Sample preparation system

Abstract

A system includes a support configured to bear a sample, a receiving area configured to receive a chamber bearing a cryogenic liquid, a translation stage configured to translate the support along a direction between a first position at a first height above a chamber disposed in the receiving area and a second position where the test sample would be disposed in the cryogenic liquid and a transfer-mechanism defining, in a first state, an opening to form and hold a suspended liquid film. The opening is disposed along the direction in the first state with the film in the opening and, following movement of the sample into contact with and through the film, causing transfer of a portion of the film to the sample, the transfer-mechanism assumes a second state, in which the opening is disrupted or moved to facilitate continued movement of the support and sample toward the second position.

Description

SAMPLE PREPARATION SYSTEMCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 665,944, filed on 28 June 2024, the disclosure of which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] This disclosure relates generally to the field of biotechnology, including the handling and processing of biological samples. More particularly, this disclosure relates to systems and methods for preparing samples for time-resolved studies of biomolecular systems. More particularly, aspects of this disclosure relate to systems and methods for rapidly initiating reactions and other chemical and structural changes in a crystalline sample and examining their progression in time using x-ray crystallography and other biophysical methods. More particularly, aspects of this disclosure relate to systems and methods for reaction initiation in a crystal followed, after a variable time interval, by quenching the reaction and the crystal’s chemical and structural state by rapid cooling of the sample to cryogenic temperature, and repeating these measurements with different time intervals between reaction initiation and quenching, in this way characterizing the progression of chemical and structural changes versus time after reaction initiation. Elements of the disclosed system are also suitable for preparation of samples for time- resolved cryo-electron microscopy.BACKGROUND

[0003] The atomic and near-atomic resolution structures of biological molecules and of small molecule organic compounds are key to understanding function and interaction within the living cell. Structures determined by x-ray crystallography have identified reaction centers, allosteric sites, mobile loops, binding domains, and other features important in design of pharmaceuticals and in engineering modified biomolecules for biotechnology. However, traditional crystallographic data collection produces time- and space-averaged molecular structures that provide limited insight into functional dynamics. When high-resolution electron density maps are available, multi-conformer models canprovide some dynamic insight, but functionally important conformers often have undetectable occupancies.

[0004] A longstanding goal of structural science has been to produce atomic resolution “movies” of biomolecules (and small molecule compounds) in action. Pioneering work in the 1980s-2000s used laser excitation synchronized with the x-ray pulse structure of synchrotron x-ray sources to study structural states of photo-active proteins on timescales following photoactivation from ~100 picoseconds (ps) to seconds (s). Development of x-ray free-electron laser (XFEL) sources with ~ 10- 100 femtosecond (fs) long x-ray pulses improved time resolutions in photo-activated experiments to ~150 fs, allowing observation of, e.g., collective motions following photolysis of the Fe-CO bond in carbonmonoxy myoglobin and application of machine learning to extract information on few-fs timescales. As impressive as these results are, less than ~1% of proteins are photoactive. Nearly all dynamics of biological interest is triggered by diffusion and binding of ligands or by diffusion of small molecules that change the ionic strength, pH, water activity, and other physico-chemical properties of the ligand solution within and surrounding the biomolecule. To study such dynamics in biomolecular crystals, the most straightforward approach — at least conceptually — is to mix crystals with ligand / small molecule solution and deliver them after a time delay tdeiay into the x-ray beam.

[0005] The most biologically interesting dynamics typically occurs on microsecond to second timescales. For example, enzyme turnover times range from about 1 microsecond (ps) to hours (h), with most turnover times being in the 1 millisecond (ms) to 1 second (s) range. Populating ligand throughout the crystal interior on the ~ 1-100 ms timescales typically required to observe biologically interesting reaction dynamics requires use of small crystals - well below 50 pm and, for low-millisecond timescales, less than 10 pm. At room temperature, such tiny crystals may sustain substantial radiation damage before a complete data set sufficient for structure determination can be obtained, and so, in embodiments, large numbers of crystals are measured for each time point.

[0006] Routine data collection with millisecond exposures from few-micrometer size crystals has become feasible in the last 15 years with the development of x-ray free- electron laser (XFEL) sources and suitable sample delivery and analysis tools, of high- brilliance synchrotron radiation sources with microfocusing beamline optics, and of x-ray detectors with kHz frame rates.

[0007] The dominant methods for obtaining time-resolved biomolecular structures are room-temperature time-resolved serial crystallography [TR-SX], performed atXFELs or synchrotrons, and time-resolved single-particle cryo-EM (TR-cryo-EM). Both techniques have limitations.

