Patent Application
20220413277
The present invention relates generally to the field of Laser Capture Microdissection (LCM).
Laser Capture Microdissection (“LCM”) (also called microdissection, laser microdissection (LMD), or laser-assisted microdissection (LMD or LAM)) is an established technology and method used to isolate a pure sample of a specific type of cells of interest (such as tumor cells) or entire areas of tissue (referred to herein generally as “target cells”) from a heterogeneous piece of tissue sample under direct microscopic visualization. The procured target cells may then be used in downstream applications, such as commercial diagnostic assays, clinical trials, and research studies by the pharmaceutical industry and academia. LCM is used by thousands of scientists worldwide and can be used in a variety of downstream applications such as genomics (DNA), transcriptomics (mRNA, miRNA), proteomics, metabolomics, gene expression and sequencing, determining molecular signatures, capillary electrophoresis, microarray analysis, polymerase chain reactions (PCR, such as qPCR or real time-PCR and proteomics), and next generation sequencing (NGS).
One form of LCM employs a laser beam or a source of radiation to heat a flat plastic film that is held against the slice of tissue sample mounted on a glass slide. The plastic film is uniformly impregnated with a dye that absorbs laser energy. The region of the plastic film positioned over the target cells is selectively heated by the radiation causing this region to melt and embed itself into the tissue segment immediately underneath. When the film is lifted off the tissue sample, the portions of the tissue adherent to the undersurface of the film are ripped free of the rest of the tissue sample (see, e.g., Espina V., et al. (2006) Nature Prot. 1(2):586-603, the entire disclosure of which is incorporated by reference herein in its entirety).
Various embodiments provide for a microscopy apparatus that comprises a microscope comprising a stage configured to hold a tissue sample, a UV laser assembly configured to emit a UV laser beam to a viewing area of the tissue sample, and an IR laser assembly configured to emit an IR laser beam to the viewing area of the tissue sample. The UV and IR laser assemblies are oriented so as to emit the respective UV and IR laser beams in a same direction.
Various other embodiments provide for a laser capture microdissection system that comprises a cap configured to adhere to target cells from a tissue sample when exposed to UV light and IR light and a microscopy apparatus. The microscopy apparatus comprises a microscope comprising a stage configured to hold the tissue sample and the cap, a UV laser assembly configured to emit a UV laser beam to a viewing area of the tissue sample, and an IR laser assembly configured to emit an IR laser beam to the viewing area of the tissue sample. The UV and IR laser assemblies are oriented so as to emit the respective UV and IR laser beams in a same direction.
Various other embodiments provide for a method of removing target cells from a tissue sample. The method comprises loading a tissue sample onto a stage of a microscope, selecting the target cells to be removed from the tissue sample, and placing a cap on the tissue sample, where the cap is configured to adhere to the target cells from the tissue sample when exposed to both the UV light and IR light. The method further comprises emitting a UV laser beam from a UV laser assembly to a viewing area of the tissue sample, emitting an IR laser beam from an IR laser assembly to the viewing area of the tissue sample, and removing the cap with the target cells adhered to the cap from a remainder of the tissue sample.
These and other features (including, but not limited to, retaining features and/or viewing features), together with the organization and manner of operation thereof, will become apparent from the following detailed description when taken in conjunction with the accompanying drawings, wherein like elements have like numerals throughout the several drawings described below.
Referring to the figures generally, various embodiments disclosed herein relate to various apparatus, systems, and methods for laser capture microdissection (“LCM”) that utilize both ultraviolet (UV) and infrared (IR) laser beams, which allows for ultra-precise laser microdissection and ultra-sensitive analysis. By providing both the UV and IR laser beams, the user is given more choices for isolating pure cell populations for a variety of different LCM applications.
As used herein and in the appended claims, singular articles such as “a” and “an” and “the” and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the claims unless otherwise stated. No language in the specification should be construed as indicating any non-claimed element as essential.
