Methods and systems for vitrified sample preparation

Abstract

In some cases, the present disclosure provides devices and methods for preparation of vitrified samples. A device may comprise a lumen configured for the preparation of the vitrified sample. In some cases, the devices can also be applied in diverse fields, including structural biology, materials analysis, thermal energy storage, and cryogenic propellant handling.

Description

WSGR Docket No. 55983-701.601METHODS AND SYSTEMS FOR VITRIFIED SAMPLE PREPARATION CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 707,005, filed October 14, 2024, which is incorporated herein by reference in its entirety.STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with the support of the United States government under Contract number W912CG-21-C-0015 by Defense Advanced Research Projects Agency.BACKGROUND

[0003] Water can, upon freezing, form ice. The formation of ice can result in an expansion of the volume of the water due to the crystal structure of the ice. Additionally, ice crystal formation can disrupt biological structures, which can cause frozen biological samples to have different geometries than the native state. Confining the water during freezing can form vitrified water, which may not have the same long range crystal order as ice and can more accurately retain the native structure of the sample.SUMMARY

[0004] Computer aided drug design may benefit from high resolution data acquired with a fast turnaround (e.g., less than 24 hours). For example, structural and spatial data with sub-nanometer precision of a drug candidate can be used in the iteration of generating new drug candidates, which can significantly reduce drug costs as well as reduce development time. Some systems to acquire such high resolution data can use costly, slow, and imprecise sample preparation techniques such as sample preparation performed ex situ of the high resolution system.

[0005] Recognized herein is a need for a fast and consistent sample preparation method and related devices. In the case of vitrified sample preparation, this can include an in situ sample freezing scheme, where a sample is frozen inside of an analysis instrument instead of ex situ (e.g., prior to loading into the instrument). In some cases, ex situ freezing can provide low reliability, increased complexity of loading the sample into an instrument (e.g., maintaining the frozen state of the sample can use additional equipment and make loading significantly more difficult and introduce a higher likelihood of damage or contamination of the sample), issues with samples aggregating at an air-water interface, or the like.

[0006] Recognized herein is a need for a ready to use (e.g., in a geometry that is usable in an imaging system without further processing) device for generating vitrified samples. Such a device may be usable in in situ or ex situ freezing systems to prepare samples and can provide increased reliability (e.g., provide samples that are in condition for imaging), decreased sampleWSGR Docket No. 55983-701.601 handling requirements, and significantly increase throughput for application such as, for example, computer aided drug design. In some cases, the methods and devices of the present disclosure can provide concentrations of samples within at least about 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or more percent of the as loaded concentration. For example, when a sample comprising a plurality of biomolecules is loaded into a device, the resultant device as sampled can have a high proportion of the biomolecules present in the device.

[0007] In an aspect, the present disclosure provides a device, comprising: a first side, wherein a widest dimension of the first side of the device is at most about 500 nanometers, wherein an inside of the first side encloses a confined lumen configured to provide a volume for the preparation of a vitrified sample, and a second side, wherein a widest dimension of the second side of the device is at least about 500 micrometers. Confinement can enable vitrification at cooling rates many orders of magnitude slower than unconfined vitrification, for example, in plunge freezing.

[0008] In some cases, the disclosed devices can be applied across diverse fields, including, but not limited to, structural biology, materials analysis, thermal energy storage, and cryogenic propellant handling.

[0009] In some embodiments, the device further comprises an enclosure, wherein the enclosure comprises the device, and wherein a pressure within the enclosure is less than about 1 bar. In some embodiments, a pressure in the device is greater than about 1 bar. In some embodiments, the device is configured to be cooled within the enclosure. In some embodiments, the device further comprises a coating disposed within the lumen. In some embodiments, the coating comprises a metal thin film. In some embodiments, the coating comprises a functionalization. In some embodiments, the functionalization comprises one or more of chemical functionalizations, biological functionalizations, or electrical functionalizations. In some embodiments, the device comprises a glass. In some embodiments, the device further comprises a sealant disposed on the device. In some embodiments, the sealant comprises silver epoxy. In some embodiments, the sealant comprises graphene. In some embodiments, the device is configured to suppress crystalline ice formation.

[0010] In another aspect, the present disclosure provides a device, comprising: a first side, wherein a widest dimension of the first side of the device is at most about 500 nanometers, a volume of the first side, wherein the volume comprises a confined volume.

[0011] In some embodiments, the device further comprises a coating disposed within the lumen. In some embodiments, the coating comprises a metal thin film. In some embodiments, the coating comprises a functionalization. In some embodiments, the functionalization comprises one or more of chemical functionalizations, biological functionalizations, or electricalWSGR Docket No. 55983-701.601 functionalizations. In some embodiments, the device comprises a glass. In some embodiments, the device further comprises a sealant disposed on the device. In some embodiments, the sealant comprises silver epoxy. In some embodiments, the sealant comprises graphene. In some embodiments, the device is configured to suppress crystalline ice formation.

[0012] A device, comprising: a first side, wherein a widest dimension of the first side of the device is at most about 500 nanometers, wherein an inside of the first side encloses a lumen configured to provide a vitrification volume for the preparation of a vitrified sample, and wherein walls of the lumen comprise a metal layer; and a second side, wherein a widest dimension of the second side of the device is at least about 500 micrometers.

[0013] In some embodiments, the metal layer seals the first side. In some embodiments, the device further comprises a coating disposed within the lumen. In some embodiments, the coating comprises a metal thin film. In some embodiments, the coating comprises a functionalization. In some embodiments, the functionalization comprises one or more of chemical functionalizations, biological functionalizations, or electrical functionalizations. In some embodiments, the device comprises a glass. In some embodiments, the device further comprises a sealant disposed on the device. In some embodiments, the sealant comprises silver epoxy. In some embodiments, the sealant comprises graphene. In some embodiments, the device is configured to suppress crystalline ice formation.

[0014] In another aspect, the present disclosure provides a device, comprising: a high vacuum system; a nanostructure comprising a volume configured to contain a sample, wherein the nanostructure is configured to prevent ice crystal formation; and a cooling element configured to cool the sample in the nanostructure while the nanostructure is in the high vacuum system.

[0015] In some embodiments, the device further comprises a coating disposed within the lumen. In some embodiments, the coating comprises a metal thin film. In some embodiments, the coating comprises a functionalization. In some embodiments, the functionalization comprises one or more of chemical functionalizations, biological functionalizations, or electrical functionalizations. In some embodiments, the device comprises a glass. In some embodiments, the device further comprises a sealant disposed on the device. In some embodiments, the sealant comprises silver epoxy. In some embodiments, the sealant comprises graphene. In some embodiments, the device is configured to suppress crystalline ice formation.

