Compositions and methods for temperature controlled sample imaging

Abstract

Systems and methods of the present disclosure improve microscope imaging of a sample via temperature control for improving the transparency of the sample in a refractive index matching medium. At least one temperature control unit is configured to transfer heat to a sample holder, the sample holder being configured to holding a sample of biological matter. At least one controller is operably connected to the at least one temperature control unit, the at least one controller being configured to modulate the at least one temperature control unit so as to control the transfer of heat to the sample holder so as to achieve a predetermined set point. The predetermined set point includes at least one temperature that exceeds room temperature, and is configured to increase transparency of the sample sufficient for the at least one optical device to view a full thickness of the sample in the sample holder.

Description

COMPOSITIONS AND METHODS FOR TEMPERATURE CONTROLLED SAMPLE IMAGINGSTATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

[0001] This embodiments was made with U.S. government support under Grant No. COBRE P20GM 104318-09, awarded by the National Institutes of Health to PI: Iain Drummond, MDIBL, and RGL: Prayag Murawala, MDIBL. The government has certain rights in the embodiments.BACKGROUND OF THE DISCLOSURE

[0002] The optical microscope, also referred to as a light microscope, typically uses visible light and a system of lenses to generate magnified images of samples, including biological samples. The object is placed on a stage or in a chamber and may be directly viewed through a set of optics. To enabling viewing, via a camera or directly through one or more eyepieces, the sample can be lit in a variety of ways. Transparent objects can be lit from any direction and solid objects can be lit with light coming through (bright field) or around (dark field) the objective lens. Typically, for larger objects, the samples are made to be transparent.SUMMARY OF THE DISCLOSURE

[0003] As described below, the present disclosure features systems, devices and methods for improving imaging of tissue-cleared samples.

[0004] The present invention is a system that includes a microscope system. The microscope system includes at least one light source configured to emit light towards a sample holder and at least one optical device configured to have the sample holder in a field of view of the at least one optical device. The system also includes at least one temperature control unit configured to transfer heat to the sample holder, the sample holder being configured to holding a sample of biological matter. The system further includes at least one controller operably connected to the at least one temperature control unit. The at least one controller is configured to modulate the at least one temperature control unit so as to control the transfer of heat to the sample holder so as to achieve a predetermined set point, wherein the predetermined set point includes at least one temperature that is established to increase transparency of the sample sufficient for the at least one optical device to view a full thickness of the sample in the sample holder. The microscope system may beC:\Documcnts and Sctings\MHuntcr-cnsor\Dcsktop\Modcls\Modcl app.docATTORNEY DOCKET NO. MDIBL-001PCT a light sheet microscope but not limited thereto. The sample holder may be configured to hold the sample in a volume of a refractive index matching medium. The sample can be a tissue cleared and / or refractive index matched sample immersed in a refractive index matching medium within the sample holder. The at least one controller can also be configured to determine the refractive index matching medium, determine a refractive index matching temperature at which the refractive index matching medium and a refractive index of the sample match, and modulate the at least one temperature control unit to as to control the transfer of heat to the sample holder to achieve the refractive index matching temperature within the sample holder. The microscope system can also include a sample holder configured to hold the sample holder in the field of view of the at least one optical device, wherein the at least one temperature control unit is attached to the sample holder so as to come into contact with the sample holder upon the sample holder being positioned on the sample holder. The at least one temperature control unit can be attached to the sample holder. The at least one controller can also be configured to determine a current temperature of the sample in the sample holder using at least one temperature sensor; determine a difference between the current temperature and the predetermined set point; and modulate, based on the difference, the at least one temperature control unit to as to control the transfer of heat to the sample holder to achieve the predetermined set point.

[0005] The present invention is also a method that includes the step of modulating, by at least one processor associated with a microscope system, at least one temperature control unit so as to control a transfer of heat to a sample holder so as to achieve a predetermined set point, wherein the sample holder is configured to hold a sample of biological matter, wherein the predetermined set point is at least one temperature that is established to increase transparency of the sample sufficient for the at least one optical device to view a full thickness of the sample in the sample holder. In the method, the sample holder may be configured to hold the sample in a volume of a refractive index matching medium. In an embodiment of the invention, the sample is a tissue cleared sample immersed in a refractive index matching medium within the sample holder. In an embodiment, the method further includes the steps of determining, by the at least one processor, the refractive index matching medium, determining, by the at least one processor, a refractive index matching temperature at which the refractive index matching medium and a refractive index of the sample match, and modulating, by the at least one processor, the at least one temperature control unit to as to control the transfer of heat to the sample holder to achieve the refractive indexATTORNEY DOCKET NO. MDIBL-001PCT matching temperature within the sample holder. In an embodiment, the microscope system includes a sample holder configured to hold the sample holder in the field of view of the at least one optical device, and wherein the at least one temperature control unit is attached to the sample holder so as to come into contact with the sample holder upon the sample holder being positioned on the sample holder. In an embodiment, the at least one temperature control unit is attached to the sample holder. In an embodiment, the method further includes the steps of determining, by the at least one processor, a current temperature of the sample in the sample holder using at least one temperature sensor, determining, by the at least one processor, a difference between the current temperature and the predetermined set point, and modulating, by the at least one processor, based on the difference, the at least one temperature control unit to as to control the transfer of heat to the sample holder to achieve the predetermined set point.

[0006] In an embodiment, the invention is an apparatus includes a sample holder associated with a microscope system, the sample holder being configured to holding a sample of biological matter;

[0007] at least one temperature control unit configured to transfer heat to a sample holder, and at least one controller operably connected to the at least one temperature control unit, with the at least one controller configured to modulate the at least one temperature control unit so as to control the transfer of heat to the sample holder so as to achieve a predetermined set point, wherein the predetermined set point is at least one temperature that is configured to increase transparency of the sample sufficient for the at least one optical device to view a full thickness of the sample in the sample holder. In this embodiment, the sample may be a tissue cleared sample immersed in a refractive index matching medium within the sample holder. In an embodiment, the at least one controller of the apparatus is further configured to determine the refractive index matching medium, determine a refractive index matching temperature at which the refractive index matching medium and a refractive index of the sample match, and modulate the at least one temperature control unit to as to control the transfer of heat to the sample holder to achieve the refractive index matching temperature within the sample holder. The at least one temperature control unit of the apparatus is attached to the sample holder in an embodiment. Further, the at least one controller of the apparatus is further configured to determine a current temperature of the sample in the sample holder using at least one temperature sensor, determine a difference between the current temperature and the predetermined set point, and modulate, based on the difference, the at leastATTORNEY DOCKET NO. MDIBL-001PCT one temperature control unit to as to control the transfer of heat to the sample holder to achieve the predetermined set point.

[0008] The embodiments provide systems, devices and methods that provide for temperature control of samples for improved tissue-cleared sample imaging. Systems and devices described herein were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of embodiment of the disclosure will be apparent from the detailed description, and from the claims.Definitions

[0009] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which these embodiments belong. The following references provide one of skill with a general definition of many of the terms used in these embodiments: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0010] By “agent” is meant a peptide, nucleic acid molecule, or small compound.

[0011] By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

[0012] By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

[0013] By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

[0014] By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.ATTORNEY DOCKET NO. MDIBL-001PCT

[0015] In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of' or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0016] “Detect” refers to identifying the presence, absence or amount of the analyte to be detected.

[0017] By "detectable label" is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.

[0018] By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include but are not limited to, polycystic kidney disease, neuro autoimmune diseases such as multiple sclerosis and neuromyelitis optica, as well any type of cancer.

[0019] By "effective amount" is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present embodiments for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

[0020] By "effective resolution" is meant the resolvable detail in an image produced by imaging equipment having particular resolution of a sensor for detecting the image, the resolution of the sensor being defined by the spatial density of pixels.

[0021] "Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.ATTORNEY DOCKET NO. MDIBL-001PCT

[0022] The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this embodiments is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0023] As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.

[0024] By ‘ ‘reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

[0025] By “refractive index matching medium” is meant a compound, solution, fluid, chemical or other substance that is transparent to the light of an illumination device, such as a microscope illumination device with a refractive index that matches to the refractive index of a transparent sample.

[0026] By “reference” is meant a standard or control condition.

[0027] By “room temperature” is meant a temperature of a room in which embodiments of the disclosed systems and devices are located and / or operated, such as but not limited to a temperature in a range of 68 to 72 degrees Fahrenheit.

[0028] By “sample” is meant a portion of an object being studied, the portion being prepared for study by one or more instruments.