The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. Additionally, the phrase “consisting essentially of” will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase “consisting of” excludes any element not specified. The expression “comprising” means “including, but not limited to.” Thus, other non-mentioned substances, additives, carriers, or steps may be present. Unless otherwise specified, “a” or “an” means one or more.
Unless otherwise indicated, all numbers expressing quantities of properties, parameters, conditions, and so forth, used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations. Any numerical parameter should at least be construed in light of the number reported significant digits and by applying ordinary rounding techniques. The term “about” when used before a numerical designation, e.g., temperature, time, amount, and concentration including range, indicates approximations which may vary by (+) or (−) 10%, 5% or 1%.
As will be understood by one of skill in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.
The purpose of the LCM technique is to provide a simple method for the procurement of certain target cells from a heterogeneous population or tissue sample contained on a typical histopathology biopsy slide. A typical tissue biopsy sample consists of a 5 to 10 micron slice of tissue that is placed on a microscope slide using techniques well known in the field of pathology. Often a pathologist desires to remove only a small portion of the tissue sample (e.g., the “target cells”) for further analysis.
The tissue sample may be a variety of different types or heterogeneous mix of cells and may be a variety of different tissue types from a variety of different organisms. For example, the tissue sample may comprise human cells and may be a cross section of the body organ that is being studied. The desired or selected regions, portions, sections, cells, or tissue of interest of the tissue sample that are to be removed from the rest of the tissue sample are referred to as the “target cells.”
In order to perform LCM, a film (such as a micropatterned thermoplastic film) is placed in contact with an upper surface of a tissue sample, while the undersurface of the tissue sample is mounted on a slide. To isolate certain target cells from the tissue sample, the film (in contact with the tissue sample) is then irradiated with electromagnetic radiation (i.e., laser energy). This film is a part of a consumable cap (as described further herein).
When the laser irradiation is directed over the desired region of the tissue sample (i.e., over the target cells), the irradiation alters the tissue sample adhesive properties. Along the upper surface of the tissue sample, the laser irradiation activates the adhesive capture along the upper surface of the tissue sample by activating the film. In particular, once the film is exposed to the focused laser beam, the exposed region of the film (and cap) that is in the beam of the laser energy is heated by the laser and locally melts. The melted area of the film embeds at the tissue sample surface, thereby adhering the exposed portion of the tissue sample (i.e., the desired target cells) to the film.
Meanwhile, the lower surface of the tissue sample, instead of directly resting on the slide, rests on a microparticle or nanoparticle coating (according to one embodiment) that can be altered in its adhesive properties by the laser energy alone or in combination with a chemical treatment of the tissue sample. The laser energy simultaneously activates the adhesive forces on the top surface of the tissue sample (as described above) while breaking or dissolving the adhesive force below the tissue sample. In particular, the adhesive capture is dissolved below the tissue sample to achieve a higher efficiency, precision of capture (with lower nonspecific capture), yield, and resolution.
After the laser irradiation, the film is then lifted from the tissue sample and removed. Since the selected target cells (immediately below the locally melted film area) are adhered to the film, the selected target cells of the tissue sample are removed with the film from the rest of the tissue sample. In particular, the target cells are torn off from and removed from the remainder of the tissue sample, which isolates the target cells from the rest of the tissue sample (or the target cells can be cut from the rest of the tissue sample). The target cells are captured and placed within a collection device to collect histologically pure, enriched cell populations from microscopic regions of tissue samples or cells.
The success of this operation is dependent on the balance of forces above the selected region of interest (i.e., between the film and the tissue sample) and below the selected region of interest (i.e., between the tissue sample and the slide). Various compositions and methods can increase the adhesive strength and improve the resolution of the film in contact with the tissue sample surface while at the same time selectively reducing the adhesive forces on the bottom of the tissue sample, where it can be tightly dried down on the slide.
As described further herein, the various embodiments disclosed herein allow the film (in contact with the tissue sample) to be irradiated with electromagnetic radiation in both the ultraviolet (UV) and infrared (IR) spectrum.