[0016] In another aspect, the present disclosure provides a device, comprising: a first side, wherein an inside of the first side encloses a confined lumen configured to provide a volume for the preparation of a vitrified sample, wherein walls of the lumen have a thickness of less than about 40% of a largest dimension of the lumen.WSGR Docket No. 55983-701.601

[0017] In some embodiments, the device further comprises a coating disposed within the lumen. In some embodiments, the coating comprises a metal thin film. In some embodiments, the coating comprises a functionalization. In some embodiments, the functionalization comprises one or more of chemical functionalizations, biological functionalizations, or electrical functionalizations. In some embodiments, the device comprises a glass. In some embodiments, the device further comprises a sealant disposed on the device. In some embodiments, the sealant comprises silver epoxy. In some embodiments, the sealant comprises graphene. In some embodiments, the device is configured to suppress crystalline ice formation.

[0018] In another aspect, the present disclosure provides a method, comprising: (a) providing a nanostructure comprising a volume containing a solvent and a sample within the solvent; (b) loading the nanostructure into a characterization system; and (c) subsequent to (b), cooling the nanostructure, thereby freezing the solvent and the sample.

[0019] In some embodiments, the freezing comprises vitrifying the solvent. In some embodiments, the method further comprises characterizing the sample using the characterization system. In some embodiments, the characterization system comprises a light microscope, a transmission electron microscope, a high resolution transmission electron microscope, a scanning electron microscope, a cryogenic electron microscope, an atom microscope by projection, an atom probe tomography instrument, a time of flight mass spectrometer, a secondary ion mass spectrometer, a field ion microscope, an x-ray spectroscopy instrument, an x-ray diffraction instrument, a free electron laser, or an x-ray tomography instrument. In some embodiments, prior to (a), the volume is primed with the solvent. In some embodiments, the sample is loaded into the solvent.

[0020] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0021] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.WSGR Docket No. 55983-701.601INCORPORATION BY REFERENCE

[0022] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0024] FIG. 1 is an example of a device, according to some embodiments.

[0025] FIGs. 2A - 2D show an example of a plurality of devices on a heating plate, according to some embodiments.

[0026] FIG. 3 shows a computer system that is programmed or otherwise configured to implement methods provided herein.DETAILED DESCRIPTION

[0027] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0028] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0029] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.WSGR Docket No. 55983-701.601

[0030] Certain inventive embodiments herein contemplate numerical ranges. When ranges are present, the ranges include the range endpoints. Additionally, every sub range and value within the range is present as if explicitly written out. The term “about” or “approximately” may mean within an acceptable error range for the particular value, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value may be assumed.

[0031] A device of the present disclosure may comprise a first side. A widest dimension of the first side of the device may be at most about 250,000, 200,000, 150,000, 100,000, 90,000, 80,000, 70,000, 60,000, 50,000, 40,000, 30,000, 25,000, 20,000, 15,000, 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, or fewer nanometers. A widest dimension of the first side may be at least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 150,000, 200,000, 250,000, or more nanometers. The widest dimension of the first side may be in a range as defined by any two of the preceding values. The first side may have a widest dimension such that imaging can occur through the first side of the device. For example, the first side of the device may be configured to be placed in an imaging device (e.g., a light microscope (e.g., a fluorescence microscope, a super-resolution microscope, etc.), a transmission electron microscope, a high resolution transmission electron microscope, a scanning electron microscope, a cryogenic electron microscope, an atom microscope by projection, an atom probe tomography instrument, a time of flight mass spectrometer, a secondary ion mass spectrometer, a field ion microscope, an x-ray spectroscopy instrument, an x-ray diffraction instrument, a free electron laser, an x-ray tomography instrument, etc.) and have a sample contained within the first side of the device be imaged. In this example, the thickness of the first side can be such that a sample placed in the first side can be imaged (e.g., is not too thick for imaging). In some cases, the first side of the device may be cylindrical. The cylindrical shape of the first side may be in a ready to use format for atom probe tomography. In some cases, the first side of the device can be rectangle shaped. The rectangle shape may be in a ready to use geometry for electron microscopy. The first side of the device may have another shape (e.g., square, rectangular, trapezoidal, triangular, oval, other geometric shapes, shapes with rounded edges, etc.). The first side may have parallel sides. The first side may have non-parallel sides (e.g., be wedge shaped).WSGR Docket No. 55983-701.601

[0032] An inside of the first side may comprise a lumen. The lumen may comprise a confined lumen (e.g., a region confined on one, two, three, four, five, or more sides of the region). For example, the lumen can have walls or other confinement (e.g., nanolayers) on three sides of the lumen. The confined lumen may be configured to provide a volume for the preparation of a vitrified sample. The lumen may have a volume of at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, or more cubic micrometers. The lumen may have a volume of at most about 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, or fewer cubic micrometers. The lumen may have a volume in a range as defined by any two of the preceding values. The lumen may comprise an opening to a second side of the device. For example, the lumen may be enclosed except for where the first side of the device and the second side of the device meet. The walls of the lumen may have a thickness of at least about 0, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more percent of a widest dimension of the lumen. The walls of the lumen may have a thickness of at most about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10 percent of a widest dimension of the lumen. The walls of the lumen may have a thickness in a range as defined by any two of the preceding values. In an example, a lumen with a 100 nanometer longest dimension can have walls that are less than about 40 nanometers in thickness. A lumen with walls that are less thick than the longest dimension of the lumen can provide decreased geometry limitations (e.g., freedom in the configuration of the device), resistance to the pressures generated within the lumen (e.g., resistance to the lower pressures generated by the vitrified solvent), and increased resolution of imaging the sample (e.g., less wall material can result in less scattering during an imaging of the sample). In some embodiments, the sealing of the device may occur spontaneously during a phase transformation. In some embodiments, the transition of a solvent or sample forms a plug during the phase transformation. In some embodiments, the lumen may self-pressurize during crystallization, vitrification, or other phase changes, with pressure increases arising from volume constraints imposed by the confined volume. In some embodiments, such confinement and pressurization may alter the thermodynamics or kinetics of a phase transition, for example suppressing nucleation, delaying crystallization, or shifting a melting or boiling point relative to bulk conditions.

[0033] The lumen may be enclosed at least partially by a nanoscale layer. For example, at least a portion of a wall of the lumen may be a material with a thickness of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, 0.003, 0.002, 0.001, 0.0005, or less micrometers. The nanoscale layer may provide, for example, confinement to the lumen (e.g., mechanical confinement configured to resist pressure applied to the nanoscale layer by a sample within theWSGR Docket No. 55983-701.601 lumen), an improved interface between the walls of the lumen and the sample (e.g., improved interfacial properties as compared to the sample interacting with a bare wall of the lumen), and improve electrical and / or thermal conductivity of the walls of the lumen. The lumen may be enclosed on at least about 1, 2, 3, 4, 5, or more sides by the nanoscale layer. The lumen may be enclosed on all sides by the nanoscale layer. The lumen may be enclosed on all sides but one by the nanoscale layer. The nanoscale layer may comprise, for example, one or more of a metal (e.g., platinum, palladium, gold, etc.), an oxide (e.g., aluminum oxide, indium tin oxide, etc.), a polymer (e.g., a conductive polymer), a silane, graphene, or the like, or any combination thereof. For example, the nanoscale layer can comprise a graphene layer disposed on the outside of the device to seal the lumen of the device. The nanoscale layer and / or the walls of the lumen may be functionalized. Examples of functionalizations include, but are not limited to, chemical functionalizations (e.g., hydrophilic treatments, hydrophobic treatments, chelation agents, click chemistry reagents, etc.), biological functionalizations (e.g., biosensor treatments, nucleic acids, antibodies, proteins, polypeptides, etc.), electrical functionalizations (e.g., conductors, insulators, etc.), or the like, or any combination thereof. The interior of the lumen may have a water contact angle of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more degrees. The interior of the lumen may have a water contact angle of at most about 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less degrees. The surface of the interior of the lumen (e.g., the nanoscale layer) may be activated. The activation may comprise use of a plasma, light illumination (e.g., ultraviolet light illumination), or the like, or any combination thereof. The activation may be used to tailor the hydrophobicity of the interior of the lumen. The lumen may be open on at least two sides of the lumen. For example, the lumen can be open to the second side of the device as well as open on another side.