[0029] By "subject" is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.ATTORNEY DOCKET NO. MDIBL-001PCT

[0030] As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0031] By “tissue cleared,” “tissue clearing” and the like is meant a group of chemical techniques used to turn tissues transparent using one or more tissue clearing methods including, but not limited to, hydrophobic clearing (e.g., organic-based, solvent-based, organic solvent-based, etc.), dehydration clearing, hydrophilic clearing (e.g., aqueous-based or water-based, etc.), hydrogelbased clearing, tissue-expansion clearing, among other clearing methods or any combination thereof.

[0032] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0033] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

[0034] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0035] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0036] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.ATTORNEY DOCKET NO. MDIBL-001PCTBRIEF DESCRIPTION OF THE DRAWINGS

[0037] Various embodiments of the present disclosure can be further explained with reference to the attached drawings, wherein like structures are referred to by like numerals throughout the several views. The drawings shown are not necessarily to scale, with emphasis instead generally being placed upon illustrating the principles of the present disclosure. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ one or more illustrative embodiments.

[0038] FIG. 1 depicts a control system for a microscope system and a temperature control unit configured to control the temperature of a sample in a sample holder to achieve improved transparency of the sample in accordance with one or more embodiment of the present disclosure.

[0039] FIG. 2 depicts a control system for a light-sheet microscope system and a temperature control unit configured to control the temperature of a sample in a sample holder to achieve improved transparency of the sample in accordance with one or more embodiment of the present disclosure.

[0040] FIG. 3 depicts a temperature control unit positioned on a sample holder to control the temperature of a sample in a sample holder to achieve improved transparency of the sample in accordance with one or more embodiment of the present disclosure.

[0041] FIG. 4 depicts a temperature control unit positioned on a stage to contact a sample holder when positioned for microscopy to control the temperature of a sample in the sample holder to achieve improved transparency of the sample in accordance with one or more embodiment of the present disclosure.

[0042] FIG. 5 depicts a flowchart illustrating a control methodology for operating a temperature control unit to control a temperature of a sample in the sample holder to achieve improved transparency of the sample in accordance with one or more embodiment of the present disclosure.

[0043] FIG. 6A, 6B, 6C, 6D, 6E, 6F, 6G and 6H depict temperature modulation unit for tissue- cleared samples in accordance with one or more embodiment of the present disclosure.

[0044] FIG. 7A, 7B, 7C and 7D depict the effect of temperature on tissue transparency and image quality in accordance with one or more embodiment of the present disclosure.ATTORNEY DOCKET NO. MDIBL-001PCT

[0045] FIG. 8A, 8B, 8C and 8D depict the effect of temperature on light transmission of tissue- cleared mouse brains in accordance with one or more embodiment of the present disclosure.

[0046] FIG. 9A, 9B and 9C depict the effect of temperature on transparency / haze of tissue-cleared mouse brain in accordance with one or more embodiment of the present disclosure, wherein “transparency” means in the context of the present invention that there is minimum deviation to very little of the incident ray of light caused by forward scattering as it transmits through a sample, whereas “haze” means that there is deviation of the incident ray of light caused by scattering as it transmits through the sample.

[0047] FIG. 10A, 10B, 10C and 10D depict the effect of temperature on light transmission of tissue-cleared mouse kidneys in accordance with one or more embodiment of the present disclosure.

[0048] FIG. 11 A, 11B and 11C depict the effect of temperature on transparency / haze of tissue- cleared mouse kidneys in accordance with one or more embodiment of the present disclosure.

[0049] FIG. 12A, 12B, 12C, 12D and 12E depict the effect of temperature on autofluorescence of tissue-cleared mouse tissue in accordance with one or more embodiment of the present disclosure.DETAILED DESCRIPTION OF THE DISCLOSURE

[0050] The embodiments feature systems, devices and methods that are useful for improving the imaging and / or detection quality of tissue cleared samples in microscopy. The improvement relates to controlling the temperature of the sample and a refractive index matching medium to achieve a temperature above room temperature that where the refractive index of the sample and of the refractive index matching medium match.

[0051] With the advanced microscopy systems, it is possible to image large biological samples (for example, whole organs or organisms) with an uninterrupted three-dimensional view of the specimen. The quality of the imaging is affected by the transparency of the sample, where the sample may be made transparent by tissue-clearing, where an opaque or translucent sample is rendered transparent. The tissue-cleared sample may then be placed in a sample holder, such as a chamber or vessel, in which the sample is immersed in a fluid, thereby suspending the sample for imaging by the microscope. However, the effective transparency of the sample while in a sample chamber can be affected by differences in the refractive index between the tissue-cleared sampleATTORNEY DOCKET NO. MDIBL-001PCT and the refractive index matching medium. Improved transparency reduces light scattering and provides an unobstructed view.

[0052] Embodiments of the present disclosure improve the effective transparency for imaging in the sample holder by controlling the temperature of the contents of the sample holder (e.g., the sample and the refractive index matching medium) because the transparency of different tissue- cleared samples and RI media is temperature-dependent. By utilizing a temperature-controlled unit, it is possible to reduce light scattering and achieve higher resolution of images.

[0053] Referring now to FIG. 1, a control system is depicted for a microscope system and a temperature control unit configured to control the temperature of a sample in a sample holder to achieve improved transparency of the sample in accordance with one or more embodiment of the present disclosure.

[0054] In some embodiments, systems and methods of the present disclosure may employ a computer system 120 operably connected to a microscope system 110 and temperature control unit 130. The computer system 120 may control one or both of the microscope system 110 and temperature control unit 130 to generate two-dimensional and / or three-dimensional views of a sample held in a sample holder 140.

[0055] In some embodiments, the computing system 120 may include hardware components such as a processor 122, which may include local or remote processing components. In some embodiments, the processor 122 may include any type of data processing capacity, such as a hardware logic circuit, for example an application specific integrated circuit (ASIC) and a programmable logic, or such as a computing device, for example, a microcomputer or microcontroller that include a programmable microprocessor. In some embodiments, the processor 122 may include data-processing capacity provided by the microprocessor. In some embodiments, the microprocessor may include memory, processing, interface resources, controllers, and counters. In some embodiments, the microprocessor may also include one or more programs stored in memory.

[0056] Similarly, the computing system 120 may include storage 123, such as one or more local and / or remote data storage solutions such as, e.g., local hard-drive, solid-state drive, flash drive, database or other local data storage solutions or any combination thereof, and / or remote data storage solutions such as a server, mainframe, database or cloud services, distributed database orATTORNEY DOCKET NO. MDIBL-001PCT other suitable data storage solutions or any combination thereof. In some embodiments, the computing system 120 may alternatively or additionally include a memory 126, such as, e.g., a suitable non-transient computer readable medium such as, e.g., random access memory (RAM), read only memory (ROM), one or more buffers and / or caches, among other memory devices or any combination thereof.

[0057] In some embodiments, the computing system 120 may implement computer engines for detection control 123, e.g., of one or more detections of the microscope system 110, illumination control 124, e.g., of one or more illumination devices of the microscope system 110, temperature control 125 of the sample in the sample holder 140 via the temperature control unit 130, among other functions or any combination thereof. In some embodiments, the terms “computer engine” and “engine” identify at least one software component and / or a combination of at least one software component and at least one hardware component which are designed / programmed / configured to manage / control other software and / or hardware components (such as the libraries, software development kits (SDKs), objects, etc.).

[0058] Examples of hardware elements may include processors, microprocessors, circuits, circuit elements (e.g., transistors, resistors, capacitors, inductors, and so forth), integrated circuits, application specific integrated circuits (ASIC), programmable logic devices (PLD), digital signal processors (DSP), field programmable gate array (FPGA), logic gates, registers, semiconductor device, chips, microchips, chip sets, and so forth. In some embodiments, the one or more processors may be implemented as a Complex Instruction Set Computer (CISC) or Reduced Instruction Set Computer (RISC) processors; x86 instruction set compatible processors, multicore, or any other microprocessor or central processing unit (CPU). In various implementations, the one or more processors may be dual-core processor(s), dual-core mobile processor(s), and so forth.

[0059] Examples of software may include software components, programs, applications, computer programs, application programs, system programs, machine programs, operating system software, middleware, firmware, software modules, routines, subroutines, functions, methods, procedures, software interfaces, application program interfaces (API), instruction sets, computing code, computer code, code segments, computer code segments, words, values, symbols, or any combination thereof. Determining whether an embodiment is implemented using hardwareATTORNEY DOCKET NO. MDIBL-001PCT elements and / or software elements may vary in accordance with any number of factors, such as desired computational rate, power levels, heat tolerances, processing cycle budget, input data rates, output data rates, memory resources, data bus speeds and other design or performance constraints.