By including both the UV laser assembly 40 and the IR laser assembly 60, the LCM system 20 provides performance improvements over conventional LCM systems. In particular, the resolution of the LCM system 20 is improved and smaller size regions of target cells can be accurately captured through microdissection within the tower of the microscope 32. By including both the UV laser assembly 40 and the IR laser assembly 60, the utility and use for additional applications of the LCM system 20 is expanded (compared to conventional LCM systems), and the LCM system 20 provides additional functionality not addressed or provided within conventional LCM systems that only use a single laser wavelength. Furthermore, higher quality images can be produced, while still being easily used to capture target cells.
The LCM system 20 allows a tissue microenvironment to be interrogated at a single cell level, provides high-fidelity visualization of the tissue (e.g., digital, full color images on a computer screen for selection by a mouse or stylus, for example), allows unique molecular signatures to be uncovered that would have otherwise been obscured in a heterogeneous cell population, and utilizes “GeckoGrip” caps with nanotechnology enhancements (as described further herein) for higher capture, extraction yields, and preservation.
Compared to conventional LCM systems, the LCM system 20 increases the speed of LCM with increased precision of targeting cells of interest, while maximizing flexibility. The LCM system 20 allows microdissected tissue samples to be maintained and in contact with a portion of the LCM system 20, while the bulk of the tissue is being imaged and cataloged for reference. The spatial orientation of the cells captured are maintained on the capture surface, thereby preserving the molecular integrity of the cells and shielding the biomolecules in the tissue sample from damage from the UV laser beam.
The LCM system 20 comprises a capture consumable or cap 24 with a film in order to perform the LCM. The cap 24 (via the film) is configured to adhere to target cells from the tissue sample when exposed to both the UV light and the IR light. The film may be a patterned (e.g., micropatterned) thermoplastic transfer film, such as those disclosed in U.S. Pat. No. 10,324,008, the entire disclosure of which is incorporated by reference herein in its entirety. The film may comprise “gecko feet” or projections, such as micropillars, micro projections, hydrogel microspheres, and/or microneedles that are attached to, continuous with, or integrally formed with a thermoplastic film, that is placed on top of the tissue sample. These projections allow for simultaneous capture and release and use micropattern surfaces for tissue and cell microdissection. The projections also improve the capture (e.g., lift) efficiency of pure populations of the target cells from the heterogeneous tissue sample by increasing the adhesive force between the top surface of the target cells and the film.
According to one embodiment, the projections may be formed on the film using a photolithography mold that is applied to the thermopolymer surface mounted on a thermopolymer extraction cap 24. The microscope 32 may include a weighted method to hold the cap 24 in place, which improves contact with the target cells. Optionally, the cap 24 may be manually placed onto the tissue sample, and the microscope 32 may comprise a cap placement system, such as a manual cap arm, to manually place the cap 24 with precision and stability. This improve the LCM polymer spot functionality (such as adherence to cells and spot diameter), allowing for smaller capture sizes.
The film (and optionally the projections) has surfaces that can be activated by selective radiant energy, such as from a laser beam, to become adhesive to an irregular tissue sample surface below. For example, the film (and optionally the projections) can be manufactured containing or impregnated with organic dyes (such as UV and/or IR absorbing dye) that are chosen to selectively absorb in the ultraviolet (UV) or infrared (IR) region of the spectrum overlapping the emission region of common laser diodes, e.g., AlGaAs laser diodes. By impregnating the projections (such as micropillars) with a gradient of laser absorbing dye, the tip of the projections can be made to swell and conform to the tissue sample surface irregularities selectively at the tip.
Additionally, LCM system 20 comprises the slide 26 that the tissue sample is mounted on. The slide 26 can also be modified by coating the slide 26 with microparticles or nanoparticles that can be altered in their tissue adhesive properties by laser irradiation. For example, the slide 26 can be coated with indium tin oxide to reduce adhesion forces between the bottom surface of the target cells and the slide 26 under the laser irradiation. The slide 26 may be constructed out of a variety of different materials, including but not limited to glass or membrane (e.g., glass membrane or metal membrane).
