Patent Grant
12135261
Tissue sections are used in a wide array of histology and pathology assays because they facilitate staining, labeling and imaging of the tissue, which often cannot be performed directly on a thick sample. The sectioning process involves embedding of the tissue in a support material such as a wax, resin, ice, or gel, and then thinly slicing it using a microtome or vibrating blade microtome to a thickness on the order of microns to hundreds of microns. The difference between a microtome and a vibrating blade microtome is that a vibrating blade microtome uses a vibrating blade submerged in fluid which allows it to controllably section softer materials, such as tissues and gels. Microtomes typically require harder embedding media that infiltrates the tissue, and can require significant post-processing to remove the infiltrated wax/resin. This post-processing can affect the sample and interfere with secondary assays. After sectioning, the collection, handling, storage, and mounting of tissue sections needs to be performed manually by an experienced technician, even in commercial automated systems. This is tedious and costly in terms of labor and throughput, and many steps of the process are subject to human error. There is a demand for a robust system for automated sectioning and storage of tissue sections to reduce cost and to improve the throughput and consistency of histology assays. These capabilities would also facilitate integration with existing and novel automated laboratory systems. One technology that provides automated capture and imaging of tissue sections is serial two-photon tomography (STPT).
STPT can quickly ex vivo image thick tissue samples with sub-micron resolution even across entire organs such as a mouse brain. Advanced molecular histology assays can annotate tissue slices with rich molecular information. However, no current techniques can produce molecularly-annotated 3D maps of thick tissues and whole organs. STPT lacks the necessary multiplexed biochemical specificity and is generally limited to light microscopy approaches, while most molecular assays lack the requisite throughput and 3D spatial extent. As such, there still exists a crucial gap in understanding the structure and composition of thick tissues and whole organs. Without such an understanding it is difficult, if not impossible, to develop effective therapeutics for many diseases and disorders.
There have been few successful efforts in histopathology to automate the tissue sectioning and storage process. While “automated” microtomes exist commercially, they tend to imply only semi-automated or assisted sectioning processes and have no capabilities for automated collection and handling of the tissue sections. These devices still require an experienced technician to manipulate and collect the slices manually or semi-manually.
Previous systems that perform automated microtomy are not compatible with soft samples and are usually not compatible with imaging between sectioning. These systems were designed with different specific target applications in mind. For example, one system sections paraffin-embedded samples with a microtome and outputs prepared slides for slide-scanning applications. Another system produces thin ultramicrotome sections of resin-embedded samples (typically C. Elegans) for serial electron microscopy. Its sample output feeds into a custom electron microscope sample holder.
Methods have been developed to capture agarose tissue sections using a six-axis robotic arm in a pick-and-place configuration. For instance, International Patent Application No. PCT/GB2014/051899 by Troy et al. (International Patent Publication WO 2014/202998), the entire contents of which is incorporated herein by reference, describes a system where the arm lowers an end effector into the bath solution, applies suction to adhere the slice to suction cups on its end effector, and then lifts the slice out of solution and deposits it in a receptacle while dispensing buffer solution. The receptacle is then loaded into a rotary carousel for storage. Cost and complexity aside, this system likely suffers from reliability issues, in particular with thinner sections, as it would not handle conformational differences among tissue slices very well, and has poor conformational control over the deposited slice. As researchers move towards thinner sections, it will be necessary to gently capture slices onto a support substrate for handling, as direct handling will damage thinner sections.
This invention relates to the field of automated histopathology, in particular automated methods for the study of biological tissue samples or the pathology of diseases and disorders in those tissues. This can include the processing, handling, and execution of a variety of methodologies on tissue sample(s) of interest.
In preferred embodiments of the present invention, multiple modalities are described for automated capture, indexing, and storage of histological tissue sections produced by a vibrating blade microtome technology to improve vibrating blade sectioning, and multiple applications of these technologies. The mechanism for capturing tissue sections applies a force to move the tissue sections or slices onto a transfer material, plate, or slide, for example. A preferred method provides an attractive force to pull a tissue slice into contact with a support substrate or pallet as the tissue slice is being sectioned from the sample block in a water bath. The substrate and the attractive force serve to guide the conformation and motion of the tissue slice such that it can be transported out of the water bath and into storage. Once out of the water bath, surface tension between the wetted tissue section and the support substrate can be used to keep the slices robustly adhered to the surface during transport without loss of conformation. Surface treatments can also be used to promote slice adhesion.
Slices can be detached from the support substrate using a fluid inertial force to overcome the adhesive surface tension force for the controllable transfer into storage or other handling systems and modalities. The slices are stored in wells or tubes, on substrates, or in some other container, with or without buffer solution depending on the application. Sections can also be stored sequentially on a film reel, or enveloped in a thin film for further automated processes. Indexing is accomplished in the control software; storage coordinates can be loaded into the software to specify how and where the slices are stored, and a metadata file tracks the physical location of each slice. The system configurations are designed for full-compatibility with many imaging modalities including serial two-photon tomography (STPT), a method for 3D tissue imaging, in order to enable the physical tissue section to be tracked relative to its 3D imaging data from STPT. In other embodiments of the invention, other imaging block face and whole specimen modalities can be used. Multiple wavelengths can be detected by a corresponding plurality of detector elements. A data processor can be used to process the spectral data generated by the detectors.
Other imaging technologies beyond STPT that will benefit from automated sectioning and collections of slices include coherent anti-Raman Stokes imaging (CARS), Stimulated Raman Scattering (SRS), Optical Coherence Tomography (OCT), Oblique Light Sheet, Light Sheet Theta Microscopy, Inverted Light Sheet Microscopy, Swept confocally-aligned planar excitation (SCAPE), selective plane illumination microscopy (SPIM), confocal Raman, confocal, spinning disk confocal, block face structured illumination, block face imaging, and optical frequency domain imaging (OFDI). In preferred embodiments, these imaging modalities are combined with physical sectioning of the tissue.
Other methods such as magnetic resonance imaging (MRI), positron emission tomography0 (PET), Optical projection Tomography (OPT) and ultrasound can be used in other embodiments, as these methods can be used to produce a 3D volume of a specimen such as a tissue (including plant tissue), organ or whole animal. In some embodiments of this invention, the specimen can be cleared by various methods (CLARITY, CUBIC, ScaleS, Sacel, 3Disco, UDisco) or labeled by whole mount procedures, iDisco for instance, or by various methods to label whole animals and organs, such as intravita labeling. The imaged specimen can then be sectioned, and the recovered sections can be analyzed by various methods, both optical and biochemical, and the information obtained from these analyses on the sections can then be aligned and registered back to the original 3D volume.