[0008] In TR-SX at synchrotrons, the amount of usable data obtained from each crystal is limited by the desired time resolution or by radiation damage and is only a tiny fraction of a complete data set when using microcrystals at room temperature. At XFELs, more x-ray photons can be scattered before a crystal is destroyed but only in a single unknown crystal orientation. In both cases, data maybe collected from a huge number of crystals — from 103to 108— to determine an electron-density map and molecular model at each time point.

[0009] AtXFELs, crystals are continuously delivered from the position where crystal and ligand are mixed to the x-ray beam. The time point after reaction initiation that is probed is set by the travel time between these positions, so changing the distance between the mixer and x-ray beam allows data collection at different time points. Crystal concentration and / or flow speed are adjusted to maximize the fraction of crystals hit by x- ray pulses. Approaches for delivering crystals in a liquid "jet" have used T-junction and coaxial mixers, yielding time resolutions of seconds, and a gas-dynamic-virtual-nozzle- based coaxial mixer that projects a jet of crystals at 10-30 m / s, yielding time resolutions of 30 ms and most recently 5 ms.

[0010] AtXFELs and synchrotrons, crystal and ligand solutions have been combined in a T-junction mixer and then deposited onto a moving x-ray transparent "conveyor belt", yielding time resolutions of seconds; and ligand solution drops have been deposited onto crystal containing drops on a conveyor belt, yielding time resolutions of >200 ms. “Fixed-target” approaches suitable for both XFEL and synchrotron sources have also been demonstrated: crystals are loaded onto a silicon wafer having an array of microfabricated wells, reactant solution is sequentially dispensed into each well, and x-ray data collected from each well a fixed time delay after dispensing, yielding time resolutions of ~30 ms. These approaches are more economical in terms of sample consumption than the microfluidic jet-based approaches, but still involve complex apparatus at the x-ray beamline. In all these approaches, optimization for a given biomolecular crystal system and efficient serial data collection requires multiple collaborators, knowledgeable beamline staff, and often extended beamline access.

[0011] All these approaches require complex set-ups at the x-ray beamline for reaction initiation and sample delivery. Development, operation, and maintenance of required hardware and software require large teams so typical TR-SX publications have 20- 40 authors. Sample consumption is enormous. In one study by Pandey et al., data was collected from ~ 100,000 crystals of size ~10 by 10 by 2 pm. The XFEL pulse structure and beam size illuminated only ~0.004% of the sample that flowed through the beam, so ~ 3xl09crystals were consumed per time point. In the fixed-target approach of Mehrabi et al., ~10, 000-40, 000 lysozyme crystals of size ~20 2 pm were measured for each time point; a comparable number were loaded onto the target wafer but were inaccessible to the x-ray beam. Suitable setups for TR-SX are available at only a handful of XFEL and synchrotron beamlines worldwide; gaining access typically requires multiple investigator collaborations; and access time per proposal is severely limited. As a result, TR-SX remains a specialist pursuit restricted to (typically well-studied) proteins whose crystals can be produced in the required quantities.

[0012] Recent excitement has focused on TR single-particle cryo-EM, due to advances in achievable resolution. In a biomolecular crystal, ligand binding and subsequent conformational motions can be inhibited by contacts between molecules in the crystal, and reactions may be completely blocked. In cryo-EM, the biomolecules are in solution and so may exhibit the full range of biologically relevant motions (although interaction with liquidair interfaces may perturb the conformational ensemble.) In TR-cryo-EM, biomolecule and ligand solution are mixed and deposited on a grid just before or during plunging into liquid ethane. Minimum reported time resolutions are ~7-10 ms, no better than in TR-SX, and have not improved substantially in the last 25 years. They are limited by mixing times, minimum feasible heights above the liquid ethane surface for sample deposition of several mm, and sample plunge speeds of a few m / s. Electron density map resolutions are typically worse than in crystallography (with a median of 3-4 A and <1% below 2 A for structures deposited in 2020), and usually insufficient to resolve mechanistically important details such as active-site side-chain conformations. Obtaining reconstructions of biomolecules smaller than ~100 kDa - which includes most enzyme monomers (average MW ~48 kDa for human proteins) - remains a serious challenge. Unlike synchrotron beamtime, which is free to academic users, cryo-EM time is expensive. However, TR-cryo-EM does not require crystallization, protein / ligand consumption is orders of magnitude smaller than in TR-SX, and samples can be prepared in the home lab and measured at any cryo-EM facility usingstandard cryo-EM instruments and protocols. As a result, it is far more accessible to the general structural biology community and applicable to far more targets than current TR- SX methods.