The microscope 32 allows the tissue sample (or the target cells) to be easily magnified, viewed or visualized, and captured (as an image) at, for example, 2×, 10×, and 40× magnification. The microscope 32 may provide digital microscopy and may include various features, such as marks for image alignment, on-screen feature selection, automatic or manual microdissection, intuitive, user-friendly operator software, and integrated telepathology options. The microscope 32 may provide high fidelity color visualization of the tissue sample by refractive index matching at the interface with capture surface.
According to one embodiment, the base of the microscope may be an Olympus ix73 inverted microscope with 2×, 10×, and 40× objectives and long-working distance condenser (and without any binoculars installed), as shown in
The microscope 32 defines an optical axis 39 that defines a path along which light propagates through the microscope 32 to the tissue sample. The optical axis 39 coincides with and extends through the middle of the field of view (or viewing area or region) of the tissue sample. The optical axis 39 passes through the center of curvature of each of the lens of the microscope 32 (and through any mirrors of the laser assemblies 40, 60). As referred to herein, the “path of light” refers to the direction that both visible light and the laser beams 42, 62 are emitted toward the tissue sample and the stage 34.
The microscope 32 may comprise an illumination or light source 38 (as shown in
The microscope 32 comprises a variety of different lens, including at least one condenser lens 36 and at least one objective lens 37 (as shown in
As shown in
As shown in
The UV laser assembly 40 and the IR laser assembly 60 of the dual laser system are both solid state and emit laser beams at different wavelengths and different energies for a wide range of applications. The UV laser assembly 40 and the IR laser assembly 60 can capture single cells or small groups of cells, microdissect complicated multicellular shapes, extract large tissue areas, and be used with a variety of tissue samples, including frozen sections, paraffin embedded sections, and live cells (and accordingly may optionally include phase contrast and Dic options for live-cell applications). According to one embodiment, the UV laser assembly 40 and the IR laser assembly 60 are both positioned outside of the optical axis 39 of the microscope 32 and have a fixed laser focus.
As shown in
In addition to tissue cutting, the UV laser assembly 40 may also be used to perform UV LCM, similar to the operation of the IR laser assembly 60. Comparatively, in conventional LCM systems that incorporate a UV laser, the UV laser is only used for tissue cutting, rather than capturing direct cap-based tissue. According to one embodiment as shown in
The UV laser assembly 40 includes a single UV mirror 44 configured to change a direction of the UV laser beam 42 emitted from the UV laser 41. The UV mirror 44 is configured to redirect the path of the UV laser beam 42 from the UV laser 41 to the viewing area of tissue sample along the stage 34. Accordingly, the UV laser beam 42 is initially emitted from the UV laser 41 in a first direction, travels in the first direction between the UV laser 41 and the UV mirror 44, bounces off and is deflected by the UV mirror 44 (which redirects the path of the UV laser beam 42), subsequently travels in a second direction between the UV mirror 44 and the tissue sample and the stage 34, and contacts the viewing area of the tissue sample while traveling in the second direction. The first and second directions may be substantially perpendicular to each other (i.e., horizontal and vertical directions, respectively, as shown in
With the UV mirror 44, the UV laser 41 can be positioned outside of the optical axis 39 of the tissue sample within the microscope 32. The first direction of the UV laser beam 42 may be substantially parallel to the top surface of the stage 34 (although, according to other embodiments, the UV laser beam 42 may initially be emitted at other angles to the stage 34 and the angle of the UV mirror 44 is adjusted accordingly).
