiment, system100also includes a second translation mechanism20coupled with the base plate12and an illumination device24. Second translation mechanism20is configured to translate the illumination device24along a second axis relative to the base plate12, with the second axis being substantially perpendicular to the first axis. For example, with the arbitrary X-Y-Z axis shown inFIG.1A, the second translation mechanism20is configured to translate the illumination device24along the T-axis relative to the base plate12. In an embodiment, illumination device24is configured to illuminate a portion of the first region21. In operation, one or more illumination source(s) of illumination device24illuminate a portion of first region21, and the portion illuminated may be scanned, e.g., by second translation mechanism20translating illumination device24along the second axis. The portion of first region21illuminated by illumination device24may form a spot, a line, a stripe or a larger area or region. Illumination device24may include a single illumination source or multiple illumination sources. For example, illumination device24may be configured with one or multiple broadband sources, and/or it may be configured with one or multiple narrowband sources, such as one or multiple LEDs operating in different frequency/wavelength bands, depending on the imaging application. For example, for optical imaging applications, one or more illumination sources emitting at one or more absorption wavelengths of one or more targets of interest may be used. Where multiple sources are included, each source may be configured to illuminate the same area (e.g., spot or line or 2-dimensional area such as a rectangle, circle, oval, etc.), or different areas, or partially overlapping areas within first region21, or portions thereof.

FIG.1Cshows a side view of the multi-modality imaging system100, according to an embodiment, andFIG.1Dillustrates shows a frontal view of the multi-modality imaging system100, according to an embodiment. As shown, system100also includes a third translation mechanism30, coupled with the base plate12and a first imaging device26, (position of first imaging device26mount position shown inFIG.1CandFIG.1D; physical representation shown inFIG.3.) and configured to translate the first imaging device26along a third axis, X. In an embodiment, the third axis, X, is parallel to the second axis and perpendicular to the first axis. For example, with the arbitrary X-Y-Z axis shown inFIG.1A, the third translation mechanism30is configured to translate the imaging device26along the X-axis relative to the base plate12. However, third axis, X, need not be parallel to the second axis and may be arranged at an angle along the X-Y plane with respect to the second axis in other embodiments. In the embodiment shown inFIG.1CandFIG.1D, imaging device26and third translation mechanism30are located proximal to the base plate12on the side opposite the sample stage14(and also on the side opposite illumination device24). In other embodiments, imaging device26and third translation mechanism30may be located proximal to the base plate12on the same side as the sample stage14(and also on the same side as illumination device24). Similarly, in the embodiment shown inFIG.1CandFIG.1D, illumination device24and second translation mechanism20are located proximal to the base plate12on the same side as the sample stage14(and also on a side opposite imaging device26); in other embodiments, illumination device24and second translation mechanism20may be located proximal to the base plate12on the side opposite to the sample stage14(and also on the same side as imaging device26or on a side opposite imaging device26). In certain embodiments, imaging device26may have one or more/multiple illumination sources. For example, the one or more/multiple illumination sources of imaging device26may include a laser, LED or other illumination source. For example, for optical imaging applications, imaging device26may include one or more illumination sources emitting at one or more absorption wavelengths of one or more targets of interest.

In an embodiment, during operation, second translation mechanism20and third translation mechanism30are configured to move together so as to translate the illumination device24along the second axis simultaneously or in step with translating the first imaging device26along the third axis, X, e.g., in response to control signals received from a control system (not shown). In other embodiments, second translation mechanism20and third translation mechanism30are configured to move separately.

In another embodiment, system100also includes a fourth translation mechanism40coupled with the third translation mechanism30and/or coupled with first imaging device26. Fourth translation mechanism40is configured to translate the first imaging device26along a fourth axis. In an embodiment, the fourth axis is substantially perpendicular to both the second axis and the first axis (and the third axis). For example, with the arbitrary X-Y-Z axis shown inFIG.1A, the fourth translation mechanism40is configured to translate the imaging device26, or a component of the imaging device26, such as a lens element, along the Z-axis relative to the base plate12. In operation, translating the imaging device26, or the component of the imaging device26, along the Z-axis enables a Z-focus or change of focal plane for imaging device26.