[0013] Freeze-quenching provides an approach to obtaining time-resolved data that has been productively used in TR cryo-EM, in spectroscopic studies (e.g., EPR) of liquid and powder samples, and also in time-resolved x-ray crystallography experiments. In this approach, ligand / small molecule containing solution is applied to a crystal. After some time interval tdeiay, the crystal is plunged into a cryogenic liquid (e.g., liquid nitrogen, liquid ethane). This stop (quenches) the reaction and, if cooling is fast enough, captures most aspects of the reaction-temperature (typically, room temperature) structure. The cryocooled crystals can then be stored, shipped to an x-ray source, and data collected remotely by a single investigator using standard cryocrystallography hardware and automated beamlines.

[0014] Unlike in time-resolved studies conducted at room / biological temperature, freeze-quenching allows the reaction and the measurement to be performed at different times and places. Measurement times can be much longer than reaction times, allowing data collection to be optimized (e.g., by first establishing the crystal position and orientation and then collecting data over an optimal range of positions and orientations) and allowing more data to be collected per crystal per time point. Radiation damage per unit dose is dramatically reduced at cryogenic temperatures, allowing far more data — roughly 50 times more data at synchrotron sources — to be collected per crystal. Furthermore, unlike in room-temperature TR-SX measurements, x-ray data can be collected from the full volume of every crystal produced, rather than from a small fraction of crystals and / or from a small fraction of each crystal’s volumes, as is typical in TR-SX. As result, complete structures at each time point can be obtained using as few as one crystal.

[0015] Freeze-quenching was used in time-resolved x-ray crystallography studies as early as the 1990s. Minimum time resolutions in these experiments ranged from seconds to minutes, limiting their usefulness to study of enzyme systems that had very slow turnover rates in crystals and other very slow processes. Time resolution was limited by available x-ray beam flux density and x-ray detectors. These limited the minimum crystal size from which x-ray data with adequate signal-to-noise could be acquired to about 50 pm and thus the minimum time required for diffusion into crystals to about 100 ms. Time resolution was also limited by modest cooling rates achieved by standard plunge incryogenic liquid methods of <103K / s, so that times required to cool from the initial reaction temperature to well below 200 K (where the reaction would be quenched) were >100 ms, longer than most reactions. Poor time resolution and lack of suitable hardware for fast and reliable sample preparation have hindered broad adoption of the mix and quench method in crystallography.

[0016] The high brilliance of both XFELs and current generation storage ring light sources combined with current x-ray detectors and analysis pipelines now enable useful data to be collected from micrometer size crystals, for which diffusion times can be below 5 ms. Advances in liquid nitrogen (LN2) cryocooling technology based on high-speed plunging and removal of cold gas layers allow even ~30 mm samples to be cooled to below 150 K in <3 ms and vitrification of cryo-EM samples. Diffusion and cooling times in the low millisecond range should allow observation of slow dynamic processes (e.g., motions of active site loops and flaps) and many (but not all) chemical intermediates, and kinetic capture of main-chain and many side chain conformations at biological temperature for low-temperature study.

[0017] Aside from X-ray crystallography, there are several other fields that could benefit from improved methods for initiating a reaction or other change in a sample and then freeze-quenching the sample for later observation. These include single particle cryoelectron microscopy, where the samples are 20-200 nm thick films containing biomolecules of interest, and study of thin slices of biological materials such as tissue slices. In both cases, the small minimum dimension of the samples allows rapid diffusion and reaction initiation, so that time resolutions in the millisecond range and even in the hundreds of microseconds are feasible.SUMMARY OF THE EMBODIMENTS

[0018] Aspects of the present disclosure relate to the design, function, and use of computer controlled and manual systems for preparing crystals of biomolecular and chemical systems for time-resolved x-ray crystallography and other time-resolved physical measurements such as spectroscopy.

[0019] Specifically, they relate to methods for time-resolved crystallography that involve initiating a reaction or other change within a crystal by rapidly bringing the crystal into contact with a solution that diffuses into the crystal and chemically triggers a change in structure. Then, after a variable time interval Dt, the crystal sample is rapidly cooled tocryogenic temperature, with the goal of capturing / quenching the room temperature state at time Dt as faithfully as possible for later examination by x-ray cryocrystallography.

[0020] The innovations described here should yield time resolutions Dt of a few milliseconds, comparable to or better than can be achieved using any other x-ray-based method, allow collection of complete structural data sets sufficient for structure determination using as few as one crystal per time point Dt, allow sample preparation for time-resolved studies with a relatively simple and inexpensive instrument, and allow x-ray data collection via routine mail-in, remote cryocrystallography at standard high-throughput cryocrystallography beamlines at storage ring (synchrotron) x-ray sources.