Depending on the arrangement of the microscopy apparatus 30, the UV mirror 44 may allow certain wavelengths of light to be transmitted through (while deflecting the UV laser beam 42). For example, as shown in
As shown in
The IR laser assembly 60 includes a single IR mirror 64 configured to change a direction of the IR laser beam 62 emitted from the IR laser 61. The IR mirror 64 is configured to redirect the path of the IR laser beam 62 from the IR laser 61 to the tissue sample to the viewing area of tissue sample along the stage 34. Accordingly, the IR laser beam 62 is initially emitted from the IR laser 61 in a first direction, travels in the first direction between the IR laser 61 and the IR mirror 64, bounces off and is deflected by the IR mirror 64 (which redirects the path of the IR laser beam 62), subsequently travels in a second direction between the IR mirror 64 and the tissue sample and the stage 34, and contacts the viewing area of the tissue sample while traveling in the second direction. The first and second directions may be substantially perpendicular to each other (i.e., horizontal and vertical directions, respectively, as shown in
With the IR mirror 64, the IR laser 61 can be positioned outside of the optical axis 39 of the tissue sample within the microscope 32. The first direction of the IR laser beam 62 may be substantially parallel to the top surface of the stage 34 (although, according to other embodiments, the IR laser beam 62 may initially be emitted at other angles to the stage 34 and the angle of the IR mirror 64 is adjusted accordingly).
Depending on the arrangement of the microscopy apparatus 30, the IR mirror 64 may allow certain wavelengths of light to be transmitted through (while deflecting the IR laser beam 62). For example, as shown in
According to one embodiment, the UV laser assembly 40 and the IR laser assembly 60 each comprise only one mirror (i.e., the UV mirror 44 and the IR mirror 64, respectively). As shown in
The UV laser assembly 40 and the IR laser assembly 60 may optionally be configured to emit the UV laser beam 42 and the IR laser beam 62, respectively, at the same time. The UV laser beam 42 and the IR laser beam 62 are emitted horizontally from the UV laser 41 and the IR laser 61, respectively, as collimated beams. The UV laser assembly 40 and the IR laser assembly 60 may optionally include multiple focusing lens to expand the collimated beams and then refocus the beams or may include a beam expander. As described further herein, the UV laser beam 42 and the IR laser beam 62 are each focused through at least one lens (such as the condenser lens 36), deflected off of a respective mirror 44, 64, and directed vertically downward to the viewing area along the stage 34.
As shown in
In some embodiments, each of the UV and IR laser controllers 49, 69 includes a central processing unit (CPU), a read only memory (ROM), and a random access memory (RAM) 103. The CPU is connected to the ROM and the RAM via a bus line. The CPU is configured to store at least one control program stored in the ROM or the memory unit in the RAM. The CPU controls operation of one or more of the laser or power supply by operating according to the control program.
For the UV laser beam 42 and the IR laser beam 62, suitable laser pulse widths may be from 0 to approximately 1 second, preferably from 0 to approximately 100 milliseconds, more preferably approximately 50 milliseconds. In at least one embodiment, the pulses from the UV laser beam 42 and IR laser beam 62 are phased such that they never overlap. The UV laser beam may be off when the IR laser beam is on, and vice versa, such that UV and IR energy are never emitted simultaneously. The spot size of the UV laser beam 42 and the IR laser beam 62 at the film located on microcentrifuge tube cap is variable from 0.1 to 100 microns, preferably from 1 to 60 microns, more preferably from 5 to 30 microns. These ranges are relatively preferred when designing the optical subsystem. From the standpoint of the clinical operator, the widest spot size range is the most versatile. A lower end point in the spot size range on the order of 5 microns is useful for transferring single cells.
The UV laser beam 42 and the IR laser beam 62 may have a wide power range. For example, a 100 milliwatt laser can be used for at least one of the UV or IR laser beam. In some embodiments, a 50 mW laser can be used for at least one of the UV or IR laser beam. The UV laser beam 42 and the IR laser beam 62 can be connected to the rest of the optical subsystem with a fiber optical coupling. Smaller spot sizes are obtainable using diffraction limited laser diodes as the emitters and/or single mode fiber optics. Single mode fiber allows a diffraction limited beam.
Changing the beam diameter of the UV laser beam 42 and the IR laser beam 62 permits the size of the portion of the sample that is acquired to be adjusted. Given a tightly focused initial condition, the beam size can be increased by defocusing. Given a defocused initial condition, the beam size can be decreased by focusing. The change in focus can be in fixed amounts. The change in focus can be obtained by means of indents on a movable lens mounting and/or by means of optical glass steps. In any event, increasing/decreasing the optical path length is the effect that is needed to alter the focus of the beam, thereby altering the spot size. For example, inserting a stepped glass prism into the beam so the beam strikes one step tread will change the optical path length and alter the spot size.