In an embodiment, sample stage14may be moved between at least a first position and a second position: for the first position, at least a first portion of sample stage14is positioned proximal to the first region21of the base plate12, and for the second position, the first portion of the sample stage is positioned proximal to the second region22of the base plate. Each of the first position and the second position may correspond to imaging positions, where a sample on the sample platform16on the sample stage14may be imaged. Additionally, each of, or both of, the first and second positions may correspond to a loading position wherein a user may access the sample stage14to load a sample platform16(with or without a sample), or to load a sample on a sample platform16already on sample stage14. A third position, such as a loading position may be provided in certain embodiments.

FIG.2Ashows a top view of the multi-modality imaging system100, with the sample stage14positioned or located in a loading position, away from first region21and second region22, according to an embodiment. Sample stage14is configured to receive and securely hold sample platform16, which is adapted to receive and hold a sample thereon. When in the loading position, at least a portion of the sample stage14is positioned proximal a loading region which provides access to load the sample platform16on the sample stage14. For example, when in the loading position the volume above the sample stage14may be completely clear of obstacles, e.g., clear of illumination device24and/or other system elements.

When sample stage14is in the loading position, in an embodiment, the area below the sample platform is blocked off by the base plate12(i.e., no imaging system is accessible directly below the glass platform, or sample area without glass). For example, the base plate12may be configured, in an embodiment, to protect system components such as the imaging system when samples, sample platform16or sample platform inserts are being positioned on the sample stage14. Also, in an embodiment, grid lines may be provided on the base plate12, such that when in the loading position, a user is able to clearly visualize the grid lines on the base plate12for accurate positioning of the sample on the sample platform16. Alternatively, this portion of the base plate12could include a removable insert that could be exchanged with other inserts displaying different grids or scales for sample positioning purposes. The removable inserts may be inserted onto (e.g., on top of) the sample platform and are not integrated with the base plate12.

Controlled movement of the sample stage14between various positions on base plate12is effected by operation of first translation mechanism10. In an embodiment, first translation mechanism10may include one or more rails and an actuator configured to move sample stage14along the one or more rails. Additionally or alternatively, the actuator may include a stepper motor, or linear screw actuator or other device capable of translating sample stage14along the Y-axis. Second translation mechanism20, third translation mechanism30, and fourth translation mechanism40may each include similar actuators or different actuators.

FIG.2Bshows a top view of the multi-modality imaging system ofFIG.2A, with sample stage14positioned in a first imaging position, according to an embodiment. In the first imaging position, at least a first portion of sample stage14is positioned proximal to the first region21of the base plate12. InFIG.2B, imaging device26and illumination device24are shown in a first position, e.g., an initial scan position; inFIG.2C, imaging device26and illumination device24are shown in a second position, e.g., a final scan position. For example, when in the first imaging position, a sample on the sample stage proximal to the first region21may be illuminated and imaged by a combination of illumination device24and image device26, and where scanning is desired, a scanned image of the sample may be acquired by scanning imaging device26and/or illumination device24across the X and T axes. A larger scan or image may also be acquired by moving sample stage14such that a slightly different portion of the sample is exposed proximal the first region21, e.g., scanning along the X, T axes and then repositioning sample to a slightly different position in first region21and then rescanning along the X, T axes to image a larger area of the sample.