[0034] In some cases, the nanoscale layer may comprise titanium nitride, platinum, or TiN / Pt bilayers, or any combination thereof. In other embodiments, coatings or functionalizations may comprise polyethylene glycol (PEG), phospholipid bilayers, or other hydrophilic or biomimetic layers to reduce biomolecule adhesion, or any combination thereof.

[0035] In some cases, an immiscible fluid boundary may be used as a seal, optionally solidifying at the interface to provide a barrier between the sample and external environment.

[0036] In some cases, the lumen may further comprise an integral filament fused to an interior wall of the lumen. The filament may be configured to promote capillary action, assist fluid filling toward the apex, or support the maintenance of a meniscus at or near the apex in open-apex embodiments, or any combination thereof.

[0037] In some cases, the lumen may be enclosed through the use of one or more nanoparticles. The one or more nanoparticles may be used as one or more plugs to an open end of the deviceWSGR Docket No. 55983-701.601(e.g., an open side of the lumen). For example, a nanoparticle can be flowed to the end of the device and lodge in an open end of the device, thereby forming the lumen. Examples of nanoparticles include, but are not limited to, metallic nanoparticles (e.g., gold nanoparticles, silver nanoparticles, etc.), semiconductor nanoparticles (e.g., quantum dots, etc.), or the like, or any combination thereof. The nanoparticle may have a surface functionalized as described elsewhere herein. The functionalization may be a same functionalization as the walls of the lumen. The functionalization may be a different functionalization as the walls of the lumen. In some embodiments, sealing may be achieved by solidified plug sealing (e.g., the liquid sample or solvent itself is changed to a solid to form a solid plug that prevents evaporation or leakage).

[0038] The device may comprise one or more wall materials. The wall materials may comprise the main structural elements of the device. For example, the device can comprise a wall material configured to provide the walls of the first and second sides. Examples of wall materials include, but are not limited to, glass (e.g., aluminosilicate glass, quartz glass, borosilicate glass, alumina, ceramic glass, titanium silicate, metallic glass, platinum-based metallic glass, iron-based metallic glass, zirconium-based metallic glass, etc.), metals (e.g., platinum, gold, aluminum, iridium, etc.), silicon (e.g., silicon nitride, doped silicon, silicon oxide, etc.), carbon (e.g., graphene, graphite, amorphous carbon, graphene oxide, reduced graphene oxide, diamond-like carbon, etc.), polymer (e.g., polydimethylsiloxane, polymethyl methacrylate, polyimide, polytetrafluoroethylene, polycarbonate, polysulfone, polyetheretherketone, polyvinylidene fluoride, insulating polymers, silanes, conductive polymers, etc.), or the like, or any combination thereof. In some cases, the walls of the device may be removed. For example, after deposition of a nanoscale layer, the walls of the device may be removed. Doing so may provide a very thin layer around the lumen, which can improve resolution of imaging the sample. In some cases, the wall material may be glass.The glass may provide unexpected material properties (e.g., thermal shock tolerance, tensile strength, etc.). For example, the device may be resilient to high thermal stress. The unexpected material properties may enable the preparation of vitrified samples with devices that use thin walls that does not result in a failure or destruction of the device.

[0039] In some cases, the first side of the lumen may be open. For example, the lumen may comprise an open side. In some cases, the device can be loaded into an imaging device or other enclosure as described elsewhere herein. The device can then be filled by, for example, a nanopipette feeding a solution comprising a sample into the lumen of the device. In some cases, the feeding nanopipette can be an additional device of the present disclosure. Upon filling, the sample and its solvent can vitrify upon contact with the device. In some cases, after filling, the open side may be sealed by application of a nanoscale layer as described elsewhere herein (e.g., with a graphene layer). In some cases, after filling, the opening may not be sealed. For example,WSGR Docket No. 55983-701.601 the opening may be left open, and the nanoscale surface tension of the solvent may retain the sample within the lumen.

[0040] In some cases, sealing may be accomplished by dip sealing and subsequent plating of an opening, thereby forming a conformal closure. In some embodiments, internal lumen seals may be formed within the channel, for example by deposition or solidification of material at an interior location.

[0041] The device may comprise a second side. A widest dimension of the second side may be at least about 1, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, or more micrometers. The widest dimension of the second side may be at most about 3,000, 2,500, 2,000, 1,500, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 10, 5, 1, or less micrometers. The widest dimension of the second side may be in a range as defined by any two of the preceding values. The second side may be configured for the loading of a sample into the device. For example, the second side may have a dimension such that a sample can be loaded into the device via a loading instrument (e.g., a pipette, tube, etc.). In this example, the second side can be large enough that the loading instrument is able to access the interior of the second side and thereby access the lumen of the first side. The first side of the device may be contiguous or in fluidic communication with the second side of the device. For example, the first side and the second side may be open to one another. The first side and the second side may together enclose a single lumen comprising the lumen of the first side. The second side may be sealed. For example, the second side can be sealed after loading a sample into the second side. The sealing may provide a device with a single lumen comprising a lumen of the first side and a lumen of the second side. For example, after sealing, the device may be entirely sealed without an opening to outside of the device. The sealing may comprise use of one or more of epoxies (e.g., metal epoxy (e.g., silver epoxy, copper epoxy, nickel epoxy, etc.), conductive acrylics, etc.), greases (e.g., vacuum greases), melting the second side of the device (e.g., fusing the second side to itself), solders, or the like, or any combination thereof.

[0042] In some cases, sealing may be achieved by a mechanical joining mechanism of materials, such as a metal, including but not limited to swaging, crimping, or other deformation techniques, to provide a closure.

[0043] In some cases, the sample may be loaded into the device through the first side (e.g., loaded through an opening in the first side). For example, the sample can be ingressed or aspirated into the device through the first side. After the loading, the first side can be blocked (e.g., by application of a graphene sheet, through use of a nanoparticle blocker, through generation of a nanoscale layer across the first side, etc.). The sample may be loaded into the device with a minimal amount of solvent. For example, the sample can be loaded into the deviceWSGR Docket No. 55983-701.601 with nanoliters of solvent, filling the tip of the device and reducing the amount of sample loaded into the device, which can enable analysis on smaller samples. By loading the device through the first side, the sample can be loaded directly into a characterization portion of the device, improving the success rate of the characterization and reducing a likelihood of the sample being at too low of a concentration for characterization.