Optionally, the microscopy system may include an extendable platform to provide optional UV laser cutting and epifluorescence.
The LCM system 20 may be set up in a variety of different configurations and arrangements, depending on the desired use and setup configuration. According to various embodiments as shown in
With this configuration, after the UV laser beam 42 and the IR laser beam 62 are each redirected by their respective mirrors 44, 64, the UV laser beam 42 and the IR laser beam 62 extend along the optical axis 39 of the microscope 32 to the stage 34, parallel to each other. Both the UV laser beam 42 and the IR laser beam 62 contact the viewing area of the tissue sample and the stage 34 at an approximately normal angle (and parallel to the optical axis 39). By arranging the UV laser beam 42 and the IR laser beam 62 to contact the viewing area along the stage 34 (and therefore extend through the cap 24) at a normal angle, the UV laser beam 42 and the IR laser beam 62 can be tightly focused. In order to position both the UV mirror 44 and the IR mirror 64 within the optical axis 39 (such that the UV laser beam 42 and the IR laser beam 62 are parallel to each other before and after their respective redirection by the mirrors 44, 64), the UV mirror 44 and the IR mirror 64 are at approximately the same angle as each other.
According to the embodiment shown in
According to the embodiment shown in
The setup of
According to the embodiment shown in
As further shown in
As also shown in
According to another embodiment, the IR laser assembly 60 may not include any IR mirrors, such as the IR mirror 64. Instead, the IR laser beam 62 may be emitted (from the IR fiber, for example) from the IR laser 61 directly parallel to and along the optical axis 39. Accordingly, the triplet collimator 162 (with its collimator mount 161) may be threaded directly onto an expander (such as, for example only, a 2× expander for an IR laser beam 62 with a width of approximately 11 mm) and vertically mounted such that the triplet collimator 162 points directly down along the optical axis 39 through the objective lens 37 (i.e., the reflective objective).
In the embodiment of
As shown in
As also shown in
In the embodiment shown in
According to one embodiment, the diameter of the UV laser beam 42 in the setup of
The achromatic doublets 243 and 263 allow the laser beams 42, 62 to be focused before contacting the respective mirrors 44, 64. Alternatively, instead of the achromatic doublets 243 and 263, aspheres (such as the Thorlabs AL50100H-B, 2 inch aspheric lens) may be used.
According to one embodiment, the microscopy apparatus 30 may not include the UV laser assembly 40, and may only include the IR laser assembly 60 as its only laser source. The IR laser assembly 60 may have any of the various configurations, components, and features described herein.
The various embodiments disclosed herein (in particular, but not limited to the embodiments shown in
In order to perform LCM in and operate the LCM system 20 (in particular to remove target cells from a tissue sample), the slide is prepared (and the tissue sample is optionally stained), and the tissue sample is loaded or placed onto the stage 34 of the microscope 32 and visually inspected through the microscope 32 (and optionally through an associated computer monitor) to identify the target cells of interest. The target cells of the tissue sample to be dissected and removed from the rest of the tissue sample through laser dissection are located, selected, outlined, and traced (on, for example, the computer monitor with freehand drawings tools) to designate which portions of the tissue sample should be microdissected. The stage 34 of the microscope 32 is moved (with the tissue sample) along a horizontal plane relative to the rest of the microscope 32 (and the rest of the microscopy apparatus 30) to position the target cells in the paths of the UV laser beam 42 and the IR laser beam 62. The operator may manually place the cap 24 (which includes the film) onto the cap holder or arm, and the cap 24 is pressed or placed onto the tissue sample (and the slide 26). The UV laser assembly 40 and the IR laser assembly 60 may optionally be test fired.