Thus, the present system removes a key barrier to obtaining 3D molecular maps by enabling an automated tissue capture device to be integrated into both 2D and 3D imaging equipment. In particular, serial blockface imaging techniques can alternate optical imaging and mechanical sectioning to construct 3D datasets of macroscopic samples. The uppermost portion of the tissue is imaged to a depth of tens of microns, for example, and then removed with a vibrating blade microtome or other sectioning device. The process then repeats until the entire tissue has been imaged. Currently, tissue sections that are generated are randomly distributed within the buffer bath and need to be manually collected, sorted, and mounted individually for secondary assays. This approach is impractical for large numbers of delicate sections. Further, thin tissue sections tend to roll and fold upon themselves making them difficult to handle. The system described in this application automatically captures tissue slices while maintaining their orientation and storing them for downstream analysis. The resulting in-depth molecular information obtained from these analyses can then be mapped back onto the high resolution 3D dataset to construct 3D molecular maps of tissue and whole organs. These secondary assays can be specifically targeted to specific regions of interest, performed on a subset of samples, for example, every Nth section, or performed on an entire sample for more exploratory assays. These secondary assays need not be known a priori and can be performed in response to the results of future assays.
Preferred embodiments of the present invention can comprise four central elements: (1) A vibrating blade microtome that can cut thin (10-1,000 micron) sections from a tissue embedded in a tissue block, for example, an agar block. (2) An inlet manifold through which the buffer solution can be drawn by an attached pump and recirculated into the buffer bath. This manifold is positioned near the vibrating blade microtome, which is also known as a vibratome, and the intake of buffer produces a fluidic force which attracts the tissue section. (3) A transfer material such as a porous conveyer belt, tape or flexible material upon which the samples are transported is driven around the inlet manifold. The manifold acts like a pulley to redirect the transport material or belt motion. The belt slides over the manifold surface and transports the tissue section out of the buffer (
Use of a transfer material such as a porous conveyor belt 114 as described above or various film as described below advantageously allows the tissue sample 120 to be quickly attracted to and immobilized upon a physical substrate. By immobilizing the tissue sample 120 on a physical substrate, the tissue sample 120 is less likely to curl or otherwise be damaged such as might occur during extended transport under fluidic or suction forces. In addition, continuous transfer materials as described herein provide the ability to immobilize a series of tissue samples 120 in an orderly fashion (e.g., in a linear fashion along the film, tape, or belt). This overcomes a typical problem with sectioning wherein loose tissue samples floating in fluid can adhere to one another or quickly move out of order.
In some embodiments as shown in
In some embodiments, the gap between support arms 471 in the vibrating blade holder 470 can reduce the weight of the arm and also allow fluid flow to pass through. Because of this gap, there is a more direct fluidic pathway between the bath solution 116 and the CIM 110 to apply a more consistent fluidic force on the tissue section during capture, at more gentle flow rates. In this arm design, the pitch angle or “angle of attack” of the blade 66 is fixed to allow more rigidity in the arm and also to ensure consistent positioning of the blade 66 relative to the conveyor 114. To adjust this angle, the blade holder component can be swapped for one with a different, set angle. This is in comparison to alternative designs in which the entire arm is able to rotate to adjust this pitch angle. Such a design causes variability in the mounting position and local fluid dynamics around the blade that can cause inconsistencies in the capture of tissue sections.
The simplest geometry for the CIM is a cylinder, but non-axisymmetric designs could be used to better-control the distribution of forces on the tissue section, as well as the belt motion. Two examples are depicted in
In some embodiments, the conveyor belt 114 can pass around the CIM 110 at a steep initial angle to clear the bath walls and bend pulleys 126 before the motion is redirected horizontally. The conveyor can be powered by a drum-style drive pulley 128. The drive pulley 128 can be crowned in order to keep the belt tracking properly or can be mounted on a tensioning bracket 127 to allow for tension adjustment of the belt 114.
The use of a capstan 114, rather than a rotating pulley, avoids the need for underwater bearings and rotary fluidic couplings which add size, cost, complexity, and failure modes to the system. The capstan 114 can provide consistency in the applied suction because the inlet holes 112 are in a fixed position relative to the vibrating blade microtome 64. This design is possible because of the lightweight loading of the conveyor. The additional belt tension required to overcome the friction on the capstan is a factor of ˜2, which is insignificant for this system. Low-friction polymer materials like Teflon or Nylon can be used for the CIM 110, and the bend pulleys 126 can be replaced with similar capstans in some embodiments. If wear or durability of the CIM 110 is a concern for a specific application, metal or ceramics can be used. The simplicity of the components means they can be easier and less expensive to swap for maintenance or in cases where cross-contamination is a concern with biological samples. The belt material can be a biocompatible, corrosion-resistant, and slightly hydrophilic polymer such as a Nylon or Polyethylene terephthalate (PET/Polyester) mesh, which is durable and inexpensive.
To prevent vibration from interfering with the sectioning process, the conveyor and its motorized components (motors, pumps, etc. . .) can be mounted independently of the vibratome 64 and imaging device or isolated with damping materials.
Instead of a conveyor belt 114, the same system configuration for tissue section capture can be used with a different transfer material in the form of a porous film 130 in a reel-to-reel configuration, with a supply reel 132 and a storage or take-up reel 133, as depicted in
Thin tissue sections have a propensity to curl into a cylindrical conformation as they are cut from the sample block, which would be difficult to unravel in an automated manner for applications like microscopy, which require sections to be flat. This phenomenon can be avoided by drawing the section into a stable, flat conformation during the sectioning process before it is able to curl. This can be accomplished by running the pump and conveyor belt 114 during sectioning to adhere the section 120 to the transfer material such as the porous conveyor belt 114. The movement of the conveyor belt 114 and periodicity of the pore spacing can serve to smooth out pulsations in the flow from displacement-style pumps, preventing suction forces from being concentrated at specific points on the tissue section.
The conveyor belt porosity can be carefully selected such that pores are large enough to not significantly disrupt the fluid intake into the CIM 110, but small enough to provide sufficient support to delicate tissue sections. Plastic filtering meshes tend to work well for this application, with pore sizes on the order of 0.1-1 mm, with the added benefit of being corrosion resistant, durable, biocompatible, and inexpensive. Polyethlylene terephthalate (PET) and Nylon can work well. Ultrasonic welding can be used to join belt ends.