[0060] In some embodiments, the computing system 120 may include one or more computer engines for any one or more of the detection control 123, illumination control 124 and / or temperature control 125. In some embodiments, the computer engine(s) may include dedicated and / or shared software components, hardware components, or a combination thereof for each control function. For example, the detection control 123, illumination control 124 and / or temperature control 125 may include a dedicated processor and storage. However, in some embodiments, the detection control 123, illumination control 124 and / or temperature control 125 may share hardware resources, including the processor 122 and storage 123 of the computing system 120 via, e.g., a bus 127.

[0061] In some embodiments, the microscope system 110 may include any microscope suitable for enabling imaging of one or more tissue samples with magnification down to the cellular level or greater (e.g., cellular nuclei, mitochondria, and other sub-cell structures). The imaging may be optical. Accordingly, the microscope system 110 may provide imagery via ocular lenses to an eyepiece or to a digital imaging device (e.g., digital camera, charge coupled device (CCD), or other electronic imaging sensor), or both. In some embodiments, the microscope system 110 may provide imagery in the visible region of the electromagnetic spectrum or by any other portion of the electromagnetic spectrum.

[0062] In some embodiments, the microscope system 110 may include a simple microscope employing a single convex lens with a small focal length for magnification. The simple microscope creates a virtual, erect, and magnified image when the sample is placed in its focus. The sample may be illuminated by an illumination source, or may be illuminated by ambient lighting, or selfilluminating (e.g., via bioluminescence or other self-illumination).

[0063] In some embodiments, the microscope system 110 may include a compound microscope, which may use multiple lenses (e.g., an objective lens and an eyepiece) to magnify the sample. The objective lens collects light from the sample, e.g., via an illumination source, and the eyepiece further magnifies the image.ATTORNEY DOCKET NO. MDIBL-001PCT

[0064] In some embodiments, the microscope system 110 may include a phase contrast microscope to enhance the visibility of transparent or unstained specimens (e.g., a tissue cleared sample) by exploiting differences in refractive index. The phase contrast microscope may convert phase shifts caused by the specimen into contrast.

[0065] In some embodiments, the microscope system 110 may include a fluorescence microscope. The fluorescence microscope may use specific fluorescent dyes to label cellular components. The dyes emit light of a different color when excited by specific wavelengths, e.g., from an illumination source of the microscope system 110.

[0066] In some embodiments, the microscope system 110 may include one or more types of fluorescence microscopes, include a selective plane illumination microscope (SPIM), also termed a “light sheet microscope”. In some embodiments, light sheet microscopy is a powerful imaging method that enables high-resolution 3D imaging of biological samples. In some embodiments, unlike other microscopes, which use the illumination source to illuminate the entire sample, light sheet microscopy selectively illuminates a thin sheet within the sample. This thin sheet of laser light allows for relatively higher spatial resolution while minimizing photo-induced damage to the specimen. To produce a full image of a sample, an electronic imaging sensor of a detection unit may image the thin sheet of the sample. As the illumination control 124 illuminates successive sheets with the sample, the detection control 123 may image each sheet and reconstruct a full image by stitching the sheets together in order.

[0067] For example, the illumination beam may be perpendicular to the detection unit, creating a sheet of light through a specific two-dimensional plane of the sample. This imaging plane coincides with the focal plane, resulting in minimal excitation above or below the focal plane (e.g., minimal out-of-focus light). By capturing fluorescence from this specific plane and stitching together all planes across the sample, light sheet microscopy provides high-quality 3D images of the sample.

[0068] In some embodiments, whether simple, confocal, fluorescence or light sheet microscopy, the quality of the resulting image is affected by the transparency of the sample and the matching of refractive indices between materials in the sample holder 140 (e.g., the material of the sample holder 140 itself, fluid suspending the sample, the sample tissue, a refractive index matching medium, among other materials and / or substances or any combination thereof. The transparencyATTORNEY DOCKET NO. MDIBL-001PCT and refractive indices of each material may be affected by the temperature of the material. For example, a refractive index matching material and a tissue cleared sample may have refractive indices that more similar at particular temperatures and / or temperature ranges. Thus, by controlling the temperature of the sample and the refractive index matching medium, the similarity of the refractive indices of the sample and the refractive index matching medium may be made more similar, thus reducing light scattering and improving the resolution of the resulting images.

[0069] Typically, microscopes capable of imaging tissue-cleared samples do not have any means of temperature control for the refractive index matching medium or the sample. Instead, the samples and refractive index matching medium are typically imaged at room temperature where microscopes are located. This lack of temperature control can cause fluctuations in the refractive index matching medium and sample depending on the geographical location of the scientific facility and the season of the year. Furthermore, many research facilities have dedicated microscopy rooms that are cooled below room temperature (e.g., below 20°C) to lower the cooling burden of laser systems and working stations. Additionally, many microscopes exhibit lower fluorescent drift under cool conditions. The low ambient temperature in such dedicated facilities can have a negative effect on tissue transparency of tissue-cleared samples (see for example, FIGs. 7-12).

[0070] In some embodiments, to address these and other technical problems in microscopy, the microscope system 110 may be paired with a temperature control system dedicated to modulating the temperature of a sample holder 140 (e.g., chamber, vessel, slide, or other holder) having the tissue-cleared sample and the refractive index matching medium. Accordingly, the temperature control 125 of the computing system 120 may control the temperature control unit 130 to transfer heat to and / or from the sample holder 140 and thereby adjust the temperature of the contents of the sample holder 140, including the sample and the refractive index matching medium. As a result, the temperature of the sample holder 140 may be controlled to maintain an optimum temperature of the refractive index matching medium and sample for the highest tissue-transparency or while taking pictures with a camera.

[0071] In some embodiments, the optimum temperature may be a predetermined temperature set point at which the refractive index of the refractive index matching medium is within a predetermined degree of similarity to the refractive index of the tissue cleared sample. In someATTORNEY DOCKET NO. MDIBL-001PCT embodiments, the predetermined degree of similarity may be defined according to the difference between the refractive index of the sample and of the refractive index matching medium. In some embodiments, the predetermined degree of similarity may be defined according to a desired degree of light scattering and / or imaging resolution. For example, a technician may experimentally determine the relationship between temperature and the difference in refractive indices, the light scattering and / or the imaging resolution for a particular combination. Upon such experimentation, the technician may identify a desired temperature set point, e.g., by balancing improvements to imaging, risk of degradation to the sample, time and / or energy to achieve and maintain the temperature, among other factors. Dependent on the sample and the amount of clearing, the temperature set point should be in a range of 0 to 50 degrees Celsius. For example, for certain combinations of material, it has been found that the temperature may be in a range of 19 to 26 degrees Celsius (see, for example, FIGS. 7-11 below.

[0072] In some embodiments, attributes of the tissue, the tissue clearing substance and / or the refractive index matching medium may be correlated to the affect of temperature on refractive index matching, light scattering, and / or transparency. Accordingly, in some embodiments, the computing system 120 may include one or more machine learning models trained on training data from study of such affects such that the machine learning model(s) may predict, for any particular combination, the optimum temperature for fluorescence and / or optical microscopy.

[0073] In some embodiments, the machine learning model(s) may predict an optimal temperature (in degrees Celsius and / or Fahrenheit) for fluorescence and / or optical microscopy associated with the user. To do so, the machine learning model(s) ingests a feature vector that encodes features representative of one or more characteristics of the tissue of the sample, the tissue clearing substance and / or the refractive index matching medium. In some embodiments, the machine learning model(s) processes the feature vector with parameters to produces a prediction of optimal temperature for fluorescence and / or optical microscopy. In some embodiments, the parameters of the machine learning model(s) may be implemented in a suitable machine learning model including a prediction machine learning model, such as, e.g., Linear Regression, Logistic Regression, Ridge Regression, Lasso Regression, Polynomial Regression, Bayesian Linear Regression (e.g., Naive Bayes regression), a convolutional neural network (CNN), a recurrent neural network (RNN), decision trees, random forest, support vector machine (SVM), K-Nearest Neighbors, or any other suitable algorithm for predicting output values based on input values. InATTORNEY DOCKET NO. MDIBL-001PCT some embodiments, for computational efficiency while preserving accuracy of predictions, the machine learning model(s) may advantageously include a random forest model.

[0074] In some embodiments, the machine learning model(s) processes the features encoded in the feature vector by applying the parameters of the prediction machine learning model to produce a model output vector. In some embodiments, the model output vector may be decoded to generate one or more numerical output values indicative of optimal temperature for fluorescence and / or optical microscopy. In some embodiments, the model output vector may include or may be decoded to reveal the output value(s) based on a modelled correlation between the feature vector and a target output. In some embodiments, the numerical output may represent optimal temperature (in degrees Celsius and / or Fahrenheit) for fluorescence and / or optical microscopy.