[0044] In some cases, the first side can be positioned within a biological cell or tissue to enable direct extraction of a sample. For example, the small size of the first side can permit insertion into a single cell to remove, for example, cytosolic fluid, organelles, or other sub-cellular material directly into the lumen. In such embodiments, the first side is advanced into the cell, the target material is aspirated into the lumen, and the first side is then retracted and optionally sealed as described elsewhere herein prior to vitrification or storage. This direct extraction workflow can reduce sample handling steps, preserve local composition, and improve the probability of obtaining a suitably concentrated sample for downstream characterization.

[0045] The device may be primed with solvent prior to a sample being introduced to the device. The priming may comprise loading the solvent into the lumen of the first side of the device. In this way, when a sample is loaded into the device, the sample can more easily diffuse into the lumen and not experience surface tension related obstruction to loading into the lumen. The priming may comprise addition of the solvent into the device and diversion of the solvent into the lumen. Examples of diversion include, but are not limited to, centrifugal diversion (e.g., use of a centrifuge to force the solvent into the lumen), thermal diversion (e.g., distillation of the solvent into the lumen from the second side), or the like, or any combination thereof.

[0046] The device may be placed within an enclosure. The enclosure may be a portion of an imaging system as described elsewhere herein. For example, the enclosure can be a sample chamber of an electron microscope. In another example, the enclosure can be a sample chamber of an atom probe tomography instrument. The enclosure may be configured to have a pressure within the enclosure of less than about 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, 0.00001, or fewer Barr. The enclosure may be configured to have a pressure within the enclosure of at least about 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, or more Barr. The enclosure may be configured to have a pressure in a range as defined by any two of the preceding values. The enclosure may be a high vacuum or ultra high vacuum chamber. The enclosure may be a portion of an imaging system described elsewhere herein. The pressure within the device (e.g., while the device is within the enclosure) may be greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, or more Barr. The pressure within the device may be less than about 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or less Barr. The pressure within theWSGR Docket No. 55983-701.601 device may be maintained even in a vacuum environment of the enclosure. For example, the device may maintain the sample at a pressure above the pressure of the enclosure. In this way, a sample comprised within the device can be imaged under conditions that more closely match ambient conditions while in a vacuum system. In some embodiments, the disclosed devices may be used in liquid-phase transmission electron microscopy (LPTEM) and / or optical microscopy, permitting in situ imaging of samples in confined geometries.

[0047] In some cases, the device may be configured to be cooled within the enclosure. For example, the enclosure may comprise a cooling element in thermal communication with the device. The cooling element may then cool the device and a sample held therein. The cooling element may be part of a cooling device. Examples of cooling devices include, but are not limited to, thermoelectric coolers, liquid cooling (e.g., using liquid nitrogen), or cryocoolers (e.g., coolers configured to use, for example, helium gas expansion and compression), or the like, or any combination thereof. For example, the enclosure can comprise a cryocooler in thermal communication with the device configured to cool the device and freeze a sample contained within the device. In this way, the device may provide for in situ cooling of the sample within the enclosure. Such in situ cooling can provide significant improvements to the sample handling of a cryogenic sample. For example, a sample can be loaded in an unfrozen state, which can reduce the complexity of handling procedures and time constraints for loading the sample, and the sample can be cooled and frozen within the enclosure.

[0048] In some cases, the disclosed devices and arrays may be configured for cryogenic propellant storage. For example, a cryogenic propellant liquid, upon heating, can form a gas that can degrade combustion quality for propulsion, change density and pressure, and complicate storage, handling, and transfer. Confining the volume of the liquid can suppress gas formation and can permit longer storage at higher temperatures.

[0049] In some cases, the device can be configured to be cooled outside of the enclosure. For example, a device can be cooled and subsequently loaded into the enclosure. The cooling may comprise use of a cooling device as described elsewhere herein. For example, the cooling can comprise use of liquid nitrogen cooler to freeze a sample in the device. The cooling may comprise use of plunge cooling. Plunge cooling may comprise providing a cryogenic liquid or gaseous atmosphere (e.g., liquid nitrogen, liquid ethane, an ice or dry ice bath, a supercooled atmosphere, etc.) and plunging the device into the cryogen. The plunging can provide for a very fast (e.g., sub millisecond) freezing of the sample within the device. In this way, the sample within the device can be frozen in a vitrified state. The device may provide an easy to manipulate and store sample holder for this kind of ex situ freezing. For example, the device can provide an easy to handle storage solution for a biological sample that is frozen and stored.WSGR Docket No. 55983-701.601

[0050] In some cases, the disclosed devices and arrays are suitable for thermal energy storage. For example, a thermal energy storage liquid, upon cooling, can form a solid, with the formation of the solid releasing latent heat energy. Confining the liquid within the lumen during cooling can prevent or delay solid formation, thereby permitting longer storage at lower temperatures and enabling controlled release of energy when desired. In some embodiments, the disclosed devices may be used with non-biological samples, including but not limited to electrolytes, catalysts, inorganic nanoparticles, or interstitial fluids.

[0051] In some cases, the lumen of the device may comprise a confined volume. For example, the lumen may be configured to withstand a pressure applied to the walls of the lumen by a sample in the lumen and not undergo a change in geometry due to the pressure. For example, when a sample freezes in the lumen, the walls of the device can contain the frozen sample. In this example, water solvating the sample may attempt to form crystalline ice and freeze, but the confined volume of the device may reduce and / or prevent crystalline ice formation in the sample by suppressing the expansion of the water, thereby providing a sample, for example, partially or fully as vitrified water (e.g., low density amorphous ice, other structures of ice, etc.). The lumen of the device may comprise a confined volume.. For example, the lumen can be formed from a material that is resistant to the pressures in and out of the lumen.. Additional techniques that can be used to reduce and / or prevent crystalline ice formation can include, but are not limited to, surface functionalization, electrical field, magnetic field, other thermodynamic suppression methods, or the like, or any combination thereof.

[0052] The device may be configured for the preparation of a vitrified sample. The vitrified sample may comprise a vitrified solvent. For example, a water solvent of the sample may be frozen in a vitrified water solvent. The dimensions and confined nature of the device may enable a frozen sample to not form crystalline ice but instead form a vitrified state. The vitrified state may preserve the structure of the sample (e.g., provide a frozen environment that does not disrupt the structure of the sample). The preparation of the vitrified sample may comprise freezing in a region configured to reduce or prevent crystalline ice formation as described elsewhere herein. The device may be configured to enable formation of vitrified samples at lower cooling rates than plunge freezing. For example, the device can enable use of a sample stage cooled by a cryocooler to form the vitrified sample by confining the sample in the confined volume. The device may enable use of a cooling rate of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 750, 1,000, 5,000, 10,000 50,000, 100,000 or more degrees Celsius per second to form a vitrified sample. The device may enable use of a cooling rate of at most about 100,000, 50,000, 10,000, 5,000, 1,000, 750, 500, 400, 300, 275, 250, 225, 200, 175, 150, 125, 100, 95, 90, 85, 80,WSGR Docket No. 55983-701.60175, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less degrees Celsius per second to form a vitrified sample. The device may enable a cooling rate in a range as defined by any two of the preceding values to form a vitrified sample. Such cooling rates may be much lower than other vitrification methods, which can provide increased cooling options as well as enable new sample handling strategies (e.g., in situ cooling, etc.).