In lieu of an attractive fluidic force that uses suction/drag to attract the tissue section, alternative physical forces could be used. These include electrical forces such as electrophoresis or dielectrophoresis, thermophoresis, magnetic forces, chemical attraction/adhesion, and direct physical contact, such as with a gentle mechanical gripper, or any combination thereof. For example, the fluid force attracts the tissue section 120, and then a chemical surface functionalization promotes short-range adhesion to the belt 114 or film 130. This functionalization entails a chemical bond or electrostatic attraction. The ionic composition and pH of the buffer solution can be modified to adjust the range and strength of the interaction between the tissue section 120 and substrate, for example using low ionic strength and low pH to facilitate longer range and stronger electrostatic interactions between a negatively charged tissue and positively charged substrate based on adjustment of the Debye length and zeta potential. As shown in
To improve the bonding of tissue to its embedding media and ensure the production of intact tissue sections for the section capture system, fixed tissue samples can be embedded in agarose (4.5-6% typical) that has been oxidized with sodium periodate to promote bond formation with proteins on the surface of the tissue. The sample block can be subsequently soaked in a solution of acrylamide and bis-acrylamide monomers overnight to infiltrate the block and the tissue. The acrylamide copolymers can then be thermally polymerized to polyacrylamide the following day with a 2 hour heat treatment at 40 C, resulting in 4-10% polyacrylamide. A HEMA copolymer can be added in similar percentages to the polyacrylamide to reduce expansion of the sample block in aqueous buffers due to water absorption. These polymers crosslink through the tissue resulting in a more homogeneous block and improvement in section quality.
The capstan design is simple and effective, but if necessary for certain belt/CIM materials or loading conditions, the capstan 110 can be replaced with a pulley 410, with a similar inlet manifold integrated within it. This pulley system requires a rotary fluid coupling and underwater bearings or bushings to allow it to rotate with the belt underwater, and may need to be made of higher-friction materials or integrate timing belt grooves to ensure that the belt does not slip (timing belt strips can be integrated into the porous belt/film in this case).
The system configurations as described so far use a vibrating blade microtome 64 in an aqueous buffer solution, cutting soft tissue embedded in a hydrogel like agar, agarose, or polyacrylamide, or hydrogel. A cryostat embodiment of the section capture system can use the same capture mechanism, except that it can be cutting frozen tissue with either a vibrating blade microtome 64 or microtome. The liquid buffer can be selected such that it can keep the tissue sample frozen during sectioning, for example using liquid nitrogen/isopentane mixture, or some other supercooled bath solution. This liquid buffer is necessary in order to take advantage of the fluidic forces to gently capture the tissue section. The tissue can be flash frozen in a standard cryo sectioning media, such as optimal cutting temperature compound (OCT). The system materials can be adjusted and insulated to operate at this lower temperature and in the presence of significant temperature gradients. The optics can be adjusted to image the frozen sample with multi-photon microscopy. The benefits to frozen sections are that they can be cut much thinner which is desirable for some assays, and also that fresh tissue can be used (not fixed) which helps preserve the biochemical composition of the tissue.
After sections detach from the sample block, they are conveyed out of the bath solution. To prevent asymmetric forces from acting on the sample and causing its orientation to shift on the conveyor, the CIM can be carefully designed to apply approximately uniform flow along its length. A symmetric design further ensures that the forces acting on the tissue section are symmetric if the sample is centered.
Once captured, it is important to be able to transfer the tissue section to a desired substrate for storage, secondary processes, or general handling. A typical example of such substrates is microscope slides, which are inexpensive, compact, and readily integrate with many standard laboratory systems. After the position of the tissue section is known, the tissue section is moved into the transfer region of the conveyor. In this region, the belt is oriented horizontal by pulleys and the conveyor frame has a cutaway to allow access to the belt. This region is designed to be on the underside of the conveyor frame and surface tension is sufficient to hold the tissue section upside-down. This allows the section to be transferred to the top surface of the slide, provides the most room for access from the slide-handling system, and also means that gravity will assist in the detachment of the tissue section, both due to its force on the section itself, and because it acts on any residual liquid on the section to promote the formation of a capillary bridge between the section and slide below it.
Once in the transfer position, the slide handling system moves the slide upwards until it gently contacts the tissue section and the section is completely conformal to the surface of the slide. Microscope slides are highly hydrophilic, and so the surface tension and gravity will tend to cause the section to preferentially adhere to the slide, compared to the plastic mesh which is typically mildly hydrophilic or even hydrophobic depending on material. At this point a gentle jet of gas, typically air, is applied to the back side of the porous conveyor to assist in detaching the section from the belt. As the gas flow impinges on the section, the slide is slowly lowered until the section is completely detached from the belt, and laying flat on the slide.
To transfer tissue sections from the section capture system 100 for storage or handling, surface tension forces are used to manipulate the tissue section 120 into position, and a gentle fluid-inertial force can be applied via a jet of gas to transfer the tissue section 120, without damage, to an adjacent well, tube, container, substrate, slide, or film. This is depicted schematically in
The gas jet may not be required in some cases, and surface tension/gravity may be sufficient alone to cleanly detach tissue sections onto a substrate. The surface tension can also be modified to adjust the adhesive properties of the section, for example by adding surfactant or adjusting the ionic composition of the capillary bridge.
The adhesion of the tissue section can also be broken by eliminating the liquid interface between the tissue section and belt. This can be done by submerging the belt and section in liquid to eliminate the interface, at which point the section will sink or float depending on the density (buoyancy) of the section and liquid as described above in relation to
In place of the gas jet to transfer tissue sections, a user can also use a liquid jet or fluid flow system 705 as shown in
In some embodiments, the substrate 710 could be coated with a thin film of liquid and moved upwards until the section is submerged in the thin film of liquid. This substrate 710 can be patterned with hydrophilic or hydrophobic surface functionalizations such that the liquid film has a consistent and controlled size and shape, or it could be constrained by a shallow well. Additional liquid can be applied to the backside of the conveyor belt to help envelop the section in liquid to eliminate the capillary bridge. This additional liquid could also be used to apply a fluid-inertial force to the section to assist in detachment. This fluid composition could also be adjusted depending on the application, for example including surfactant, histological stain, or various buffers, and need not be an aqueous solution. One of the potential benefits of trapping a tissue section in a thin film of fluid is that the fluid interface may help constrain the conformation of the tissue section, preventing it from curling or folding.
An alternative to the transfer system described can be to use a reel-to-reel arrangement as described previously and depicted in
Tissue sections 120 can be stored in a wide assortment of different containers or substrates depending on the application of interest. The two most widely desirable storage methods would be directly on glass microscope slides 614 or in well plates 612. The gas-jet transfer technique is highly robust because it can reliably transfer tissue sections 120 to flat substrates 614 without loss of conformation, but is general enough to deposit the section into open containers 612 as well. As an alternative, a fluid flow system can be used to detach the tissue section 120 from the conveyer and onto the slide 614 or collection chamber 612. As depicted in
In addition to slides and well arrays, tissue sections can be deposited into tissue storage tubes or custom well arrays. Tissue storage tubes are desirable in cases where the sections are to be frozen for future analysis. If a researcher wished to perform a secondary assay on a single tissue section, he/she need not defrost the entire batch of tissue samples, just the specific tube of interest. Custom wells, substrates, or arrays of wells/substrates can be desirable in cases where custom storage features are desirable, such as integrated fluidic channels, higher storage densities, custom geometry, or compatibility with specific systems.