In an embodiment, as shown inFIG.3, for example, a second imaging device or system28is located proximal to the second region22of base plate12. Second imaging system28may image a sample proximal to the second region21of the base plate12and may utilize illumination from illumination device24, or it may utilize illumination from a separate source or sources. For example, in an embodiment, wherein the second region22is an exposed region in the base plate12, the second imager28may be located proximal to the base plate12and configured to image a region proximal to the second exposed region in the base plate. Second imager28may be located proximal to the base plate12on the side opposite sample stage14, or on the same side as sample stage14. In another embodiment, second imaging system28may not require illumination to induce a reaction. For example, second imaging system28may include a chemiluminescence imaging system with detected light being generated from a chemically exothermic reaction or induced by an electrochemical stimulus.

FIG.2Dshows a top view of the multi-modality imaging system ofFIG.2A, with sample stage14positioned in a second imaging position, according to an embodiment. In the second imaging position, at least a first portion of sample stage14is positioned proximal to the second region22of the base plate12. InFIG.2D, illumination device24is shown in a first position, e.g., an initial scan position; inFIG.2E, illumination device24is shown in a second position, e.g., a final scan position. For example, when in the second imaging position, a sample on the sample stage14proximal to the second region22may be illuminated by illumination device24, and where scanning is desired, illumination device24may be scanned across the T-axis.FIGS.2B-2Eshow the sample stage14, illumination device24, and imaging system26at extents of the area that is scannable by the imaging system26.FIG.2DandFIG.2Eboth show that a portion of the sample stage may be placed over the second region22during the course of imaging with imager26; the second imaging system28(seeFIG.3) can be placed such that it can image a portion of the sample platform16without requiring any additional range of motion in any of the actuators. This spatial efficiency is an advantage that the present embodiments provide over prior systems.

The present embodiments advantageously enable multi-modal imaging using the same system platform. For example, a line-scan imaging system may be combined with area imaging system on the same system platform; the line-scan imaging system may implement angled reflective imaging, trans-illumination imaging, epi-illumination imaging and/or fluorescence imaging and the area imaging system may implement chemiluminescence imaging as examples. For the line-scan imaging system, the resulting scanned image, e.g., following angled reflective imaging and/or fluorescence imaging, that is displayed to the user may be comprised of one, or more, parallel offset scanned stripes that may be stitched/directionally assembled together. The stripes may be generated when the sample is moved, relative to the imaging system, along the Y-axis. Subsequent stripes are created, parallel to the first stripe, after repositioning (e.g., indexing to the subsequent stripe; which may be the adjacent stripe to the previously scanned stripe or it may be the same stripe (in whole or in part) being re-scanned) the imaging system, relative to the sample, along the X-axis. Focus may be adjusted by moving a component, such as a lens, of the imaging system in a direction normal to the sample plane, relative to the sample, e.g., along the Z-axis. For sample scans that require trans-illumination, the illumination device (e.g., trans-illumination device) is moved along the T-axis parallel to the X-axis, located opposite the imaging system relative to the sample plane (as shown inFIG.3andFIG.4to be positioned above the sample plane).FIG.3shows a side-view of the multi-modality imaging system including imaging system26and imaging system28, and includes a cutaway showing a sample laying on the sample platform16between the first imaging system26and the illumination device24(i.e. trans-illumination configuration), according to an embodiment.FIG.4shows the same view asFIG.3, but without the cutaway.

In an embodiment, the second imager28may include a wide-field or area-imaging device, such as a chemiluminescence imaging system and the first imager26is a line-scanning imaging system. For example, the second area-imaging system28could capture images of samples that have more time-sensitive characteristics than some other samples which can be scanned. Since the sample would be placed on the same sample stage for both imaging systems, both imaging system26and imaging system28could be used sequentially to capture various types of signals (e.g., chemiluminescence, which is relatively time-sensitive, and fluorescence or RGB reflected light) on the same sample. These multiple acquired images could then be overlaid onto each other, providing more information from a single sample than any one of the signal types could on its own.