[0053] In some cases, the device may be a standalone device. For example, the device can be an independent device with a single lumen. In some cases, the device may be a portion of an array of devices. The array of devices can comprise a plurality of devices each configured as described elsewhere herein. For example, a single substrate can comprise a plurality of microscale devices.

[0054] In some cases, the devices of the present disclosure can be ready to use in an imaging system. For example, after loading the sample into the device and vitrifying the sample, the device can be loaded into the imaging system without further processing (e.g., cutting, milling, coating, forming, shaping, welding, etc.). The form of the ready to use device can be tailored to the imaging system. For example, a device configured for use in a transmission electron microscope can have a lumen less than about 100 nanometers thick to enable imaging through the lumen. In another example, a device configured for atom probe tomography can have a lumen with an apex diameter of about 100 nanometers. The ready to use geometry of the device can provide reduced preparation time and cost as compared to a geometry that is not ready to use. Additionally, the ready to use geometry can reduce contamination from post-processing the device.

[0055] In some cases, the devices of the present disclosure can provide storage for a biological sample. For example, a biological sample can be suspended in a vitrified solvent and stored at cryogenic temperatures. In this way, the structure of the sample can be maintained during storage (e.g., as the vitrified solvent does not disrupt the structure as much as crystalline ice from a frozen solvent), even at cryogenic temperatures. In this example, the structure of the biological sample can be maintained during storage and investigated upon removal of the biological sample from storage. In some cases, the biological sample can be placed in the device and frozen in a freezer without further preparation. The device can reduce or prevent the formation of crystals and provide easily prepared vitrified samples for storage.

[0056] The methods and / or devices of the present disclosure may be suitable for a variety of sequencing, storage, mapping, and imaging applications and for sequencing biomolecules (e.g., nucleic acid molecules) derived from any of a variety of samples and sources. Biomolecules (e.g., nucleic acids), in some cases, may be extracted from any of a variety of biological samples, e.g., blood samples, saliva samples, urine samples, cell samples, tissue samples, and the like. The samples of the present disclosure may be extracted from one or more sources. For example, aWSGR Docket No. 55983-701.601 tissue sample can be extracted from a subject. The samples of the present disclosure may be extracted from other samples. For example, a plurality of cells can be extracted from a tissue. In another example, a biomolecule (e.g., a nucleic acid molecule) can be extracted from a cell.

[0057] For example, the disclosed devices and systems may be used for the analysis of biomolecules (e.g., nucleic acids molecules) derived from any of a variety of different cell, tissue, or sample types. For example, nucleic acids may be extracted from cells, or tissue samples comprising one or more types of cells, derived from eukaryotes (such as animals, plants, fungi, protista), archaebacteria, or eubacteria. In some cases, biomolecules (e.g., nucleic acids) may be extracted from prokaryotic or eukaryotic cells, such as adherent or non-adherent eukaryotic cells. Biomolecules (e.g., nucleic acids) can be variously extracted from, for example, primary or immortalized rodent, porcine, feline, canine, bovine, equine, primate, or human cell lines. Biomolecules (e.g., nucleic acids) may be extracted from any of a variety of different cell, organ, or tissue types (e.g., white blood cells, red blood cells, platelets, epithelial cells, endothelial cells, neurons, glial cells, astrocytes, fibroblasts, skeletal muscle cells, smooth muscle cells, gametes, or cells from the heart, lungs, brain, liver, kidney, spleen, pancreas, thymus, bladder, stomach, colon, or small intestine). Biomolecules (e.g., nucleic acids) may be extracted from normal or healthy cells. Alternately or in combination, the biomolecules can be extracted from diseased cells, such as cancerous cells, or from pathogenic cells that are infecting a host. Some nucleic acids may be extracted from a distinct subset of cell types, e.g., immune cells (such as T cells, cytotoxic (killer) T cells, helper T cells, alpha beta T cells, gamma delta T cells, T cell progenitors, B cells, B-cell progenitors, lymphoid stem cells, myeloid progenitor cells, lymphocytes, granulocytes, Natural Killer cells, plasma cells, memory cells, neutrophils, eosinophils, basophils, mast cells, monocytes, dendritic cells, and / or macrophages, or any combination thereof), undifferentiated human stem cells, human stem cells that have been induced to differentiate, rare cells (e.g., circulating tumor cells (CTCs), circulating epithelial cells, circulating endothelial cells, circulating endometrial cells, bone marrow cells, progenitor cells, foam cells, mesenchymal cells, or trophoblasts). The biomolecules (e.g., nucleic acids) may optionally be attached to one or more moieties (e.g., non-nucleotide moieties) such as labels and other small molecules, large molecules (such as proteins, lipids, sugars, etc.), and / or solid or semi-solid supports, for example through covalent or non-covalent linkages with either end of a biomolecule (e.g., the 5' or 3' end of the nucleic acid). Labels can include any moiety that is detectable using any of a variety of detection methods known to those of skill in the art, and thus renders the attached oligonucleotide or nucleic acid similarly detectable. Some labels, e.g., fluorophores, can emit electromagnetic radiation that is optically detectable or visible. The labelsWSGR Docket No. 55983-701.601 may comprise one or more barcode molecules (e.g., molecules configured to provide a detectable signal corresponding to an identity of the molecule).

[0058] In some cases, a plurality of devices as described herein can be arranged in an array.Arrays may comprise multiple confined-volume lumens on a single substrate or as a collection of individual devices coupled thermally or fluidically. Arrays can provide modular scaling of capacity, improved control of phase transformation events across multiple confined volumes, and redundancy for reliability. For example, arrays may be configured for scalable thermal energy storage by charging and discharging multiple phase-change lumens in parallel or series, or for cryogenic propellant storage by distributing propellant across multiple lumens to reduce bulk boil-off and improve feedline stability. Unless otherwise stated, the term “device” as used herein encompasses both single devices and arrays of devices.

[0059] The one or more samples may comprise one or more label moi eties. Examples of label moieties include, but are not limited to, optical barcodes, nanoparticles, magnetic moieties, or electrical moieties, or any combination thereof. For example, the one or more samples may comprise a plurality of barcode tags. For example, the one or more samples can be treated with a plurality of barcode tags configured to bind to a plurality of analytes (e.g., nucleic acids, proteins, carbohydrates, etc.) contained within the one or more samples.