The storage process can be further automated, and several examples are depicted schematically in
This section capture technology is designed to work directly with STPT to capture tissue sections that are produced in the process of 3D tissue imaging. These sections can be used in secondary assays in order to map multiplexed biomolecular information, such as protein distributions, onto the subcellular 3D dataset produced by STPT. This 3D molecular mapping concept is depicted in the flow chart in
These assays can be semi- or fully-automated in conjunction with the output of the section capture and storage system. For example, well plates can be fed into automated liquid handling systems to perform free floating staining/labeling and rinses. Microscope slides can be used in automated staining systems, cover slippers, and slide scanning systems, for fully automated secondary labeling and imaging.
One of the more interesting applications of this molecular mapping technology is the ability to perform expansion microscopy on a specific region of tissue that is identified via STPT. A user can image an entire organ, identify a section of interest, and then infiltrate and embed the tissue in an expansion microscopy media and expand the tissue to image a region of interest in high resolution, for example to look at the dendritic spines of neurons.
As a standalone system, section capture and storage is valuable for any histology or pathology lab that works with tissue samples that require a vibrating blade microtome for sectioning. Manual sectioning and handling of tissue sections is a major source of human error and variability among laboratories, and this technology can assist to standardize section preparation with increased consistency and throughput, and provide a technological bridge to further automation of histopathology methods.
It is important that sectioning technology be used in conjunction with our section capture system to ensure the production of high-quality, intact sections, and to enable the consistent production of thinner sections which are desirable for many secondary assays. For example, thinner sections can be stained more rapidly, or used in imaging modalities that are incompatible with thick sections. There are several techniques to improve the quality of sections produced by the sectioning system.
The first technique involves double-embedding of the tissue sample as depicted in
Another method for production of thinner sections as depicted in
Another simple method to improve section quality is to increase the relative velocity of the sample transverse to the blade. Typical vibrating blade microtome cutting moves the sample into the blade at a constant normal velocity, while the blade vibrates in the transverse direction sinusoidally which applies shear. A good balance between the normal and transverse cutting velocity components is necessary for optimal sectioning Reyssat E, Tallinen T, Le Merrer M, Mahadevan L. Slicing Softly with Shear. Physical Review Letters. 2012; 109(24):244301, the contents of this publication being incorporated herein by reference. Because our transverse velocity component is sinusoidal, there are points along the velocity vs. time curve where the transverse velocity is momentarily near zero, which is undesirable for cut quality. One method to improve the transverse velocity component is to angle the blade relative to the direction of motion of the sample (
The method can include a step 1508 of transporting the cut section using the conveyor belt or transport system. The transporting of the cut section can be done as described above using the conveyor belt with reference to
The method can include an optional step 1512 of protecting the cut section by envelopment in a film. In some embodiments, the sections can be individually encapsulated as described above with reference to
The method can include a step 1516 of repeating the prior steps iteratively to create a series of images of a plurality of consecutive cut sections. Serial sectioning and transport of cut sections can be performed as described, for example, previously with reference to
In some embodiments, the tissue section 120 can include external fiduciary markers 1602, internal fiduciary markers 1604, or a combination of both as shown in
Identification of the location of fiduciary markers 1602, 1604 within the tissue section 120 can aid in determining whether the tissue section 120 is aligned with on the conveyor belt 114 and whether it is lying flat or in an otherwise desirable configuration. In some embodiments, the conveyor belt 114 can transport the tissue section 120 past an illumination or excitation source 1606. The illuminated tissue section 120 can then be imaged using a camera 1610 attached to a processing unit 1612. In some embodiments, an emission filter 1616 can be placed between the camera 1610 and the tissue section 120 to filter the light from the tissue section 120. In some embodiments, the processing unit 1612 can include an object recognition module 1610 to isolate and identify the fiduciary markers 1602, 1604 within the image acquired by the camera 1610. The processing unit 1612 can then apply an analysis module 1612 to the image to identify the positions of the fiduciary markers 1602, 1604 within the image. For example, a centroid can be computed for each fiduciary marker and assigned coordinates in an x-y plane. Each identified fiduciary marker 1602, 1604 can be assigned its own coordinates until the cth marker is assigned the coordinates (xc,yc). The processing unit 1612 can also include an output or system feedback module 1614. The output or system feedback module 1614 can identify or compute characteristics for each imaged fiduciary marker 1602, 1604 including area, shape, orientation, centroid, and local deformations. The characteristics of the fiduciary markers 1614 can be analyzed to determine if the vibrating blade microtome 64 or the section capture system 100 need to be adjusted to return to normal operation. For example, if a threshold number of tissue sections 120 include deformed, missing, or incorrectly sized or shaped fiduciary markers 1602, 1604, adjustments may need to be made to the microtome or section capture system 100.
Tissue sections are thin, transparent, and wet, which can be difficult to sense against a mesh background. For a digital detection signal, there are several types of standard automation sensors that will work when properly tuned, including:
Custom photoelectric sensors 1660 will also work and can be tuned to the specific application as shown in
To reduce the incidence of false positive sensor signals, the detection sensor 1660 can be “gated” such that the sensor status is only monitored when the tissue section 120 is in close proximity. An initial movement of the conveyor 114 can serve as coarse positioning, and the sensor edge-finding routine can serve as fine positioning to determine the exact location of the tissue section 120. Having multiple redundant sensors can also be used to reduce the incidence of false-positives in various embodiments.
Several steps can be taken to improve contrast of the tissue sections against the background, including the use of different mesh materials, different colors, and modifications to the embedding material as noted previously.
Computer vision can also be used to identify the position and orientation of tissue sections on the conveyor belt. In some embodiments, a camera can image the belt under specific illumination conditions, which may include diffuse light, dark-field, UV, IR, polarized, or low-angle light sources (or any combination thereof), with a variety of filters, such as standard band-pass emission or polarizing filters. Once the section is clearly illuminated with good contrast, thresholding and object recognition algorithms can be used to identify the section. Computer vision can be used as the sensor itself, to detect the presence and location of the tissue section 120, or it can be used in addition to a digital sensor to provide the system with additional information. For example, orientation data can be fed back into the system such that it can be corrected in the transfer process. Large deviations in orientation, size, or shape of the tissue section may also be used as an indication of the sectioning quality and may trigger errors in the system.