Via software, the operator activates the UV laser assembly 40 and the IR laser assembly 60, which emits the UV laser beam 42 and the IR laser beam 62, respectively, to the viewing area of the tissue sample (via a diascopic illumination pillar according to one embodiment). As described further herein, the UV mirror 44 and the IR mirror 64 change the direction of the UV laser beam 42 and the IR laser beam 62, respectively, as the UV laser beam 42 and the IR laser beam 62 travel from the UV laser 41 and the IR laser 61, respectively, to the viewing area of the tissue sample. The UV laser beam 42 and the IR laser beam 62 melt or soften the cap surface, which adheres exposed portions (i.e., the target cells) of the tissue sample to the cap 24, thereby enabling the capture the selected portions (i.e., the target cells) of the tissue sample onto the cap 24. The cap 24 (with the target cells attached or adhered to the cap 24) is then manually removed from the tissue sample, which lifts off or removes the target cells from the remainder of the tissue sample. The microdissected material on the cap is then inspected for positive identification of the captured material and subsequent downstream analysis.
The cap 24 (with the target cells attached) can subsequently be fit onto a Eppendorf tube (such as a 0.5 millileter (mL) Eppendorf tube), which can then be used with a Reagents Kit for downstream applications (e.g., extraction of DNA or RNA for downstream molecular analysis such as gene expression or DNA sequencing).
The microscopy apparatus 30 may further comprise various user input and control devices, such as a computer system including a computer, software, and a display (such as a touch-sensitive screen interface (e.g., a wireless tablet)). All of the components of the microscopy apparatus 30 may be fully integrated with the software of the computer system. The microscope 32, the UV laser assembly 40, and the IR laser assembly 60 may connect to and be controlled by the computer system.
The computer system may allow the user to control the LCM system. For example, through the computer system, the user can take a photo or image from the microscope 32 with the camera 35 (using, for example, Spinnaker Software Development Kit (SDK) for a FLIR camera).
The user may also use the computer system to simply select (via, for example, free hand or with simple shapes, such as a rectangle or circle) the target cells on the tissue sample through a still image or in real-time. The computer system allows the user to place single IR spots that correspond to the laser diameter on the target cell(s) and incorporates a measuring tool to measure the area captured and the diameter of the lasers. The computer system also allows the user to choose which cutting option for the microscopy apparatus 30 to perform on the selected target cells. In particular, through the computer system, the user can instruct the microscopy apparatus 30 to perform an IR LCM, a UV cutting, and/or a UV LCM.
The computer system allows the size of the UV laser beam 42 to be manually assessed and allows both the IR laser beam 62 and the UV laser beam 42 to be located, manually or automatically.
Once the user has manually set up the slide 26, positioned the cap 24, and calibrated the lasers beams 42, 62 to be directed to the slide 26, the user can use the software to select what type of slide 26 (e.g., glass, glass membrane, or metal membrane) has been loaded onto the microscopy apparatus 30.
The user can also use the computer system control both the UV and IR laser assemblies 40, 60 via a USB Relay Controller. The UV and IR laser assemblies 40, 60 may use certain switches of the controller. For example, the IR laser assembly 60 may use switches 1-7 of the controller, and the UV laser assembly 40 may only use switch 8 as an on/off switch. The computer system may also control the power levels and duration of the IR laser beam 62 with single power operations. The computer system may move the stage 34, fire the IR laser beam 62 to facilitate adhesion of the target cells to the cap 24, and fire the UV laser beam 42 to cut along the selected outline of the tissue sample. In particular, the adhesion of the target cells to the cap may be accomplished by patterning the caps with a micro-patterned surface which utilizes Van der Waals forces to serve as gecko-like feet that readily grip the tissue topography of the target cells.