[0060] In some cases, the devices of the present disclosure can be used to hold a sample without vitrification of the solvent. For example, a sample can be placed into the device and imaged in a liquid state. In this example, the device can provide an environment closer to an ambient environment than the environment of the imaging system. For example, the device can protect the sample from the high vacuum of a transmission electron microscope.Computer systems

[0061] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 3 shows a computer system 301 that is programmed or otherwise configured to implement the methods of the present disclosure. The computer system 301 can regulate various aspects of the present disclosure, such as, for example, generation or filling of devices, use of illumination systems, temperature control, etc. The computer system 301 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0062] The computer system 301 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 305, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 301 also includes memory or memory location 310 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 315 (e.g., hard disk), communication interface 320 (e.g., network adapter)WSGR Docket No. 55983-701.601 for communicating with one or more other systems, and peripheral devices 325, such as cache, other memory, data storage and / or electronic display adapters. The memory 310, storage unit 315, interface 320 and peripheral devices 325 are in communication with the CPU 305 through a communication bus (solid lines), such as a motherboard. The storage unit 315 can be a data storage unit (or data repository) for storing data. The computer system 301 can be operatively coupled to a computer network (“network”) 330 with the aid of the communication interface 320. The network 330 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that is in communication with the Internet. The network 330 in some cases is a telecommunication and / or data network. The network 330 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 330, in some cases with the aid of the computer system 301, can implement a peer-to-peer network, which may enable devices coupled to the computer system 301 to behave as a client or a server.

[0063] The CPU 305 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 310. The instructions can be directed to the CPU 305, which can subsequently program or otherwise configure the CPU 305 to implement methods of the present disclosure. Examples of operations performed by the CPU 305 can include fetch, decode, execute, and writeback.

[0064] The CPU 305 can be part of a circuit, such as an integrated circuit. One or more other components of the system 301 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0065] The storage unit 315 can store files, such as drivers, libraries and saved programs. The storage unit 315 can store user data, e.g., user preferences and user programs. The computer system 301 in some cases can include one or more additional data storage units that are external to the computer system 301, such as located on a remote server that is in communication with the computer system 301 through an intranet or the Internet.

[0066] The computer system 301 can communicate with one or more remote computer systems through the network 330. For instance, the computer system 301 can communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 301 via the network 330.

[0067] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 301, such as, for example, on the memory 310 or electronic storage unit 315. The machine executableWSGR Docket No. 55983-701.601 or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 305. In some cases, the code can be retrieved from the storage unit 315 and stored on the memory 310 for ready access by the processor 305. In some situations, the electronic storage unit 315 can be precluded, and machine-executable instructions are stored on memory 310.

[0068] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as- compiled fashion.

[0069] Aspects of the systems and methods provided herein, such as the computer system 301, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and / or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk.“Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0070] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmissionWSGR Docket No. 55983-701.601 media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0071] The computer system 301 can include or be in communication with an electronic display 335 that comprises a user interface (UI) 340. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0072] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 305. The algorithm can, for example, control cooling equipment configured to cool a device comprising a sample.

[0073] The devices and arrays can be operated under programmatic control using one or more processors of the central processing unit 305 executing instructions stored in the memory 310. Such control can regulate temperature, pressure, fluidic actuation, and timing during filling, priming, vitrification, storage, or release operations, or any combination thereof. The control can be coordinated over the bus 315, and can be supported by firmware and higher-level software layers.

[0074] The central processing unit 305 can execute closed-loop thermal control algorithms using sensor input received through the I / O interface 320, for example from thermistors, resistance temperature detectors, or thermocouples. The CPU 305 can provide control signals over the bus 315 to actuators such as Peltier elements, cryocoolers, resistive heaters, or valves in a heatexchange system. In this way, the computer system 301 can implement rate-of-cooling control, ramp profiles, setpoint transitions, and safety interlocks to achieve vitrification or thermalcycling protocols.

[0075] Fluidic and filling operations can be automated or semi-automated by the CPU 305 in communication with liquid-level sensors, pressure transducers, or optical detectors interfaced through the I / O interface 320. The computer system can drive microinjectors, vacuum sources,WSGR Docket No. 55983-701.601 immiscible-fluid dispensers, or mechanical valves to perform controlled fill, displacement, and sealing sequences.

[0076] Arrayed devices can be managed by supervisory control logic executed by the CPU 305. The supervisory logic can sequence per-device operation, coordinate parallel or staggered operation across devices, and interface with higher-level scheduling or batch-processing software.

[0077] The CPU 305 can execute application-specific protocols for TES or propellant arrays. For TES, instructions in memory 310 can direct charge scheduling, state-of-charge estimation based on telemetry, and discharge triggers such as nucleation stimuli or thermal drawdown. For propellant arrays, protocols can include boil-off mitigation routines, pooling logic to aggregate liquid from selected lumens, and controlled equalization and feedline coupling steps.

[0078] Instrumentation such as temperature, pressure, and acoustic sensors can provide diagnostic data through the VO interface 320 to the CPU 305. The CPU can log this data to memory 310, run diagnostics to detect anomalous nucleation or seal failure, and issue alerts or safety actions such as isolating lumens or opening relief pathways.

[0079] The computer system 301 can present a user interface or programmatic API via the VO interface 320 to configure protocols, review telemetry, and export logged data. The interface can support secure remote access, role-based control, and export of time-stamped metadata for archiving.

[0080] The CPU 305 can run closed-loop control algorithms complemented by model-based features (e.g., predictive thermal models, nucleation likelihood estimators) stored in the memory 310 and adapted based on measured state and predicted risk.

[0081] Safety and redundancy features can be enforced by the CPU 305 in combination with hardware interlocks connected through the VO interface 320, including redundant sensors, hard limits on heater / cooler power, hardware-enforced pressure relief, and secure firmware update procedures.

[0082] The computer system 301 can automate sample processing pipelines, with the CPU 305 and memory 310 executing instructions to integrate filling, priming, vitrification, imaging scheduling, and recording of per-sample provenance (e.g., device identifier, fill composition, and timestamps for each operation).EXAMPLES

[0083] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.Example 1 - Device parameter calculationsWSGR Docket No. 55983-701.601

[0084] In some examples, to suppress crystallization of a water solvent, a device may have a pressure resistance of about 200 megapascals. For an example aluminosilicate glass device with aP'V10 nanometer lumen, using the formula for determination of wall thickness of t = — where t is the wall thickness, P is the internal pressure, r is the internal radius of the lumen, and o is the tensile strength of the wall material (700 megapascals in the case of the example aluminosilicate glass), provides a wall thickness of 10 nanometers. This can provide a lower bound of the wall thickness. For example, a device with a 15 nanometer wall can have a safety margin over the 10 nanometer minimum.

[0085] The tensile strength of a material (e.g., a wall material) can increase as the length scale of the material is decreased. For example, at the micro and nanoscale, a reduction in defects (e.g., reduction in intrinsic defect density), increased surface to volume ratio, more uniform stress distribution during mechanical load, decrease in internal defects, and the like, can result in improved material properties for wall materials of the present disclosure.