In various embodiments, a slide storage carousel 1800 can be used to store slides as depicted in
The gripper tooling is custom designed for handling microscope slides and consists of two slender fingers with grooves into which the sides of the microscope slides fit. These grooves are sized to the tolerances of typical slides (1.1 mm) and have radiused edges to guide the slide edges in without chipping or damage in case of slight misalignment. The grooves terminate at a specific distance along the length of the finger, to provide a hard stop for the end of the slides.
Slides can be stored in compact polyceramic racks that are compatible with commercial staining and coverslipping systems and are autoclavable and reusable. The racks can be oriented vertically with the slides stored flat. These racks can be stationary, or mounted to a rotary carousel, actuator, or stage to move them into the robot envelope or into a more desirable position for the robot to deposit slides, or for the user to access the racks. They can also be enclosed for humidification, using a sponge or active humidifier.
To grab a slide from the rack, the robotic gripper can open wide and move into position a few millimeters from the target grab location on the slide. The gripper can then close until it is spaced just slightly wider than the slide, such that the slide rests within the grooves, but no gripping force is being applied. In some embodiments, this spacing can be on the order of ˜1 mm. If the slide is misaligned, this initial motion serves the purpose of roughly aligning the slide. Next, the gripper can be moved to the target grab location on the slide. The tooling is closed enough such that if the slide is protruding too far from the rack, it will catch on the hard stop at the end of the groove and be pushed gently into position. Once in position, the gripper can be fully activated to apply a force to grip the slide, which will further force the slide into the grooves in case of any misalignment. This gripper motion and tooling design can allow the robot to compensate for any misalignment of the slide within its rack position. Once grabbed, the slide is moved into position for the tissue section to be transferred.
Once the section is transferred to the slide, the robot inserts the slide back into the rack, opens fully to release the slide, and then retracts. The gripper can have sufficient range to handle 25 mm or 50 mm glass slides. The gripper tooling can be slender enough to not interfere with adjacent slides in the racks, allowing for a good storage density that reduces the size and footprint of the storage system.
Slide rack positions can be taught to the robot using a defined local coordinate system such that only one point needs to be taught for each rack. A capacitive proximity sensor, inductive sensor, or photoelectric sensor can be integrated into the end effector and synchronized with the robot to provide semi-automated positioning feedback for the teach routine, or for use in detecting the presence or absence of a slide in the rack. The electric gripper can also provide feedback to indicate if it reached its target position and target gripper trigger force which can be used as an indication if a slide was grabbed or not.
The automated section capture and storage system includes several elements functioning in combination to controllably section and mount tissue sections onto flat substrates such as microscope slides. These elements include the following features shown generally in
The system shown in
While this invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various equivalents thereof or changes in form and details can be made without departing from the scope of the invention encompasses by the appended claims.
This application is a 35 U.S.C. § 371 national stage filing of International Application No. PCT/US2017/062428, filed on Nov. 17, 2017, which claims priority to U.S. Provisional Application No. 62/424,270, filed Nov. 18, 2016, the entire contents of the above applications being incorporated herein by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/062428 | 11/17/2017 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/094290 | 5/24/2018 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 4190472 | Slonicki | Feb 1980 | A |
| 4960330 | Kerschmann | Oct 1990 | A |
| 5048904 | Montagu | Sep 1991 | A |
| 5109149 | Leung | Apr 1992 | A |
| 5139338 | Pomerantz et al. | Aug 1992 | A |
| 5156019 | McCormick | Oct 1992 | A |
| 5233197 | Bowman et al. | Aug 1993 | A |
| 5583342 | Ichie | Dec 1996 | A |
| 5633695 | Feke et al. | May 1997 | A |
| 5691839 | Kobayashi | Nov 1997 | A |
| 5700346 | Edwards | Dec 1997 | A |
| 5740708 | Tabone | Apr 1998 | A |
| 5783814 | Fairley et al. | Jul 1998 | A |
| 6020591 | Harter et al. | Feb 2000 | A |
| 6028306 | Hayashi | Feb 2000 | A |
| 6219179 | Nielsen et al. | Apr 2001 | B1 |
| 6248988 | Krantz | Jun 2001 | B1 |
| 6387653 | Voneiff et al. | May 2002 | B1 |
| 6392795 | Okada | May 2002 | B2 |
| 6423960 | Engelhardt et al. | Jul 2002 | B1 |
| 6449039 | Bouzid | Sep 2002 | B1 |
| 6496267 | Takaoka | Dec 2002 | B1 |
| 6603537 | Dietz et al. | Aug 2003 | B1 |
| 6623977 | Farquharson et al. | Sep 2003 | B1 |
| 7003143 | Hewitt et al. | Feb 2006 | B1 |
| 7009699 | Wolleschensky et al. | Mar 2006 | B2 |
| 7092557 | Eisfeld et al. | Aug 2006 | B2 |
| 7110118 | Unlu et al. | Sep 2006 | B2 |
| 7115885 | Hell | Oct 2006 | B2 |