[0075] In some embodiments, the parameters of the machine learning model(s) may be trained based on known outputs. For example, the one or more characteristics of the tissue of the sample, the tissue clearing substance and / or the refractive index matching medium may be paired with a target value or known value to form a training pair, such as a historical one or more characteristics of the tissue of the sample, the tissue clearing substance and / or the refractive index matching medium and an observed result and / or human annotated value representing a data point in the relationship between the historical one or more characteristics of the tissue of the sample, the tissue clearing substance and / or the refractive index matching medium and optimal temperature (in degrees Celsius and / or Fahrenheit) for fluorescence and / or optical microscopy. In some embodiments, the one or more characteristics of the tissue of the sample, the tissue clearing substance and / or the refractive index matching medium may be provided to the machine learning model(s), e.g., encoded in a feature vector, to produce a predicted output value. In some embodiments, an optimizer associated with the machine learning model(s) may then compare the predicted output value with the known output of a training pair including the historical one or more characteristics of the tissue of the sample, the tissue clearing substance and / or the refractive index matching medium to determine an error of the predicted output value. In some embodiments, the optimizer may employ a loss function, such as, e.g., Hinge Loss, Multi-class SVM Loss, Cross Entropy Loss, Negative Log Likelihood, or other suitable classification loss function to determine the error of the predicted output value based on the known output.ATTORNEY DOCKET NO. MDIBL-001PCT

[0076] In some embodiments, the known output may be obtained after the machine learning model(s) produces the prediction, such as in online learning scenarios. In such a scenario, the machine learning model(s) may receive the one or more characteristics of the tissue of the sample, the tissue clearing substance and / or the refractive index matching medium and generate the model output vector to produce an output value representing optimal temperature (in degrees Celsius and / or Fahrenheit) for fluorescence and / or optical microscopy. Subsequently, a user may provide feedback by, e.g., modifying, adjusting, removing, and / or verifying the output value via a suitable feedback mechanism, such as a user interface device (e.g., keyboard, mouse, touch screen, user interface, or other interface mechanism of a user device or any suitable combination thereof). The feedback may be paired with the one or more characteristics of the tissue of the sample, the tissue clearing substance and / or the refractive index matching medium to form the training pair and the optimizer may determine an error of the predicted output value using the feedback.

[0077] In some embodiments, based on the error, the optimizer may update the parameters of the machine learning model(s) using a suitable training algorithm such as, e.g., backpropagation for a prediction machine learning model. In some embodiments, backpropagation may include any suitable minimization algorithm such as a gradient method of the loss function with respect to the weights of the prediction machine learning model. Examples of suitable gradient methods include, e.g., stochastic gradient descent, batch gradient descent, mini-batch gradient descent, or other suitable gradient descent technique. As a result, the optimizer may update the parameters of the machine learning model(s) based on the error of predicted labels in order to train the machine learning model(s) to model the correlation between one or more characteristics of the tissue of the sample, the tissue clearing substance and / or the refractive index matching medium and optimal temperature for fluorescence and / or optical microscopy in order to produce more accurate output values based on one or more characteristics of the tissue of the sample, the tissue clearing substance and / or the refractive index matching medium.

[0078] As detailed above, the optimum temperature may depend on the refractive index matching medium, the tissue of the sample, and / or the substance used to tissue clear the sample, or any combination thereof. Accordingly, the temperature control 125 may be preconfigured to achieve the predetermined temperature set point associated with a particular combination of tissue cleared sample and refractive index matching medium, e.g., based on prior study of the combination to obtain the desired light scattering and resolution of imaging of the combination, machine learning-ATTORNEY DOCKET NO. MDIBL-001PCT based prediction, and / or any other algorithmic and / or statistical determination or any combination thereof. In some embodiments, the predetermined temperature set point may be a user- configurable parameters that a user may establish before or during imaging of the sample.

[0079] For example, temperature configurations for one or more samples, refractive index matching media, or other materials or any combination therefore may be stored in the storage 123 and / or memory 126 of the computing system 120. Thus, when microscope imaging is initiated, the computing system 120 may initiate temperature control 125 via the temperature control unit 130 based on attributes of the microscope imaging task, including, e.g., the sample being imaged, the tissue clearing substance and / or the refractive index matching medium. Based on the temperature configurations stored in memory 126 and / or storage 123, the temperature control 125 may use the attributes of the microscope imaging task to determine the predetermined set point that is optimal for the sample. As a result, the temperature control 125 may automatically determine the predetermined temperature set point based on the attributes.

[0080] In some embodiments, the attributes may be provided by user input, e.g., via a computer input mechanism such as touch screen, mouse, keyboard, touch pad, gesture control, voice control (e.g., artificial intelligence or other voice control), among other input mechanisms or any combination thereof. In some embodiments, the attributes may be provided via encoded indicia associated with the sample and / or sample holder 140. For example, a machine readable code may be printed or otherwise affixed to the sample holder or a container holding the sample prior to initiating the microscope imaging. The machine readable code may encode the attributes and / or one or more references that are configured to be used to query for the attributes, e.g., in the storage 123 and / or memory 126 or other database. For example, the machine readable code may be a bar code, quick reference (QR) code, near field communication (NFC) tag, radio frequency identification (RFID) tag, or other medium of encoding data or any combination thereof. Thus, the computing system 120 may read the machine readable code, e.g., via a code reader such a barcode or QR code scanning device (e.g., image sensor, digital imaging or camera software functionality, among others or any combination thereof), an NFC and / or RFID reader (passive, active or any combination thereof), etc. As a result, the temperature control 125 may decode the machine readable code to obtain the attributes and / or one or more references, and thereby determine the predetermined temperature set point for the microscope imaging task.ATTORNEY DOCKET NO. MDIBL-001PCT

[0081] In some embodiments, the temperature control unit 130 may include a temperature feedback mechanism such a temperature sensor, thermometer, thermistor, or other temperature detection device for feeding back a current temperature to the computing system 120. In some embodiments, the temperature feedback mechanism may be attached to, inserted in or otherwise positioned so as to detect the temperature of the sample holder 140, sample, and / or refractive index matching medium. In some embodiments, the temperature feedback mechanism may be a part of the temperature control unit 130, e.g., as an integrated device, or may be separate from the temperature control unit 130 to result in two or more separate components.

[0082] Accordingly, the temperature control 125 may receive from the temperature feedback mechanism a current temperature of the sample holder 140, sample, and / or refractive index matching medium. The temperature control 125 may obtain the current temperature continuously, periodically, intermittently, or on any regular or irregular cadence to update the current temperature of the sample holder 140, sample, and / or refractive index matching medium. Based on the current temperature or a difference between the current temperature and the predetermined temperature set point, the temperature control 125 may determine an amount of heat to set the temperature control unit 130 to produce so as to module the temperature control unit 130 to control the transfer of heat to the sample chamber to achieve the predetermined temperature set point.

[0083] In some embodiments, the temperature control 125 may modulate the temperature control unit 130 using control signals. The control signals may define an intensity of heat, an energy or power of heat, a frequency, or other control parameters or any combination thereof. For example, the temperature control unit 130 may include a device configured to output heat in response to an electrical control signal. The device may include, e.g., a resistive heating element, a positive temperature coefficient (PTC) element, an inductive heating component, a radiative heating device (e.g., an infrared lamp, a laser, or other radiation-based heating device or any combination thereof), a gas powered heating apparatus or other flame or combustion based heating apparatus, among other heat sources controllable via electronic signaling, or any combination thereof.

[0084] The computing system 120 may employ the temperature control 125 to send and / or modulate control signals to the temperature control unit 130 to adjust the heat output by the temperature control unit 130, and thereby modulate the temperature of the contents of the sample holder 140 (e.g., the sample and / or refractive index matching medium). In some embodiments, theATTORNEY DOCKET NO. MDIBL-001PCT control signals may include pulse width modulation, frequency modulation, amplitude modulation, or other suitable modulation technique.

[0085] In some embodiments, the control methodology may include providing a control signal instructing the temperature control unit 130 to output heat at full power until the predetermined temperature set point is achieved. In some embodiments, the control signal may instruct the temperature control unit 130 to reduce heat output as the predetermined temperature set point is approached in power steps and / or a continuous progression reducing power. In some embodiments, the control signal may instruct the temperature control unit 130 to produce heat at a power dependent on the difference between the current temperature and the predetermined temperature set point. Other control methodologies are contemplated.