Moving the imaging system26only on a slower step-axis (X-axis) and focus-axis (fourth axis), the motion requirements of any moving mass and cable cycling stress is advantageously simplified. Also, having the imaging system remaining stationary, during scanning, alleviates possible functionality and stress related issues that could impact the scan quality. For example, in an embodiment, the imaging system26only moves in one axis (X-axis), and remains stationary while the actual scanning motion is done by actuator10moving the sample stage14. The imager26can then be moved between scan passes to position it to collect the next parallel offset scan stripe that is assembled to produce the complete image.

Moving the illumination device24on a separate axis from imaging system26allows the illumination components to be minimally sized similar to the imaging system, simplifying uniformity calibration, and adds the potential flexibility for additional illumination sources or components that can be shifted opposite of the imaging system.

The embodiments herein are useful for point-scan imaging, line-scan imaging, and area imaging applications. For line-scan imaging, a line is scanned (swept) along an axis to generate an image stripe, the imaging system is indexed relative to the sample and scanned again, and so on, until finished at which point all stripes are stitched (e.g., directionally assembled) or combined together to form a complete image.

FIG.5illustrates a method500of imaging a sample using one or multiple imaging modalities according to an embodiment. In step510, a sample is received. Receiving the sample may include receiving sample platform16on sample stage14. The sample may already be located on sample platform16when the user positions sample platform16on sample stage14. Alternatively, sample platform16may be positioned on sample stage14and then the sample may be placed, and secured, onto sample platform16. In an embodiment, sample stage14and sample platform16may be configured such that when sample platform16is positioned on sample stage14, the sample is located in a first portion of sample stage16and such that when sample stage16is moved to a first imaging position, e.g., proximal to the first exposed region21in base plate12, the first portion of the sample stage is positioned proximal to the first exposed region21in the base plate12. Guidelines or markers on the baseplate may facilitate placement by the user. Proper placement of the sample on the sample platform helps ensure proper registration of the sample relative to imagers26,28when in an imaging position. In an embodiment, sample stage14may be repositioned, if needed, to the loading position so that sample platform16may be received without interference from other system components. For example, first translation mechanism10may be activated to translate or move sample stage14to the loading position as shown inFIG.1B.

In step520, sample stage14is moved to an imaging position. For example, first translation mechanism10may be activated to translate or move sample stage14to a first imaging position proximal to first exposed region21of base plate12or proximal to second region22of base plate12. In step530, the sample is imaged using a first imaging system. For example, with sample stage14positioned such that the sample is proximal to region21of base plate12, the sample may be imaged using first imaging device26. Depending on the imaging modality of first imaging device26, illumination device24may be simultaneously activated.

In one embodiment, for example, imaging device26includes a line scanning imaging system including a detector and one or more illumination sources. As an example, one useful imaging system is provided in US Patent Application Publication No. 2020/0220332 A1, titled “LASER LINE ILLUMINATION USING COMBINED SINGLE-MODE AND MULTI-MODE LASER SOURCES”, which is hereby incorporated by reference in its entirety. In this embodiment, third translation mechanism30may be activated to scan imaging device26along the third axis so as to scan the line generated by the one or more illumination sources and detect light reflected and/or emitted from the sample. In another embodiment, imaging device26includes a line-scanning imaging system including a detector without illumination sources. In this embodiment, second translation mechanism20may be activated to scan illumination device24along the second axis so as to scan the line generated by the one or more illumination sources of illumination device24, and third translation mechanism30may be simultaneously activated to scan imaging device26along the third axis to detect light emitted from the sample (e.g., a trans-illumination-based imaging system). Where desirable, fourth translation mechanism40may be activated to refocus imaging device30along the Z-axis when acquiring one or more images of the sample. In step530, one or more images of the sample may be acquired.

In step540, sample stage14is moved to another imaging position. For example, first translation mechanism10may be activated to translate or move sample stage14to a second imaging position from the first imaging position, e.g., proximal to second region22of base plate12or proximal to first exposed region21of base plate12.