[0086] FIG. 1 is an example of a device 100, according to some embodiments. The device may comprise a first side 110 and a second side 120. The lumen of the first side 110 can have a 100 nanometer longest dimension (e.g., diameter) and can comprise a nanoscale layer 130. The second side 120 can be used to fill the device with, for example, a sample in a solvent. The lumen can be configured to, upon cooling of the sample, form a vitrified solvent around the sample. Example 2 Device filling

[0087] In some examples, a device with a closed first side (e.g., a closed tip of a nanopipette) can be primed for loading. The priming may enable loading of the volume of the closed first side by removing gas and overcoming hydrophobic forces within the lumen. The priming may comprise loading the device through the second side with solvent (e.g., water). Applying heat to the second side (e.g., through use of a hotplate, radiation, etc.) can distil the solvent from the second side into the closed first side. FIGs. 2A - 2D show an example of a plurality of devices 210 on a heating plate 220, according to some embodiments. Over the course of the priming process, the bubble 230 seen at 0 minutes (FIG. 2B) can be seen moving away from the first side as the priming occurs at 4 minutes (FIG. 2C) and 8 minutes (FIG. 2D). In this way, the first side of the device can be primed, and a sample later added to the device can more easily diffuse into it.Example 3 Thermal energy storage (TES)

[0088] In some examples, an array of devices may be loaded with a phase-change material (PCM) and thermally cycled. For example, a thermal energy storage liquid, upon cooling, can form a solid, with the formation of the solid releasing latent heat energy. Confining the liquidWSGR Docket No. 55983-701.601 within the lumen during cooling can prevent or delay solid formation, thereby permitting longer storage at lower temperatures and enabling controlled release of energy when desired.

[0089] In one example, a single device comprises a confined lumen filled with a PCM and arranged within a thermally conductive mounting that forms part of a modular array. The array may comprise a plurality of discrete devices in thermal communication with a common heat exchanger or distributed heat collection channels, and the devices may be arranged in serial or parallel flow architectures depending on desired charge / discharge characteristics.

[0090] Charging of the TES may comprise heating the PCM (e.g., via external heat input, electrical resistive heating, or through a heat-exchange fluid) to move the PCM into a higher- energy state, and storage may comprise maintaining the PCM in a confined, non-equilibrium liquid state by virtue of the lumen geometry and sealing. Discharge (release of stored energy) may be accomplished by permitting or inducing a controlled phase transformation, for example by locally applying heat removal, introducing a nucleation stimulus, mechanically altering confinement (e.g., removing or altering a seal), or otherwise triggering crystallization, with the released latent heat captured via the same heat-exchange architecture used for charging.

[0091] In some examples, suitable PCM materials include, but are not limited to, organic PCMs (e.g., paraffins), salt hydrates, eutectic mixtures, or other materials with useful latent heat characteristics. The devices and arrays may further include thermal insulation, thermal bridging features, integrated sensors (e.g., temperature and pressure sensors), and control electronics configured to monitor state of charge and to execute charge / discharge protocols.

[0092] In some examples, sealing and filling options described herein (e.g., solidified plug sealing, immiscible fluid boundaries, dip-sealing and plating, or mechanical joining mechanisms) can be employed to prevent vapor ingress or unintended nucleation during long term storage. In addition, embodiments may include safety and control features such as pressure relief pathways, redundant sealing, and diagnostic telemetry to detect and manage anomalous phase changes.

[0093] In some examples, systems built from arrays of such devices provide modular scaling, allowing the storage capacity and peak power handling to be tuned by changing array size, device geometry, or interconnection topology. Methods for making and using such TES arrays include filling the lumens with a selected PCM, sealing and priming the array, thermally cycling during commissioning, monitoring stability during storage, and triggering controlled release when energy recovery is required.Example 4 Cryogenic propellant storage

[0094] In some examples, an array of devices may be filled with a cryogenic propellant and exposed to storage and handling. For example, a cryogenic propellant liquid, upon heating, can form a gas. Formation of gas can degrade combustion quality for propulsion, change density andWSGR Docket No. 55983-701.601 pressure, and complicate storage, handling, and transfer. Confining the volume of the liquid during storage and handling can suppress gas formation and can permit longer storage at higher temperatures.

[0095] In one example, a device or array comprises a plurality of confined lumens sized and sealed to stabilize a cryogenic liquid within each lumen. Confinement may retard nucleation and bubble growth by limiting the volumetric expansion pathways available to the propellant, and sealing techniques disclosed herein (e.g., graphene sheets, solidified plugs, immiscible boundary seals, or plated dip seals) may be used to maintain the confined state during storage and handling.

[0096] Filling and service methods may include controlled filling at low temperature, use of immiscible buffer fluids or menisci to isolate the propellant plug, integration of filaments or wicking elements to maintain menisci at open apexes, and careful selection of materials and coatings to minimize boil-off, permeation, and chemical reactivity. Release or delivery of propellant for propulsion may be affected by controlled warming, pressure equalization, actuation of release valves, or selective removal of confinement (e.g., mechanical actuation or melting of a localized seal) to reconnect the lumen contents to feedlines.

[0097] Examples of propellants include, but are not limited to, oxygen, methane, hydrogen, or other cryogenic liquids; the devices and arrays may be configured and constructed from materials and coatings compatible with the chosen propellant to avoid undesirable chemistry, embrittlement, or permeability. The devices may include integrated sensors (e.g., pressure transducers, level sensors, thermometry), safety features (e.g., burst discs, controlled venting paths), and thermal management (e.g., multilayer insulation, active cryocoolers) as appropriate to the application.

[0098] In some examples, arrays of confined lumens offer system-level benefits such as reduced bulk boil-off, improved feedline stability (reduced gas ingestion), and finer-grained control over propellant thermodynamic state prior to combustion. Methods of use include filling arrays under controlled conditions, monitoring during storage, selectively pooling liquids from multiple lumens to supply a combustion system, and controlled regeneration or purging cycles to ready the array for subsequent refill or launch activities.

[0099] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shallWSGR Docket No. 55983-701.601 be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WSGR Docket No. 55983-701.601CLAIMSWHAT IS CLAIMED IS:

1. A device, comprising: a first side, wherein a widest dimension of said first side of the device is at most about 500 nanometers, wherein an inside of said first side encloses a confined lumen configured to provide a volume for the preparation of a vitrified sample, and a second side, wherein a widest dimension of said second side of the device is at least about 500 micrometers.

2. The device of claim 1, further comprising an enclosure, wherein said enclosure comprises said device, and wherein a pressure within said enclosure is less than about 1 bar.

3. The device of claim 1, wherein a pressure in said device is greater than about 1 bar.

4. The device of claim 1, wherein said device is configured to be cooled within said enclosure.

5. The device of claim 1, further comprising a coating disposed within said lumen.

6. The device of claim 5, wherein said coating comprises a metal thin film.

7. The device of claim 5, wherein said coating comprises a functionalization.

8. The device of claim 7, wherein said functionalization comprises one or more of chemical functionalizations, biological functionalizations, or electrical functionalizations.

9. The device of claim 1, wherein said device comprises a glass.

10. The device of claim 1, further comprising a sealant disposed on said device.

11. The device of claim 10, wherein said sealant comprises silver epoxy.

12. The device of claim 10, wherein said sealant comprises graphene.

13. The device of claim 1, wherein said device is configured to suppress crystalline ice formation.

14. A device, comprising: a first side, wherein a widest dimension of said first side of the device is at most about 500 nanometers, a volume of said first side, wherein said volume comprises a confined volume.