[0086] In some embodiments, the temperature control 125 and the temperature control unit 130 may be configured to maintain the optimal temperature for the use in the microscope system 110, in light transmittance and haze-measuring machines such as a haze meter to measure and determine the optimum transparency for different tissue-cleared samples, or in any other device and / or system to controllably adjust the refractive indices of materials via temperature control, or any combination thereof.

[0087] Thus, the temperature control 125 of the computing system 120 may control the temperature control unit 130 to transfer heat to the sample holder 140 so as to achieve a predetermined temperature set point in the sample and / or refractive index matching medium. As a result, the microscope imaging, whether via fluorescence and / or optical illumination, may be improved via improved transparency with reduced light scattering, thus resulting in increased effective resolution of the resulting images.

[0088] Referring now to FIG. 2, a control system and temperature control unit are depicted for a light-sheet microscope system with control of the temperature of a sample in a sample holder are depicted to achieve improved transparency of the sample in accordance with one or more embodiment of the present disclosure.

[0089] In some embodiments, the microscope system 110 may include a light sheet microscope. Thus, the microscope system 110 uses light-sheet microscopy technology that, because only a thin section (for example, on the order of a micrometer (pm) wide taken along the z axis) of the sampleATTORNEY DOCKET NO. MDIBL-001PCT201 is illuminated at a time with a scanned sheet of laser light while a detector records the part of the sample 201 that is being illuminated, damage to the sample 201 is reduced.

[0090] In general, the microscope system 110 is made up of a plurality of light sheets (for example, light sheet 202) that illuminate the sample 201 from a distinct direction along a respective light sheet axis, and a detection mechanism 216 collects the resulting fluorescence along a plurality of detection views. In particular, an illumination mechanism 212 may direct a light sheet 202 to illuminate the sample 201 from a light sheet axis, and the detection mechanism 216 collects the resulting fluorescence along a detection view 203.

[0091] Each light sheet 202 is generated with the illumination mechanism 212 including, e.g., a static light sheet or a laser scanner that moves a thin (for example, a pm-thick or less) beam of laser light along an illumination axis, which is perpendicular to the detection axis of the detection view 203, to form a light beam that extends generally along or parallel with a plane to form the sheet 202. The laser beam in the form of the light sheet 202 illuminates the sample 201 along the illumination axis on a side of the sample 201. Rapid scanning of a thin volume and fluorescence detection at a right angle to the illumination axis provides an optically sectioned image. The light sheet 202 excites fluorophores within the sample 201 into higher energy levels, which then results in the subsequent emission of a fluorescence photon P, and the fluorescence photons P are detected by the detectors within the detection mechanism 216. In some embodiments, the excitation is one- photon excitation, or it is multi-photon (for example, two-photon) excitation.

[0092] The fluorophores that are excited in the specimen can be labels that are attached to the cells, such as, for example, genetically-encoded fluorescent proteins such as GFP or dyes such as Alexa-488. However, the fluorophores can, in some implementations that use second-harmonic generation or third-harmonic generation, be actual or native proteins within the cells that emit light of specific wavelengths upon exposure with the light sheet 202.

[0093] The light sheet 202 passes through the sample 201 and excite the fluorophores. However, the light sheet 202 is subject to light scattering and light absorption along their respective paths through the sample 201. Moreover, very large (large compared with the image volume IV or the field-of-view (FOV)) or fairly opaque specimens can absorb energy from the light sheet 202.

[0094] Moreover, if the light sheet 202 are implemented in a two-photon excitation scheme, then only the central region 103 of the overlapping light sheet 202 may have a high enough powerATTORNEY DOCKET NO. MDIBL-001PCT density to efficiently trigger the two-photon process, and it is possible that only (the close) half of the sample 201 emits fluorescence photons P in response to exposure to two-photon light sheet 202.

[0095] Accordingly, the light sheet 202 may more effectively excite fluorophores throughout a thickness of the sample 201 where the sample 201 is transparent. Indeed, improving the transparency may improve the excitation of fluorophores. Moreover, improved transparency may make the detection of the fluorophores by the detection mechanism 216 more effective. As a result, improved transparency results in improved fidelity of the detection of the fluorophores by improving the excitation of the fluorophores and improving the ability to detect the fluorophores.

[0096] In some embodiments, as detailed above, the sample 201 may be transparent as a result of tissue clearing of a macroscopic object, such as an organ or anatomical part of an animal under study. The sample 201 may be immersed in a refractive index matching medium within a sample holder 140 including a chamber or container. The refractive index matching medium is selected to have a refractive index matching to the tissue of the sample 201 to provide uniform refractive indices throughout all materials in the sample holder 140. Nevertheless, there may be differences in the refractive index of the refractive index matching medium and the refractive index of the tissue of the sample. Even where the differences may seem negligible, such differences may impose limits on the effective resolution of an image of the sample 201 by the detection mechanism 216 that is less than the resolution that the detection mechanism 216 is capable of detecting because of light scattering resulting from the differences in refractive indices.

[0097] As detailed above, the transparency and refractive indices of each material may be affected by the temperature of the material. For example, a refractive index matching material and a tissue cleared sample may have refractive indices that are more similar at particular temperatures and / or temperature ranges. Thus, by controlling the temperature of the sample and the refractive index matching medium, the similarity of the refractive indices of the sample and the refractive index matching medium may be made more similar, thus reducing light scattering and improving the resolution of the resulting images.

[0098] Typically, microscopes capable of imaging tissue-cleared samples do not have any means of temperature control for the refractive index matching medium or the sample. Instead, the samples and refractive index matching medium are typically imaged at room temperature whereATTORNEY DOCKET NO. MDIBL-001PCT microscopes are located. This lack of temperature control can cause fluctuations in the refractive index matching medium and sample depending on the geographical location of the scientific facility and the season of the year. Furthermore, many research facilities have dedicated microscopy rooms that are cooled below room temperature (e.g., below 20°C) to lower the cooling burden of laser systems and working stations. Additionally, many microscopes exhibit lower fluorescent drift under cool conditions. The low ambient temperature in such dedicated facilities can have a negative effect on tissue transparency of tissue-cleared samples.

[0099] In some embodiments, to address these and other technical problems in microscopy, the microscope system 110 may be paired with a temperature control system dedicated to modulating the temperature of a sample holder 140 (e.g., chamber, vessel, slide, or other holder) having the tissue-cleared sample and the refractive index matching medium. Accordingly, the temperature control 125 of the computing system 120 may control the temperature control unit 130 to transfer heat to and / or from the sample holder 140 and thereby adjust the temperature of the contents of the sample holder 140, including the sample 201 and the refractive index matching medium. As a result, the temperature of the sample holder 140 may be controlled to maintain an optimum temperature of the refractive index matching medium and sample for the highest tissuetransparency or while taking pictures with a camera.

[0100] In some embodiments, the optimum temperature may be a predetermined temperature set point at which the refractive index of the refractive index matching medium is within a predetermined degree of similarity to the refractive index of the tissue cleared sample. In some embodiments, the predetermined degree of similarity may be defined according to the difference between the refractive index of the sample and of the refractive index matching medium. In some embodiments, the predetermined degree of similarity may be defined according to a desired degree of light scattering and / or imaging resolution. For example, a technician may experimentally determine the relationship between temperature and the difference in refractive indices, the light scattering and / or the imaging resolution for a particular combination. Upon such experimentation, the technician may identify a desired temperature set point, e.g., by balancing improvements to imaging, risk of degradation to the sample, time and / or energy to achieve and maintain the temperature, among other factors.ATTORNEY DOCKET NO. MDIBL-001PCT

[0101] Thus, as detailed above, the temperature control 125 of the computing system 120 may control a temperature control unit 130 associated with microscope system 110 to provide heat to the sample 201 and improve transparency.

[0102] The microscope system 110 also includes or is otherwise associated with the computing system 120. The computing system 120 includes a detection control 123 and an illumination control 124 that provide synchronized control of all opto-mechanical components within the microscope 110 with millisecond precision over long periods of time, including that of the detection mechanism 216 and the illumination mechanism, respectively. Thus, the computing system 120 performs the optical alignment on the sample 201, and provides a robust pipeline for simultaneous high-speed image acquisition with a plurality of detectors (or cameras) within the detection mechanism 216 for efficient automated image processing to register and reconstruct the image data arising from every experiment.

[0103] The detection mechanism 216 may include a respective detector or camera in addition to a set of detection optical devices arranged to collect and record the fluorescence emitted from the sample 201. Each detection mechanism 216 may also include a set of actuators that are coupled to one or more of the detectors and the detection optical devices.