| 7139415 | Finkbeiner | Nov 2006 | B2 |
| 7197193 | Li et al. | Mar 2007 | B2 |
| 7209287 | Lauer | Apr 2007 | B2 |
| 7215469 | Nakata et al. | May 2007 | B2 |
| 7217573 | Oshida et al. | May 2007 | B1 |
| 7274446 | Wolleschensky et al. | Sep 2007 | B2 |
| 7329860 | Feng et al. | Feb 2008 | B2 |
| 7366394 | Takamatsu et al. | Apr 2008 | B2 |
| 7372985 | So et al. | May 2008 | B2 |
| 7374907 | Boneiff | May 2008 | B1 |
| 7502107 | Mohanty et al. | Mar 2009 | B2 |
| 7561326 | Funk et al. | Jul 2009 | B2 |
| 7724937 | So et al. | May 2010 | B2 |
| 7749754 | Sherwood et al. | Jul 2010 | B2 |
| 7767414 | Smith et al. | Aug 2010 | B1 |
| 7772985 | Kobayashi et al. | Aug 2010 | B2 |
| 7863585 | Hell et al. | Jan 2011 | B2 |
| 7869132 | Saito et al. | Jan 2011 | B2 |
| 7966091 | Fujimoto | Jun 2011 | B2 |
| 8238632 | Wilson et al. | Aug 2012 | B2 |
| 8384907 | Tearney et al. | Feb 2013 | B2 |
| 8728799 | Schmitt | May 2014 | B2 |
| 8771978 | Ragan | Jul 2014 | B2 |
| 8839700 | Chen et al. | Sep 2014 | B2 |
| 8967024 | Magavi et al. | Mar 2015 | B2 |
| 8995733 | Van Dijk et al. | Mar 2015 | B2 |
| 9527681 | Ito | Dec 2016 | B2 |
| 9530204 | Morimoto | Dec 2016 | B2 |
| 9551658 | Hell et al. | Jan 2017 | B2 |
| 9719928 | Hell | Aug 2017 | B2 |
| 9901295 | Iverson et al. | Feb 2018 | B2 |
| 9983134 | Ragan | May 2018 | B2 |
| 10054519 | Chen et al. | Aug 2018 | B2 |
| 10197476 | Gong et al. | Feb 2019 | B2 |
| 10627316 | Sawada | Apr 2020 | B2 |
| 20020190212 | Boas et al. | Dec 2002 | A1 |
| 20020191884 | Letant et al. | Dec 2002 | A1 |
| 20030132394 | Wolleschensky et al. | Jul 2003 | A1 |
| 20030223107 | Olszak et al. | Dec 2003 | A1 |
| 20040032650 | Lauer | Feb 2004 | A1 |
| 20040076319 | Fauver et al. | Apr 2004 | A1 |
| 20040110206 | Wong et al. | Jun 2004 | A1 |
| 20040125372 | Walla et al. | Jul 2004 | A1 |
| 20040228568 | Letant et al. | Nov 2004 | A1 |
| 20040257562 | Wachsmuth | Dec 2004 | A1 |
| 20040257646 | Wachsmuth | Dec 2004 | A1 |
| 20050024637 | Olschewski | Feb 2005 | A1 |
| 20050036667 | So et al. | Feb 2005 | A1 |
| 20050046836 | Olschewski | Mar 2005 | A1 |
| 20050046848 | Cromwell et al. | Mar 2005 | A1 |
| 20050259319 | Brooker | Nov 2005 | A1 |
| 20050260764 | Grigsby et al. | Nov 2005 | A1 |
| 20060012875 | Wolleschensky | Jan 2006 | A1 |
| 20060014287 | Sherwood et al. | Jan 2006 | A1 |
| 20060139637 | Cho et al. | Jun 2006 | A1 |
| 20060158655 | Everett et al. | Jul 2006 | A1 |
| 20060179992 | Kermani | Aug 2006 | A1 |
| 20060211752 | Kohn et al. | Sep 2006 | A1 |
| 20070038121 | Feldman et al. | Feb 2007 | A1 |
| 20070057211 | Bahlman et al. | Mar 2007 | A1 |
| 20070091428 | Wilson et al. | Apr 2007 | A1 |
| 20070229801 | Tearney et al. | Oct 2007 | A1 |
| 20070254280 | Lexow et al. | Nov 2007 | A1 |
| 20070258122 | Chamgoulov et al. | Nov 2007 | A1 |
| 20070260138 | Feldman et al. | Nov 2007 | A1 |
| 20080102006 | Kram et al. | May 2008 | A1 |
| 20080130093 | Silberberg et al. | Jun 2008 | A1 |
| 20080154128 | Milner | Jun 2008 | A1 |
| 20080192231 | Jureller et al. | Aug 2008 | A1 |
| 20090091566 | Turney et al. | Apr 2009 | A1 |
| 20090093709 | Patel et al. | Apr 2009 | A1 |
| 20090225399 | Zhao et al. | Sep 2009 | A1 |
| 20100000383 | Koos et al. | Jan 2010 | A1 |
| 20100028978 | Angros | Feb 2010 | A1 |
| 20100081190 | Hyde et al. | Apr 2010 | A1 |
| 20100081915 | Hyde et al. | Apr 2010 | A1 |
| 20100093022 | Hayworth et al. | Apr 2010 | A1 |
| 20100214404 | Chen et al. | Aug 2010 | A1 |
| 20100229702 | Fujimoto et al. | Sep 2010 | A1 |
| 20100265554 | Shirakura et al. | Oct 2010 | A1 |
| 20100294949 | Sasaki et al. | Nov 2010 | A1 |
| 20100323445 | Hayworth et al. | Dec 2010 | A1 |
| 20110058178 | Tearney et al. | Mar 2011 | A1 |
| 20110137126 | French et al. | Jun 2011 | A1 |
| 20110320174 | Ragan et al. | Dec 2011 | A1 |
| 20120163680 | Lefebvre | Jun 2012 | A1 |
| 20120208184 | Ragan | Aug 2012 | A1 |
| 20130010283 | Villiger et al. | Jan 2013 | A1 |
| 20130019725 | Magavi et al. | Jan 2013 | A1 |
| 20130045167 | Melles | Feb 2013 | A1 |
| 20130142413 | So et al. | Jun 2013 | A1 |
| 20130149734 | Ammar et al. | Jun 2013 | A1 |
| 20130203100 | Otter et al. | Aug 2013 | A1 |
| 20140041500 | Isagawa | Feb 2014 | A1 |
| 20140146376 | Kleppe et al. | May 2014 | A1 |
| 20140232848 | Schwedt et al. | Aug 2014 | A1 |
| 20140297199 | Osten | Oct 2014 | A1 |
| 20140356876 | Ragan | Dec 2014 | A1 |
| 20150008096 | Ito | Jan 2015 | A1 |
| 20150168276 | Orfield | Jun 2015 | A1 |
| 20150276563 | Carrigan et al. | Oct 2015 | A1 |
| 20150300924 | Miyatani et al. | Oct 2015 | A1 |
| 20160003715 | Torres et al. | Jan 2016 | A1 |
| 20160264973 | Aceto et al. | Sep 2016 | A1 |
| 20160290895 | Daniel | Oct 2016 | A1 |
| 20160377546 | Ragan et al. | Dec 2016 | A1 |
| 20170199104 | Gradinaru et al. | Jul 2017 | A1 |
| 20180045623 | Ragan et al. | Feb 2018 | A1 |
| 20180202935 | Bahlman et al. | Jul 2018 | A1 |
| 20190064070 | Ragan | Feb 2019 | A1 |
| 20210325308 | Kannan et al. | Oct 2021 | A1 |
| 20230296484 | Ragan et al. | Sep 2023 | A1 |
| Number | Date | Country |
|---|---|---|
| 101395465 | Mar 2009 | CN |
| 105842000 | Aug 2016 | CN |
| 1580586 | Sep 2005 | EP |
| 2249194 | Nov 2010 | EP |
| H06-323967 | Nov 1994 | JP |
| 2000-193889 | Jul 2000 | JP |
| 2001-235401 | Aug 2001 | JP |
| 2001-249124 | Sep 2001 | JP |
| 2004-028965 | Jan 2004 | JP |
| 2005-017127 | Jan 2005 | JP |
| 2005-508489 | Mar 2005 | JP |
| 2005-275206 | Oct 2005 | JP |