15. The device of claim 14, further comprising a coating disposed within said lumen.

16. The device of claim 15, wherein said coating comprises a metal thin film.

17. The device of claim 15, wherein said coating comprises a functionalization.

18. The device of claim 17, wherein said functionalization comprises one or more of chemical functionalizations, biological functionalizations, or electrical functionalizations19. The device of claim 14, wherein said device comprises a glass.

20. The device of claim 14, further comprising a sealant disposed on said device.

21. The device of claim 20, wherein said sealant comprises silver epoxy.

22. The device of claim 20, wherein said sealant comprises graphene.WSGR Docket No. 55983-701.60123. The device of claim 14, wherein said device is configured to suppress crystalline ice formation.

24. A device, comprising: a first side, wherein a widest dimension of said first side of the device is at most about 500 nanometers, wherein an inside of said first side encloses a lumen configured to provide a vitrification volume for the preparation of a vitrified sample, and wherein walls of said lumen comprise a metal layer; and a second side, wherein a widest dimension of said second side of the device is at least about 500 micrometers.

25. The device of claim 24, wherein said metal layer seals said first side.

26. The device of claim 24, further comprising a coating disposed within said lumen.

27. The device of claim 26, wherein said coating comprises a metal thin film.

28. The device of claim 26, wherein said coating comprises a functionalization.

29. The device of claim 28, wherein said functionalization comprises one or more of chemical functionalizations, biological functionalizations, or electrical functionalizations.

30. The device of claim 24, wherein said device comprises a glass.

31. The device of claim 24, further comprising a sealant disposed on said device.

32. The device of claim 31, wherein said sealant comprises silver epoxy.

33. The device of claim 31, wherein said sealant comprises graphene.

34. The device of claim 24, wherein said device is configured to suppress crystalline ice formation.

35. A device, comprising: a high vacuum system; a nanostructure comprising a volume configured to contain a sample, wherein said nanostructure is configured to prevent ice crystal formation; and a cooling element configured to cool said sample in said nanostructure while said nanostructure is in said high vacuum system.

36. The device of claim 35, further comprising a coating disposed within said lumen.

37. The device of claim 36, wherein said coating comprises a metal thin film.

38. The device of claim 36, wherein said coating comprises a functionalization.

39. The device of claim 38, wherein said functionalization comprises one or more of chemical functionalizations, biological functionalizations, or electrical functionalizations.

40. The device of claim 35, wherein said device comprises a glass.

41. The device of claim 35, further comprising a sealant disposed on said device.

42. The device of claim 41, wherein said sealant comprises silver epoxy.WSGR Docket No. 55983-701.60143. The device of claim 41, wherein said sealant comprises graphene.

44. The device of claim 35, wherein said device is configured to suppress crystalline ice formation.

45. A device, comprising: a first side, wherein an inside of said first side encloses a confined lumen configured to provide a volume for the preparation of a vitrified sample, wherein walls of said lumen have a thickness of less than about 40% of a largest dimension of said lumen.

46. The device of claim 45, further comprising a coating disposed within said lumen.

47. The device of claim 46, wherein said coating comprises a metal thin film.

48. The device of claim 46, wherein said coating comprises a functionalization.

49. The device of claim 48, wherein said functionalization comprises one or more of chemical functionalizations, biological functionalizations, or electrical functionalizations.

50. The device of claim 45, wherein said device comprises a glass.

51. The device of claim 45, further comprising a sealant disposed on said device.

52. The device of claim 51, wherein said sealant comprises silver epoxy.

53. The device of claim 51, wherein said sealant comprises graphene.

54. The device of claim 45, wherein said device is configured to suppress crystalline ice formation.

55. A method, comprising:(a) providing a nanostructure comprising a volume containing a solvent and a sample within said solvent;(b) loading said nanostructure into a characterization system; and(c) subsequent to (b), cooling said nanostructure, thereby freezing said solvent and said sample.

56. The method of claim 55, wherein said freezing comprises vitrifying said solvent.

57. The method of claim 55, further comprising characterizing said sample using said characterization system.

58. The method of claim 55, wherein said characterization system comprises a light microscope, a transmission electron microscope, a high resolution transmission electron microscope, a scanning electron microscope, a cryogenic electron microscope, an atom microscope by projection, an atom probe tomography instrument, a time of flight mass spectrometer, a secondary ion mass spectrometer, a field ion microscope, an x-ray spectroscopy instrument, an x-ray diffraction instrument, a free electron laser, or an x-ray tomography instrument.

59. The method of claim 55, wherein, prior to (a), said volume is primed with said solvent.

60. The method of claim 59, wherein said sample is loaded into said solvent.WSGR Docket No. 55983-701.60161. The device of claim 1, wherein the device is configured for use as a thermal energy storage(TES) element, the confined volume containing a phase-change medium stabilized in a nonequilibrium state.

62. The device of claim 61 wherein the phase-change medium comprises a phase-change material.

63. The device of claim 62, wherein the phase-change material is a salt hydrate, eutectic mixture, or an organic phase-change material.

64. The device of claim 1, wherein the device is configured for storage of a cryogenic propellant, the confined volume reducing vapor formation during storage and handling.

65. The device of claim 64, wherein the cryogenic propellant comprises oxygen, methane, hydrogen, ammonia, or combinations thereof.

66. The device of claim 45, wherein the device is configured for use as a thermal energy storage (TES) element, the confined volume containing a phase-change medium stabilized in a nonequilibrium state.

67. The device of claim 66, wherein the phase-change medium comprises a phase-change material.

68. The device of claim 67, wherein the phase-change material is a salt hydrate, eutectic mixture, or an organic phase-change material.

69. The device of claim 45, wherein the device is configured for storage of a cryogenic propellant, the confined volume reducing vapor formation during storage and handling.

70. The device of claim 69, wherein the cryogenic propellant comprises oxygen, methane, hydrogen, ammonia, or combinations thereof.

71. The device of claim 61 or 66, wherein a plurality of the devices are arranged in an array configured for thermal energy storage.

72. The device of claim 64 or 69, wherein a plurality of the devices are arranged in an array configured for cryogenic propellant storage.

73. A method of storing thermal energy, comprising:(a) providing a device according to claim 61 or 66;(b) charging the device by adding energy to said phase-change medium, including by altering confinement; and(c) discharging stored energy by releasing latent heat from said phase-change medium, including by altering confinement.

74. The method of claim 73, further comprising operating a plurality of devices as an array to provide scalable charge and discharge of stored thermal energy.

75. A method of storing and handling a cryogenic propellant, comprising:WSGR Docket No. 55983-701.601(a) providing a device according to claim 64 or 69;(b) filling the device with said cryogenic propellant; and(c) maintaining said cryogenic propellant within the device during storage and handling.

76. The method of claim 75, further comprising selectively releasing said cryogenic propellant, including by altering confinement.

77. The method of claim 75, further comprising operating a plurality of devices as an array to provide scalable cryogenic propellant storage and delivery.

78. The method of claim 76, further comprising operating a plurality of devices as an array to provide scalable cryogenic propellant storage and delivery.

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