[0104] Referring now to FIG. 3, a temperature control unit is depicted positioned on a sample holder to control the temperature of a sample in a sample holder to achieve improved transparency of the sample in accordance with one or more embodiment of the present disclosure.

[0105] In some embodiments, the temperature control unit 130 may transfer heat to the sample holder 140 via contact with the sample holder 140. For example, the temperature control unit 130 may include a resistive heating element and / or PTC element. Thus, the temperature control unit 130 may include materials such as a metal coil or strip, a ceramic coil or strip, or other element for converting electrical energy to heat, e.g., via resistance through the element. Such a temperature control unit 130 may be most effective via direct contact with the sample holder 140. Accordingly, the temperature control unit 130 may be attached directly or indirectly to the sample holder 140.

[0106] In some embodiments, the temperature control unit 130 may be attached to the sample holder 140 via an adhesive, such as a high temperature adhesive such as, a temperature resistant epoxy, flue tape, silicone adhesive among other high-temperature adhesive materials. TheATTORNEY DOCKET NO. MDIBL-001PCT temperature control unit 130 may adhered to a surface of the sample holder 140 using the high temperature adhesive.

[0107] In some embodiments, the temperature control unit 130 may be attached to the sample holder 140 via a holder, clamp or frame in which the coil is housed. The holder, clamp or frame may then be attached to the sample holder 140, e.g., using mechanical fasteners, such as, using screws, clamps, or other types of fasteners or any combination thereof.

[0108] In some embodiments, the temperature control unit 130 may be attached to the sample holder 140 embedding the temperature control unit 130 into a portion of the sample holder 140. For example, during formation of the sample holder 140, such as the injection molding of a plastic or molding of a plastic or glass, the temperature control unit 130 may be incorporated into an area within the material of the sample holder 140. Thus, the temperature control unit 130 may be made integral with the sample holder 140.

[0109] In some embodiments, as depicted in FIG. 3, the temperature control unit 130 may be attached to an underside of the sample holder 140. In addition or in the alternative, the temperature control unit 130 may be attached to a side of the sample holder 140, such as one or more sides of a polygonal sample holder, or in one or more positions on a cylindrical wall of the sample holder 140. In some embodiments, the temperature control unit 130 is depicted in FIG. 3 as being a single unit, though more than one temperature control unit 130 may be attached to the sample holder 140. For example, multiple temperature control units 130 may be attached to the sample holder 140 so as to reduce convective currents within the refractive index matching medium by more uniformly heating the contents of the sample holder 140.

[0110] In some embodiments, by attaching the temperature control unit 130 to the sample holder 140, the sample holder 140 may be produced as a modified component for a standard microscope system 110, thereby reducing the costs of incorporating the temperature control unit 130 into the microscope system 110. The sample holder 140 may be placed on a stage 341 of the microscope system 110 so as to bring the sample holder 140 and sample into alignment with, e.g., the illumination mechanism 212 and the detection mechanism 216 and / or other microscope imaging components for imaging.

[0111] Referring now to FIG. 4, a temperature control unit is depicted positioned on a stage to contact a sample holder when positioned for microscopy to control the temperature of a sample inATTORNEY DOCKET NO. MDIBL-001PCT the sample holder to achieve improved transparency of the sample in accordance with one or more embodiment of the present disclosure.

[0112] In some embodiments, the temperature control unit 130 may transfer heat to the sample holder 140 via contact or proximity with the sample holder 140. For example, the temperature control unit 130 may include a resistive heating element and / or PTC element that may conduct heat into the sample holder 140 via contact. Thus, the temperature control unit 130 may include materials such as a metal coil or strip, a ceramic coil or strip, or other element for converting electrical energy to heat, e.g., via resistance through the element. Such a temperature control unit 130 may be most effective via direct contact with the sample holder 140. Accordingly, the temperature control unit 130 may be attached directly or indirectly to the sample holder 140.

[0113] In another example, the temperature control unit 130 may be an inductive heating element that may excite electrical energy within the sample holder 140, such as by inducting a current within a metal component, plate or coil in a portion of the sample holder 140. Thus, the temperature control unit 130 may be positioned such that during imaging the temperature control unit 130 is in sufficiently near proximity to the sample holder 140 to induce the electrical current and produce heat in the sample holder.

[0114] In some embodiments, the temperature control unit 130 may be attached to a stage 442 of the microscope system 110 so as to position the temperature control unit 130 to be in a position matching the placement of the sample holder 140 during imaging. Accordingly, the temperature control unit 130 may be attached to the stage 442 via an adhesive, such as a high temperature adhesive such as, a temperature resistant epoxy, flue tape, silicone adhesive among other high- temperature adhesive materials. The temperature control unit 130 may adhered to a surface of the sample holder 140 using the high temperature adhesive.

[0115] In some embodiments, the temperature control unit 130 may be attached to the stage 442 via a holder, clamp or frame in which the coil is housed. The holder, clamp or frame may then be attached to the stage 442, e.g., using mechanical fasteners, such as, using screws, clamps, or other types of fasteners or any combination thereof.

[0116] In some embodiments, the temperature control unit 130 may be attached to the stage 442 by embedding the temperature control unit 130 into a portion of the stage 442. For example, during formation of the stage 442, such as the injection molding of a plastic or molding of a plastic, metalATTORNEY DOCKET NO. MDIBL-001PCT or glass, the temperature control unit 130 may be incorporated into an area within the material of the stage 442. Thus, the temperature control unit 130 may be made integral with the stage 442.

[0117] In some embodiments, as depicted in FIG. 4, the temperature control unit 130 may be attached to a surface of the stage 442. In addition or in the alternative, the temperature control unit 130 may be attached to an underside of the stage 442. In some embodiments, the temperature control unit 130 is depicted in FIG. 4 as being a single unit, though more than one temperature control unit 130 may be attached to the stage 442. For example, multiple temperature control units 130 may be attached to the stage 442 so as to reduce convective currents within the refractive index matching medium by more uniformly heating the contents of the sample holder 140.

[0118] The sample holder 140 may be placed on a stage 442 of the microscope system 110 so as to bring the sample holder 140 and sample into alignment with, e g., the illumination mechanism 212 and the detection mechanism 216 and / or other microscope imaging components for imaging while bringing the sample holder 140 into proximity and / or contact with the temperature control unit 130 so as to enable the temperature control unit 130 to transfer heat to the sample holder 140 before and / or during imaging.

[0119] Referring now to FIG. 5, a flowchart is depicted illustrating a control methodology for operating a temperature control unit to control a temperature of a sample in the sample holder to achieve improved transparency of the sample in accordance with one or more embodiment of the present disclosure.

[0120] As detailed above, a temperature control 125 of a computing system 120 may provide control signals according to a control methodology to actuate a temperature control unit to provide heat to a sample, sample holder and / or refractive index matching medium. The control methodology may be configured to achieve a predetermined temperature set point associated with improved or optimal refractive index matching between a tissue cleared sample and the refractive index matching medium.

[0121] In some embodiments, the temperature control methodology may include performing one or more operations to modulate the heat transferred from the temperature control unit 130 to the sample, sample holder and / or refractive index matching medium.ATTORNEY DOCKET NO. MDIBL-001PCT

[0122] Step 501 of the control methodology includes identifying the sample material and / or refractive index matching medium.

[0123] Step 502 of the control methodology includes determining a temperature set point to match refractive indices. Such temperature set point may be material-specific, in that the refractive index of the sample and / or the refractive index matching medium may behave differently in response to temperature. Thus, the predetermined temperature set point may be the temperature at which the refractive index of the refractive index matching medium is within a predetermined degree of similarity to the refractive index of the tissue cleared sample. In some embodiments, the predetermined degree of similarity may be defined according to the difference between the refractive index of the sample and of the refractive index matching medium. In some embodiments, the predetermined degree of similarity may be defined according to a desired degree of light scattering and / or imaging resolution. For example, a technician may experimentally determine the relationship between temperature and the difference in refractive indices, the light scattering and / or the imaging resolution for a particular combination. Upon such experimentation, the technician may identify a desired temperature set point, e.g., by balancing improvements to imaging, risk of degradation to the sample, time and / or energy to achieve and maintain the temperature, among other factors.

[0124] Step 503 of the control methodology optionally includes, upon determining the temperature set point, determining a current temperature of contents of the sample holder. The control methodology may achieve the temperature set point based on feedback from a temperature feedback mechanism, or may be achieve the temperature by other techniques, e.g., an algorithm modelling the change in temperature of the contents of the sample holder 140 in response to a particular heat output of the temperature control unit 130.