In step550, the sample is imaged using a second imaging system. For example, with sample stage14positioned such that the sample is proximal to second region22of base plate12, the sample may be imaged using the second imaging device28. Depending on the imaging modality of second imaging device28, illumination device24may be simultaneously activated (e.g., in some embodiments, illumination device24may be configured to illuminate second region22in addition to first exposed region21or instead of illuminating first exposed region21). In one embodiment, for example, second imaging device28includes an area imaging system. For example, second imaging system28may include a detector configured to detect a chemiluminescence signal emitted from the sample. Alternatively, second imaging system28may include a different area imaging system configuration.

In step560, image(s) acquired in step530and/or in step550are processed. For example, where multiple line-scan images are acquired, the line-scan images may be stitched, assembled, combined or compiled together to form a composite image of a region of the sample or the entire sample. Where area/wide-field images are acquired, these may be processed as desired. In step570, one or more images may be visually presented, e.g., on a display monitor or other display device. Where multiple imaging modalities are used to acquire images of the sample, e.g., line-scan imaging in step530and area imaging in step550, the resulting images may be provided separately for visualization or they may be combined to form a composite image that is viewable on a display device.

It should be appreciated that steps540and550are optional; in an embodiment, sample stage14may be moved to a single imaging location in step520, and imaging performed in step530and processing of the image(s) acquired in step560may be performed without imaging in a second imaging location.

In certain embodiments, the T-axis carries illumination source24. Since the T-axis can move independently of the X-axis, various other devices or components may be coupled with the second translation mechanism20and moved on the T-axis simultaneously with illumination source24, to any desired position. For example, in an embodiment where at least one of the X- or T-axis is on the same side of the sample platform as the sample, regardless of the position of the other of the two axes, the T-axis (second translation mechanism20) may be configured to carry a system to position (e.g., move, manipulate, or dispense) a sample or other material or substance which interacts physically with the sample on the sample platform, (e.g., a system for dispensing an activator or chemical agent onto the sample, an arm to grip and move microtiter plates, an automated pipette, or a dispensing head, etc.). In an embodiment where the X-axis (third translation mechanism30) carries an imaging system, and the T-axis is on the opposite side of the sample platform as the X-axis, the T-axis could carry one or multiple different optical backgrounds for imaging. In certain embodiments, the X- and T-axes could both carry imaging systems, adding additional imaging modes available for use (e.g., one images in the UV spectrum, and the other in the visible and/or infrared and/or near-infrared, thus circumventing the challenge in selecting optical components compatible with a larger wavelength range).

The embodiments described herein are particularly useful for implementing one or multiple imaging modalities using the same system platform. For example, the various imaging modalities may include point scanning and line-scanning imaging systems and area imaging or wide-field imaging systems, including but not limited to fluorescence imaging systems, optical imaging systems, Confocal Laser Scanning Microscopy (CLSM) systems, and/or a combination of imaging systems. Additionally, illumination device24and imaging device26may operate in a trans-illumination mode where optical elements are configured to direct illumination (e.g., illumination beam or light beam) from the illumination source24toward the sample platform16from a side opposite the imaging system26. For example, the illumination source of illumination device24may include a laser, LED or other illumination source.

To image in fluorescence, a one or more targets of interest (e.g., within or on a sample where such targets of interest may contain a fluorescent material, which may be located on sample platform16) is illuminated by an optical signal having a first spectral content (excitation light) where a portion of such a signal is absorbed by at least part of the target of interest and emitted as optical signal of a second spectral content (emission light). The emission light is then detected as a measure of the amount of the fluorescent material present in or on the one or more targets of interest within or on a sample at the designated, illuminated location. Imaging an area of a sample containing one or more targets of interest comprising fluorescent material, therefore, requires excitation light delivered to the one or more targets of interest within or on a sample, an imaging system that collects light from the one or more targets of interest and projects the collected light onto an optical detector (e.g., detector array), and a means to separate the emitted fluorescence light from the portion of excitation light that makes its way through the imaging system. The latter, typically, includes one or more optical interference filters. In certain aspects, relevant filter wavelengths may be anywhere within the ultra-violet to visible to far-red spectrum.