| 2006-010620 | Jan 2006 | JP |
| 2006-031004 | Feb 2006 | JP |
| 2006-178472 | Jul 2006 | JP |
| 2007-057255 | Mar 2007 | JP |
| 2007-187603 | Jul 2007 | JP |
| 2007-212388 | Aug 2007 | JP |
| 2008-076251 | Apr 2008 | JP |
| 2008-164521 | Jul 2008 | JP |
| 2008-197127 | Aug 2008 | JP |
| 2009-517662 | Apr 2009 | JP |
| 2009-150864 | Jul 2009 | JP |
| 2010-008989 | Jan 2010 | JP |
| 2010-532878 | Oct 2010 | JP |
| 2010-250191 | Nov 2010 | JP |
| 2010-271569 | Dec 2010 | JP |
| 2011-527218 | Oct 2011 | JP |
| 2012-229994 | Nov 2012 | JP |
| 2013-045078 | Mar 2013 | JP |
| 2013-113689 | Jun 2013 | JP |
| 2013-160718 | Aug 2013 | JP |
| 2016-191708 | Nov 2016 | JP |
| 426146 | Apr 1974 | SU |
| 426164 | Apr 1974 | SU |
| 199802851 | Jan 1998 | WO |
| 200042417 | Jul 2000 | WO |
| 200140769 | Jun 2001 | WO |
| 200142796 | Jun 2001 | WO |
| 2002084265 | Oct 2002 | WO |
| 2006127967 | Nov 2006 | WO |
| 2006135769 | Dec 2006 | WO |
| 2007099382 | Sep 2007 | WO |
| 2007149971 | Dec 2007 | WO |
| 2010045400 | Jul 2010 | WO |
| 2011163484 | Dec 2011 | WO |
| 2012068142 | May 2012 | WO |
| 2012069076 | May 2012 | WO |
| 2012172024 | Dec 2012 | WO |
| 2013045078 | Apr 2013 | WO |
| 2013126762 | Aug 2013 | WO |
| 2014202998 | Dec 2014 | WO |
| 2015046518 | Apr 2015 | WO |
| 2015100421 | Jul 2015 | WO |
| 2016145366 | Oct 2016 | WO |
| 2016163385 | Oct 2016 | WO |
| 2018094290 | May 2018 | WO |
| Entry |
|---|
| Thorlabs, Galvanometer Mirror Systems Overview. Life Science Catalog. pp. 317-323, (2013). |
| Allen, Bringing CLARITY to the study of the brain. Retrieved online at: http://membercentral.aaas.org/blogs/qualia/bringing-clarity-study-brain. 3 pages. Mar. 11, 2013. |
| Andresen et al., Time-multiplexed multifocal multiphoton microscope. Opt Lett. Jan. 15, 2001;26(2):75-7. |
| Augustinack et al., MRI parcellation of ex vivo medial temporal lobe. Neurolmage. Article in Press, May 2013, 8 pages. |
| Bewersdorf et al., Multifocal multiphoton microscopy. Opt Lett. May 1998;23(9):655-7. |
| Bird et al., Fibre-optic two-photon scanning fluorescence microscopy. J Microsc. Oct. 2002;208(Pt 1):35-48. |
| Bird et al., Two-photon fluorescence endoscopy with a micro-optic scanning head. Opt Lett. Sep. 1, 2003;28(17):1552-4. |
| Buist et al., Real time two-photon absorption microscopy using multi point excitation. Journal of Microscopy. Nov. 1998;192(2):217-226. |
| Cambridge Technology, Inc., Model 6240H Galvanometer Optical Scanner, Instruction Manual. www.cambridgetechnology.com. 22 pages, Sep. 23, 2004. |
| Chasles et al., Optimization and characterization of a structured illumination microscope. Opt Express. Nov. 26, 2007;15(24):16130-40. |
| Denk et al., Two-photon Laser Scanning fluorescence microscopy. Science. Apr. 6, 1990;248:73-76. |
| Dubois et al., High-resolution full-field optical coherence tomography with a Linnik microscope. Appl Opt. Feb. 1, 2002;41(4):805-12. |
| Ducros et al., Efficient large core fiber-based detection for multi-channel two-photon fluorescence microscopy and spectral unmixing. J Neurosci Methods. Jun. 15, 2011;198(2):172-80. |
| Goldman, Lightning strikes twice: Optogenetics pioneer Karl Deisseroth's newest technique renders tissues transparent, yet structurally intact. Scope, Stanford Medicine. Retrieved online at: http://scopeblog.stanford.edu/2013/04/10/lightning-strikes-twice-optogenetics-pioneer-karl-deisseroths-newest-technique-renders-tissues-transparent-yet-structurally-intact/ 2 pages. Apr. 10, 2013. |
| Helmchen et al., A miniature head-mounted two-photon microscope: High-resolution brain imaging in freely moving animals. Neuron. Sep. 27, 2001;31(6):903-12. |
| Helmchen et al., Enhanced two-photon excitation through optical fiber by single-mode propagation in a large core. Appl Opt. May 20, 2002;41(15):2930-4. |
| Hillman et al., Depth-resolved optical imaging and microscopy of vascular compartment dynamics during somatosensory stimulation. Neuroimage. Mar. 2007;35(1):89-104. |
| Jeong et al., Combined two-photon microscopy and optical coherence tomography using individually optimized sources. Opt Express. Jul. 4, 2011;19(14):13089-96. |
| Jung et al., Multiphoton endoscopy. Opt Lett. Jun. 1, 2003;28(11):902-4. |
| Kim et al., High speed handheld multiphoton multifoci microscopy. Proc. SPIE Int. Soc. Opt. Eng.; Progress in Biomedical Optics and Imaging—Multiphoton microscopy in the Biomedical Sciences IV. Jun. 2004;5353:267-272. |
| Kim et al., Multifocal multiphoton microscopy based on multianode photomultiplier tubes. Optics Express. Sep. 3, 2007;15(18):11658-11678. |
| Kim et al., Optical biopsy in high-speed handheld miniturized multifocal multiphoton microscopy. Progr. Biomed. Opt. Imaging Proc. SPIE: Multiphoton Microscopy in the Biomedical Sciences V. Mar. 2005; 5700:14-22. |
| Kim et al., Usage of multi anode PMT on the multi-photon fluorescence spectroscopy and video rate microscopy. 24th Annual Conference and the Annual Fall Meeting of the Biomedical Engineering Society EMBS/BMES Conference. Proceedings of the Second Joint Engineering in Medicine and Biology, p. 1206. 2002. |
| Leveque-Fort et al., Fluorescence lifetime imaging with multifocal two-photon microscope. Proceedings of SPIE—The International Society for Optical Engineering. Jun. 2004;5323(1):99-107. |
| Leveque-Fort et al., Time-resolved multifocal multiphoton microscopy. Proceedings of SPIE—The International Society for Optical Engineering. Jun. 2003;5139:173-179. |