[0125] Step 504 of the control methodology includes modulating temperature control unit to transfer heat to the sample holder. In some embodiments, the modulation may be performed based on feedback from the temperature feedback mechanism where used, or may omit the temperature feedback mechanism.

[0126] As detailed above, to modulate the temperature control unit, the control methodology may include defining an intensity of heat, an energy or power of heat, an electrical energy or electrical power provided to the temperature control unit, a frequency of activation of the temperature controlATTORNEY DOCKET NO. MDIBL-001PCT unit, or other control parameters or any combination thereof of the temperature control unit. In some embodiments, the control signals may include pulse width modulation, frequency modulation, amplitude modulation, or other suitable modulation technique.

[0127] For example, the temperature control unit 130 may include a device configured to output heat in response to an electrical control signal. The device may include, e.g., a resistive heating element, a positive temperature coefficient (PTC) element, an inductive heating component, a radiative heating device (e.g., an infrared lamp, a laser, or other radiation-based heating device or any combination thereof), a gas powered heating apparatus or other flame or combustion based heating apparatus, among other heat sources controllable via electronic signaling, or any combination thereof.

[0128] Step 505 of the control methodology includes controlling the temperature control unit to maintain the temperature for the duration of imaging. Maintaining the temperature may include providing a constant heat output, e.g., via the control parameters of the temperature control unit where the heat output is associated with the temperature set point (e.g., based on an algorithm as detailed above). In some embodiments, the control methodology may include adjusting the heat output based on a current state of the temperature control unit and / or sample holder, such as, e.g., a temperature measured at or in the sample holder. Based on the measured temperature, the control methodology may include further modulating (e.g., at step 504) the temperature control unit to adjust to any changes in the temperature.

[0129] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the embodiments, and are not intended to limit the scope of what the inventors regard as their embodiments.EXAMPLES

[0130] One or more examples of embodiment of the present disclosure are depicting in FIGs. 6 through 12. FIGs. 6 through 12 depict the signal-to-noise ratio being transparency-dependent (see FIGs. 7A, 7B, 7C and 7D). Furthermore, FIGs. 6 through 12 depict evidence that the optimum transparency and light transmission of a refractive index matching medium and tissue specimens are temperature-dependent (see FIGs. 7 through 11). Finally, FIGs. 6 through 12 illustrate thatATTORNEY DOCKET NO. MDIBL-001PCT autofluorescence of a tissue-cleared sample can be influenced by altering the temperature (see Fig. 12), which can also significantly improve imaging outcomes.

[0131] Referring now to FIGs. 6A-H, a temperature modulation unit for tissue-cleared samples is depicted in accordance with one or more embodiment of the present disclosure. FIG. 6A shows a Custom chamber holder. FIG. 6B shows Heat-Cold-Pad mounted on a copper heatsink. FIG. 6C shows a Heat-Cold-Pad from the heat-cold pad mounted in the chamber holder. FIG. 6D shows a temperature modulation chamber. The arrow is pointing at the thermo-sensor adhered to the chamber (1) and temperature control unit (2). FIG. 6F shows a mesoSPIM light-sheet. Inset shows tissue-cleared Thyl-EGFP-M mouse brain, with light-sheet illuminating a single optical layer. FIG. 6G shows a Zeiss AxioZoom Stereoscope with a tissue-cleared moused brain. FIG. 6H shows a haze meter with a tissue-cleared sample. FIGs. 6F-G show a system equipped with the temperature-controlled unit depicted in FIG. 6E. Dashed rectangle areas show the corresponding parts of the apparatus shown in FIG. 6E.

[0132] Referring now to FIGs. 7A-D, the effect of temperature on tissue transparency and image quality is depicted in accordance with one or more embodiment of the present disclosure. FIG. 7A shows a brightfield and darkfield image of the same non-uniform tissue-cleared Thyl-EGFP-M mouse brain imaged on Zeiss AxioZoom stereoscope (Zoom 11, 2x; N.A. 0,2). Dashed rectangle areas indicate regions with different tissue transparency which (1) has high transparency and (2) low transparency. Inset (1) and (2) show high resolution light-sheet images, recorded on a temperature controlled mesoSPIM (12x, N.A. 0,53), of rectangle areas in xy (axial resolution) and two different areas in xz (lateral resolution). FIG. 7B shows a brightfield (left) and darkfield (right) images of the same uniformly tissue-cleared Thyl-EGFP-M mouse brain imaged on Zeiss AxioZoom stereoscope (Zoom 11, 2x; N.A. 0,2) showing impact of temperature on tissue transparency. FIG. 7C and 7D show light-sheet images, recorded on a temperature controlled mesoSPIM (12x, N.A. 0,53) of the mouse brain displayed in FIG. 7B from all spatial direction xy (lateral resolution), xz and yz (axial resolution) showing impact of temperature on transparency and image quality in respect to sharpness, signal to noise ratio and signal brightness. Insets in FIG. 7C show higher magnification of Dashed rectangle areas.

[0133] Referring now to FIGs. 8A-D, the effect of temperature on light transmission of tissue- cleared mouse brains is depicted in accordance with one or more embodiment of the presentATTORNEY DOCKET NO. MDIBL-001PCT disclosure. FIG. 8 A show a tissue-cleared mouse brains under different temperature, Refractive Index (RI) matching media- and used dehydration agent conditions. FIG. 8B illustrates the effect of temperature on total transmission of tissue-cleared brain samples. All values are mean ± SD; statistical significance was determined by non-parametric paired t-test. FIG. 8C depicts quantification and comparison of best performing dehydration agent and RI matching media from the respective group in FIG. 8B and the optimum temperature for highest total transmission. All values are mean ± SD; statistical significance was determined by non-parametric unpaired t-test. FIG. 8C shows the effect of temperature on Red-Green-Blue (RGB)-Transmission (same color code on Heatmap) of tissue-cleared brains. In FIGs. 8C and 8D, ns P > 0.05, * P < 0.05. ** P < 0.01, *** P < 0.001, **** P < 0.0001 Total Transmission (TT), Haze (H), Ethyl Cinnamate (ECi), Dibenzylether (DBE), 1 :2 ratio of Benzyl alcohol in Benzoate (BABB), Methanol (MeOH), Ethanol (EtOH), tertiary-Butanol (tert-But), Tetrahydrofuran (THF).

[0134] Referring now to FIGs. 9A-C, the effect of temperature on transparency / haze of tissue- cleared mouse brain is depicted in accordance with one or more embodiment of the present disclosure. FIG. 9A shows a tissue-cleared mouse brains under different temperature-, Refractive Index (RI) matching media- and used dehydration agent conditions. FIG. 9B shows the effect of temperature on transparency of tissue-cleared brain samples. All values are mean ± SD; statistical significance was determined by non-parametric paired t-test. FIG. 9C shows a quantification and comparison of best performing dehydration agent and RI matching media from the respective group in FIG. 9B and the optimum temperature for highest transparency. All values are mean ± SD; statistical significance was determined by non-parametric unpaired t-test in FIGs. 9B and 9C, ns P > 0.05, * P < 0.05. ** P < 0.01, *** P < 0.001, **** P < 0.0001 Total Transmission (TT), Haze (H), Ethyl Cinnamate (ECi), Dibenzylether (DBE), 1 :2 ratio of Benzyl alcohol in Benzoate (BABB), Methanol (MeOH), Ethanol (EtOH), tertiary-Butanol (tert-But), Tetrahydrofuran (THF).

[0135] Referring now to FIGs. 10A-D, the effect of temperature on light transmission of tissue- cleared mouse kidneys is depicted in accordance with one or more embodiment of the present disclosure. FIG. 10A shows tissue-cleared mouse kidneys under different temperature-, Refractive Index (RI) matching media- and used dehydration agent conditions. FIG. 10B shows the effect of temperature on total transmission of tissue-cleared brain samples. All values are mean ± SD; statistical significance was determined by non-parametric paired t-test. FIG. 10C shows a quantification and comparison of best performing dehydration agent and RI matching media fromATTORNEY DOCKET NO. MDIBL-001PCT the respective group in FIG. 10B and the optimum temperature for highest total transmission. All values are mean ± SD; statistical significance was determined by non-parametric unpaired t-test. FIG. 10D shows a temperature impact on Red-Green-Blue (RGB)-Transmission (same color code on Heatmap) of tissue-cleared brains. In FIG. 10B and 10C, ns P > 0.05, * P < 0.05. ** P < 0.01, *** p < 0.001, **** p < 0.0001. Total Transmission (TT), Haze (H), Ethyl Cinnamate (ECi), Dibenzylether (DBE), 1 :2 ratio of Benzyl alcohol in Benzoate (BABB), Methanol (MeOH), Ethanol (EtOH), tertiary-Butanol (tert-But), Tetrahydrofuran (THF).