The precision of the LCM system 20 is the amount of material (i.e., target cells) actually captured versus the area outlined for capture and can be correlated to the resolution of the laser spot size. To provide precise results, the LCM system 20 captures the target cells with 1 μm of precision of which target cells were initially selected compared to where the lasers were actually fired. According to one embodiment, the diameter of the laser spot size of the IR laser beam 62 is approximately 5 μm, and the diameter of the laser spot size of the UV laser beam 42 is approximately 1 μm or less. Furthermore, the stage 34 of the microscope 32 has greater than approximately 1 μm accuracy to ensure the correct target cells are captured. The precision of the LCM system 20 is measured via image analysis with tools such as ImageJ or a ruler measuring and is greater than approximately 95% capture of the target cells within the capture boundaries, as determined by ImageJ analysis.
The reliability of the LCM system 20 is how consistently a given amount of target cells are extracted from a given tissue sample type using a given microdissection setting and extraction area. The reliability also refers to the ability of the lasers to fire in the correct position as selected by the user. The reliability of the LCM system 20 can be measured using a standard tissue sample type, such as a fixed monolayer culture cells or a block of fixed cultured cells, to reduce the effects of inter-patient sample variability on reliability measurements and to standardize measurements using this tissue. When extracting this standard tissue sample type at a given setting with a given extraction area, the quantity of captured material does not vary by greater than approximately 5%.
The sensitivity of the LCM system 20 is the quantifiable amount of DNA, RNA, or protein from a given amount of microdissected tissue. Sensitivity can be measured by quantifying the total amount of extracted DNA, RNA, or protein content from a given number of extracted cells and performing a total protein content analysis using a Bradford assay. The total extracted protein from multiple tissue types of at least 1000 cells will be greater than a predetermined cutoff-value.
The tissue total yield per region of the LCM system 20 is more than approximately 30% better yield of tissue captures per spot compared to IR LCM by Thermo Fischer and approximately equal to UV cutting by Leica, Zeiss, and Thermo Fischer.
Table 1 provides exemplary specifications of various values that may be used within the LCM system 20, according to various embodiments.
Table 2 also provides exemplary specifications of various values that may be used within the LCM system 20, according to various embodiments.
Table 3 provides exemplary parts (and their respective model and brand) that may be used within the LCM system 20, according to various embodiments. As shown, the exemplary parts may be included to perform various functions within the LCM system 20.
As utilized herein, the terms “approximately,” “about,” “substantially” and similar terms are intended to have a broad meaning in harmony with the common and accepted usage by those of ordinary skill in the art to which the subject matter of this disclosure pertains and should be interpreted as including at least a de minimis level of variance from the identified value. It should be understood by those of skill in the art who review this disclosure that these terms are intended to allow a description of certain features described without restricting the scope of these features to the precise numerical ranges provided. Accordingly, these terms should be interpreted as indicating that insubstantial or inconsequential modifications or alterations of the subject matter described and are considered to be within the scope of the disclosure.
The terms “coupled,” “connected,” “attached,” and the like as used herein mean the joining of two members directly to one another. Such joining may be stationary (e.g., permanent) or moveable (e.g., removable or releasable).
References herein to the positions of elements (e.g., “top,” “bottom,” “above,” “below,” etc.) are merely used to describe the orientation of various elements in the FIGURES. It should be noted that the orientation of various elements may differ according to other exemplary embodiments, and that such variations are intended to be encompassed by the present disclosure.
It is important to note that the construction and arrangement of the various exemplary embodiments are illustrative only. Although only a few embodiments have been described in detail in this disclosure, those skilled in the art who review this disclosure will readily appreciate that many modifications are possible (e.g., variations in sizes, dimensions, structures, shapes and proportions of the various elements, values of parameters, mounting arrangements, use of materials, colors, orientations, etc.) without materially departing from the novel teachings and advantages of the subject matter described herein. For example, the position of elements may be reversed or otherwise varied, and the nature or number of discrete elements or positions may be altered or varied. The order or sequence of any process or method steps may be varied or re-sequenced according to alternative embodiments. Other substitutions, modifications, changes and omissions may also be made in the design, operating conditions and arrangement of the various exemplary embodiments without departing from the scope of the present invention.
The present application claims priority to U.S. provisional application No. 62/937,624 filed Nov. 19, 2019, the entire contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/060892 | 11/17/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62937624 | Nov 2019 | US |