Wide-Field imaging, as considered herein, includes collecting light from a contiguous area and projecting it onto a detector array, such as a CCD or other detectors having an array of sensing locations or pixels, at the same time in a way that preserves the relative locations of each point within the contiguous area. Wide-field imaging is different from collecting light from one point (or line) at a time and sequentially scanning to a different point (or line) in order to cover a larger area, i.e. point-scan (or line-scan) imaging. Another imaging modality includes collecting light from a large area and condensing the total amount of light onto a detector and reading it as total signal; such measurement techniques do not require specific location information.

A “target of interest” may include a material or molecule of interest such as a biomolecule. Biomolecules are molecules of a type typically found in a biological system, whether such molecule is naturally occurring or the result of some external disturbance of the system (e.g., a disease, poisoning, genetic manipulation, etc.), as well as synthetic analogs and derivatives thereof (e.g. recombinant). Non-limiting examples of biomolecules include amino acids (naturally occurring or synthetic), peptides, polypeptides, glycosylated and unglycosylated proteins (e.g., polyclonal and monoclonal antibodies, receptors, interferons, enzymes, etc.), nucleosides, nucleotides, oligonucleotides (e.g., DNA, RNA, PNA oligos), polynucleotides (e.g., DNA, cDNA, RNA, etc.), carbohydrates, hormones, haptens, steroids, toxins, liposomes, micelles and vesicles, etc. and any combination thereof. Biomolecules may be isolated from natural sources, or they may be synthetic. The target of interest may be, for example, an enzyme or other protein. The target of interest may be a peptide or a polypeptide. The target of interest may be an antibody, antibody-like or a fragment of an antibody. The target of interest may be a nucleic acid molecule. The target of interest may include deoxyribonucleic acids (DNA) or ribonucleic acids (RNA). The target of interest may be a polynucleotide or other polymer. The target of interest may thus be, for example, proteins, nucleic acids, carbohydrates, lipids, or any other type of molecule or any combination thereof.

The target of interest may be unmodified or the target of interest may be modified to contain one or more labels. An unmodified target of interest may be visualized through its inherent auto-fluorescent spectral properties during optical imaging. An unmodified target of interest comprising of non-fluorescent or non-excitable material may be visualized through the administration of one or more chemical agents, such as reagents, dyes, stains and like agents to the sample comprising such unmodified target of interest prior to or during optical imaging. The target of interest may be modified to contain one or more labels through physical conjugation, chemical conjugation, genetic expression, etc. The one or more labels of the modified target of interest may comprise an excitable material. Non-limiting examples of labels include fluorescent materials (e.g. fluorophores or other like materials), phosphorescent materials (e.g. porphyrin or other like materials), bioluminescent materials (e.g. Luciferase expression or other like materials), chromophoric materials (e.g. chromophores or other like materials), etc. Embodiments of label materials of a target of interest may refer to any liquid, solid, or other type of material that absorbs light and re-emits at least a portion of what is absorbed as an optical signal (light) of a different spectral content as a measure of the amount present of that target of interest at that location.

Embodiments of the present invention with optical imaging systems address to imaging targets of interest contained in or on a sample. A “sample” includes and may refer to any liquid, solid, or other type of material that may be comprised of or as, in or on a cell or cells (e.g. in whole or lysed); a slurry or an extraction of cellular components; a tissue or tissues; an organ, organs, organoid or other organ-like materials; organisms such as but not limited to invertebrate or vertebrate organisms (i.e. in whole or in part); substrates such as but not limited to western blots, membranes, woven networks of fibers, gels, plastic media, glass media or other media in the form of a plate, dish, tube, slide, etc.; or any combination thereof.