[0136] Referring now to FIGs. 11A-C, the effect of temperature on transparency / haze of tissue- cleared mouse kidneys is depicted in accordance with one or more embodiment of the present disclosure. FIG. 11 A shows tissue-cleared mouse kidneys under different temperature-, Refractive Index (RI) matching media- and used dehydration agent conditions. FIG. 11B shows the effect of temperature on transparency of tissue-cleared kidney samples. All values are mean ± SD; statistical significance was determined by non-parametric paired t-test. FIG. 11C shows a quantification and comparison of best performing dehydration agent and RI matching media from the respective group in FIG. 1 IB and the optimum temperature for highest transparency. All values are mean ± SD; statistical significance was determined by non-parametric unpaired t-test. In (B) and (C) ns P > 0.05, * P < 0.05. ** P < 0.01, *** P < 0.001, **** P < 0.0001. Total Transmission (TT), Haze (H), Ethyl Cinnamate (ECi), Dibenzylether (DBE), 1 :2 ratio of Benzyl alcohol in Benzoate (BABB), Methanol (MeOH), Ethanol (EtOH), tertiary-Butanol (tert-But), Tetrahydrofuran (THF).

[0137] Referring now to FIGs. 12A-E, the effect of temperature on autofluorescence of tissue- cleared mouse tissue is depicted in accordance with one or more embodiment of the present disclosure. FIGs. 12A, 12B, 12C and 12D are associated with autofluorescence recorded on a Zeiss AxioZoom stereoscope (Zoom 1 l,2x; N.A. 0,2) with Zeiss eGFP filter set 38HE (Excitation: Zeiss BP 470 / 40 (HE), Mirror: Zeiss FT 495, Emission: Zeiss BP 525 / 50) and mCherry filter set 63HE (Excitation: Zeiss BP 572 / 25, Mirror: Zeiss FT 590, Emission: Zeiss BP 629 / 62). FIG. 12A shows a tissue-cleared mouse brain and kidneys under different temperature, Refractive Index (RI) matching media- and used dehydration agent conditions, eGFP filter (green) and mCherry filter (magenta). FIG. 12B shows a temperature impact on mouse brain autofluorescence shown on heatmap, eGFP filter (green) and mCherry filter (magenta). FIG. 12C shows a temperature impact on mouse kidney autofluorescence shown on heatmap, eGFP filter (green) and mCherry Filter (magenta). FIG. 12D shows a comparison of temperature impact on autofluorescence levels inATTORNEY DOCKET NO. MDIBL-001PCT differently cleared mouse tissues with eGFP filter. Kidney (yellow), Brain (magenta). FIG. 12D shows a comparison of temperature impact on autofluorescence levels in differently cleared mouse tissues with mCherry filter. Kidney (orange), Brain (cyan). Total Transmission (TT), Haze (H), Ethyl Cinnamate (ECi), Dibenzylether (DBE), 1 :2 ratio of Benzyl alcohol in Benzoate (BABB), Methanol (MeOH), Ethanol (EtOH), tertiary-Butanol (tert-But), Tetrahydrofuran (THF).

[0138] From the foregoing description, it will be apparent that variations and modifications may be made to the embodiments described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0139] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0140] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

1. ATTORNEY DOCKET NO. MDIBL-001PCTCLAIMSWhat is claimed is:

1. A system comprising: a microscope system comprising: at least one light source configured to emit light towards a sample holder; and at least one optical device configured to have the sample holder in a field of view of the at least one optical device; at least one temperature control unit configured to transfer heat to the sample holder, the sample holder being configured to holding a sample of biological matter; and at least one controller operably connected to the at least one temperature control unit, the at least one controller configured to: modulate the at least one temperature control unit so as to control the transfer of heat to the sample holder so as to achieve a predetermined set point; wherein the predetermined set point comprises at least one temperature that is established to increase transparency of the sample sufficient for the at least one optical device to view a full thickness of the sample in the sample holder.

2. The system of claim 1, wherein the microscope system comprises a light sheet microscope.

3. The system of claim 1, wherein the sample holder is configured to hold the sample in a volume of a refractive index matching medium.

4. The system of claim 1, wherein the sample comprises a tissue cleared sample immersed in a refractive index matching medium within the sample holder.

5. The system of claim 4, wherein the at least one controller is further configured to: determine the refractive index matching medium; determine a refractive index matching temperature at which the refractive index matching medium and a refractive index of the sample match; and modulate the at least one temperature control unit to as to control the transfer of heat to the sample holder to achieve the refractive index matching temperature within the sample holder.ATTORNEY DOCKET NO. MDIBL-001PCT6. The system of claim 1, wherein the microscope system comprises a sample holder configured to hold the sample holder in the field of view of the at least one optical device; and wherein the at least one temperature control unit is attached to the sample holder so as to come into contact with the sample holder upon the sample holder being positioned on the sample holder.

7. The system of claim 1, wherein the at least one temperature control unit is attached to the sample holder.

8. The system of claim 1, wherein the at least one controller is further configured to: determine a current temperature of the sample in the sample holder using at least one temperature sensor; determine a difference between the current temperature and the predetermined set point; and modulate, based on the difference, the at least one temperature control unit to as to control the transfer of heat to the sample holder to achieve the predetermined set point.

9. A method comprising: modulating, by at least one processor associated with a microscope system, at least one temperature control unit so as to control a transfer of heat to a sample holder so as to achieve a predetermined set point; the sample holder being configured to hold a sample of biological matter; wherein the predetermined set point comprises at least one temperature thatis established to increase transparency of the sample sufficient for the at least one optical device to view a full thickness of the sample in the sample holder.

10. The method of claim 9, wherein the sample holder is configured to hold the sample in a volume of a refractive index matching medium.

11. The method of claim 9, wherein the sample comprises a tissue cleared sample immersed in a refractive index matching medium within the sample holder.ATTORNEY DOCKET NO. MDIBL-001PCT12. The method of claim 11, further comprising: determining, by the at least one processor, the refractive index matching medium; determining, by the at least one processor, a refractive index matching temperature at which the refractive index matching medium and a refractive index of the sample match; and modulating, by the at least one processor, the at least one temperature control unit to as to control the transfer of heat to the sample holder to achieve the refractive index matching temperature within the sample holder.

13. The method of claim 9, wherein the microscope systrem comprises a sample holder configured to hold the sample holder in the field of view of the at least one optical device; and wherein the at least one temperature control unit is attached to the sample holder so as to come into contact with the sample holder upon the sample holder being positioned on the sample holder.

14. The method of claim 9, wherein the at least one temperature control unit is attached to the sample holder.

15. The method of claim 9, further comprising: determining, by the at least one processor, a current temperature of the sample in the sample holder using at least one temperature sensor; determining, by the at least one processor, a difference between the current temperature and the predetermined set point; and modulating, by the at least one processor, based on the difference, the at least one temperature control unit to as to control the transfer of heat to the sample holder to achieve the predetermined set point.

16. An apparatus comprising: a sample holder associated with a microscope system, the sample holder being configured to holding a sample of biological matter; at least one temperature control unit configured to transfer heat to a sample holder; and at least one controller operably connected to the at least one temperature control unit, the at least one controller configured to:ATTORNEY DOCKET NO. MDIBL-001PCT modulate the at least one temperature control unit so as to control the transfer of heat to the sample holder so as to achieve a predetermined set point; wherein the predetermined set point comprises at least one temperature that is established to increase transparency of the sample sufficient for the at least one optical device to view a full thickness of the sample in the sample holder.

17. The apparatus of claim 16, wherein the sample comprises a tissue cleared sample immersed in a refractive index matching medium within the sample holder.

18. The apparatus of claim 17, wherein the at least one controller is further configured to: determine the refractive index matching medium; determine a refractive index matching temperature at which the refractive index matching medium and a refractive index of the sample match; and modulate the at least one temperature control unit to as to control the transfer of heat to the sample holder to achieve the refractive index matching temperature within the sample holder.

19. The apparatus of claim 16, wherein the at least one temperature control unit is attached to the sample holder.

20. The apparatus of claim 16, wherein the at least one controller is further configured to: determine a current temperature of the sample in the sample holder using at least one temperature sensor; determine a difference between the current temperature and the predetermined set point; and modulate, based on the difference, the at least one temperature control unit to as to control the transfer of heat to the sample holder to achieve the predetermined set point.

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