| Lim et al., Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy. Opt Lett. Aug. 15, 2008;33(16): 1819-21. |
| Makhlouf et al., A dual modality fluorescence confocal and optical coherence tomography microendoscope. Endoscopic Microscopy V. Guillermo J. Tearney (Ed.), Proceedings of SPIE BiOS. Feb. 24, 2010;7558:75580K-1-75580K-8. |
| Nielsen et al., High efficiency beam splitter for multifocal multiphoton microscopy. J Microsc. Mar. 2001;201(Pt 3):368-76. |
| Osten, CSHL team introduces automated imaging to greatly speed whole-brain mapping efforts. Cold Spring Harbor, N.Y. 4 pages, Jan. 12, 2012. |
| Qin et al., 1D-Scanning Adressable Multiregional Multifocal Multiphoton Microscopy. Ultrafast Imaging and Spectroscopy. Proceedings of SPIE. Sep. 28, 2013;8845:884517. 9 pages. |
| Ragan et al., Serial two-photon tomography: an automated method for ex-vivo mouse brain imaging. Nat Methods. Sep. 1, 2012;9(3):255-258. |
| Reyssat et al., Slicing softly with shear. Phys Rev Lett. Dec. 14, 2012;109(24):244301, 5 pages. |
| Sacconi et al., Microphoton multifocal microscopy exploiting a diffractive optical element. Opt Lett. Oct. 15, 2003;28(20):1918-20. |
| Shao et al., Multifocal multiphoton microscopy based on a spatial light modulator. Appl Phys B. Jun. 2013;107(3):653-657. |
| So et al., High-throughput nonlinear optical microscopy. Biophys J. Dec. 17, 2013;105(12):2641-54. |
| Tang et al., Combined multiphoton microscopy and optical coherence tomography using a 12-fs broadband source. J Biomed Opt. Mar.-Apr. 2006;11(2):020502. 3 pages. |
| Wang et al., Reconstructing micrometer-scale fiber pathways in the brain: multi-contrast optical coherence lomography based tractography. Neuroimage. Oct. 15, 2011;58(4):984-92. |
| Webb et al., A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning. American Institute of Physics, Review of Scientific Instruments. Mar. 2002;5139:1898-1907. |
| European Office Action for Application No. 11790823.6, dated Sep. 19, 2018, 8 pages. |
| International Preliminary Report on Patentability for Application No. PCT/EP2011/066246, dated Mar. 28, 2013. 10 pages. |
| International Preliminary Report on Patentability for Application No. PCT/US2014/072368, dated Jun. 28, 2016, 9 pages. |
| International Search Report and Written Opinion for Application No. PCT/US2017/062428, dated Apr. 12, 2018, 14 pages. |
| International Search Report for Application No. PCT/EP2011/066246, dated Jan. 23, 2012. 5 pages. |
| International Search Report for Application No. PCT/US2011/060831, dated Jul. 17, 2012. 7 pages. |
| International Search Report for Application No. PCT/US2016/022106, dated Jun. 17, 2016. 3 pages. |
| Japanese Office Action for Application No. 2016-543202, dated Dec. 26, 2018. 11 pages. |
| Bewersdorf et al., Multifocal Multi-Photon Microscopy. Handbook of Biological Confocal Microscopy, Third Edition. James H. Pawley (Ed.). Springer Science+Business Media, LLC. pp. 550-551, (2006). |
| Cha et al., Non-descanned multifocal multiphoton microscopy with a multianode photomultiplier tube. AIP Advances. 2015;5:084802, 14 pages. |
| Economo et al., A platform for brain-wide imaging and reconstruction of individual neurons. Elife. Jan. 20, 2016;5:e10566, 34 pages. |
| Engelbrecht et al., Miniaturized selective plane illumination microscopy for high-contrast in vivo fluorescence imaging. Opt Lett. May 1, 2010;35(9):1413-5. |
| Helmchen et al., Miniaturization of two-photon microscopy for imaging in freely moving animals. Cold Spring Harb Protoc. Oct. 1, 2013;2013(10):904-13. |
| Helmchen, Miniaturization of fluorescence microscopes using fibre optics. Exp Physiol. Nov. 2002;87(6):737-45. |
| Kim et al., High-speed, two-photon scanning microscope. Appl Opt. Oct. 1, 1999;38(28):6004-9. |
| Kim et al., Three-Dimensional Image Cytometer Based on a High-Speed Two-Photon Scanning Microscope. Proc SPIE. Apr. 2001,4262:238-46. |
| Konig, Multiphoton microscopy in life sciences. J Microsc. Nov. 2000;200(Pt 2):83-104. |
| Moneron et al., Two-photon excitation STED microscopy. Optics Express. Aug. 17, 2009;17(17):14567-14573. |
| So et al., Two-Photon Excitation Fluorescence Microscopy. Annu Rev Biomed Eng. Aug. 2000;20:399-429. |
| U.S. Appl. No. 11/442,702, filed May 25, 2006, 2007-0057211, Abandoned. |
| U.S. Appl. No. 15/515,536, filed Nov. 16, 2017, U.S. Pat. No. 10,598,597, Issued. |
| U.S. Appl. No. 13/297,035, filed Nov. 15, 2011, U.S. Pat. No. 8,771,978, Issued. |
| U.S. Appl. No. 14/300,425, filed Jun. 10, 2014, U.S. Pat. No. 9,983,134, Issued. |
| U.S. Appl. No. 15/989,886, filed May 25, 2018, U.S. Pat. No. 10,908,087, Issued. |
| U.S. Appl. No. 15/192,345, filed Jun. 24, 2016, 2016-0377546, Published. |
| U.S. Appl. No. 15/556,862, filed Sep. 8, 2017, U.S. Pat. No. 10,788,403, Issued. |
| U.S. Appl. No. 17/035,040, filed Sep. 28, 2020, 2021-0199545, Published. |
| U.S. Appl. No. 15/515,536, filed Nov. 16, 2017, 2018-0202935, Published. |
| U.S. Appl. No. 15/989,886, filed May 25, 2018, 2019-0064070, Published. |
| U.S. Appl. No. 15/556,862, filed Sep. 8, 2017, 2018-0045623, Published. |
| Matsumoto et al., Modulation of the pupil function of a microscope objective lens for multifocal multi-photon microscopy using a spatial light modulator. Proc SPIE. Feb. 28, 2014;8948:89481Q-1-89481Q-8. |
| Number | Date | Country | |
|---|---|---|---|
| 20190301980 A1 | Oct 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62424270 | Nov 2016 | US |