[0021] These innovations will make time-resolved x-ray crystallography, which until now has been a specialist pursuit suitable for only a tiny subset of biomolecular systems, broadly accessible to the structural biology community.

[0022] Although focused on chemical-based triggering of changes / reactions in biomolecular crystals and then cryoquenching for later examination by x-rays, the innovations described here can also be applied to non-biological crystals and also to cells, tissues, and other small biological samples that, once cryocooled, may be examined using x- rays and other probes including various spectroscopies and optical (including nonlinear and fluorescent) imaging methods. The innovations described here can also be used for study of structural changes triggered by other mechanisms such as illumination with light / electromagnetic radiation.

[0023] As used herein, the term “low temperature” may be defined to include a temperature that leads to full or partial vitrification of a solvent within a sample, which may depend on the solvent composition and solutes present. For example, a typical low temperature in this application is the temperature of boiling liquid nitrogen (77 K = -196 °C). Temperatures less than about 140 K (-123 °C) may completely inhibit ice crystal formation in samples held at those temperatures.

[0024] As used herein, the term “cryogenic liquid” may refer to any substance with a freezing temperature below 150 K and may be below 120 K, including nitrogen, propane, and ethane. In most TR-SX applications, the cryogenic liquid may be liquid nitrogen (LN2).

[0025] As used herein, the term “ligand solution” may refer to a solution of any molecule or molecules that may interact and bind with a biomolecule, and that may thereby cause a change in structure or dynamics of the biomolecule and / or of a ligand, trigger achemical reaction involving the biomolecule and possibly also a ligand, or modulate a chemical reaction. Use here will also include a solution including solutes — small molecule compounds and polymers — that may change the ionic strength, pH, water activity, or viscosity, among other properties, of the solvent within the biomolecular sample or the pKas of amino acids within the protein, and that may thereby cause a change in structure or dynamics of the biomolecule or trigger a chemical reaction involving the biomolecule.

[0026] As used herein, the term “goniometer base” may refer to a base, usually of magnetic steel, that attaches to goniometer stages that hold samples in x-ray beams, and that accepts and holds a rod to which a loop or microfabricated film sample support may be attached.

[0027] As used herein, “crystallography sample support” may refer to a polymer loop or a microfabricated thin film attached to a rod or to a frame, that is inserted into a goniometer base, and that is used to hold crystals in x-ray beams for data collection.

[0028] As used herein, the terms “cryocrystallography sample storage cassette”, “sample storage cassette”, “cassette”, and Unipuck” refer to an assembly or cassette into which multiple crystallography sample supports attached to standardized goniometer bases can be inserted and be held for storage, shipping, and automated handling at an x-ray source.SYSTEM COMPONENTS AND SPECS

[0029] Aspects of this disclosure include structures, apparatuses and approaches to facilitate handling, chemical / diffusion based triggering of changes / reactions, and then rapid cooling to cryogenic temperature of biomolecular crystals and other small (millimeter or smaller and in embodiments, 200 micrometer and smaller) samples so as to capture the state of the sample at its initial temperature some time Dt after triggering for study at low / cryogenic temperatures below 200 K and preferably below 150 K.

[0030] Aspects of this disclosure include structures, apparatuses, and approaches that allow rapid translation of samples from a reaction initiation point into a cryogenic liquid, where rapid includes speeds between about 0.1 m / s and 10 m / s and, in embodiments, between about 1 and 4 m / s.

[0031] Aspects of this disclosure include structures, apparatus, and approaches that facilitate cooling of the samples at the largest possible cooling rates, and preferably atrates greater than 15,000 K / s, so that sample cooling from 273 K to below 150 K occurs in less than 10 milliseconds.

[0032] Aspects of this disclosure include structures, apparatus, and approaches that allow rapid application of a ligand-containing liquid to a sample, including while the sample is translating at high speed toward a cryogenic liquid, where rapid application is in a time less than 50 ms and , in embodiments, less than 10 ms.

[0033] Aspects of this disclosure include structures, surface treatments, and approaches that facilitate transfer of crystals and of ligand solution to a crystallography sample support to provide uniform coverage of samples on a crystal-accepting portion of a crystallography sample support.

[0034] Aspects of this disclosure include structures, apparatus, and approaches for storing samples after reaction initiation and cryocooling and facilitating their automated handling at an x-ray source.Basic System

[0035] According to aspects of this disclosure, a system for preparing samples for time-resolved crystallography experiments may be comprised of a crystallography sample support, a motor- or gravity-driven high-speed sample vertical translation stage or a robot capable of high speed vertical motion, a sample carriage attached to the stage or robot that accepts and holds the sample support, a sample cryocooling and storage unit holding a first volume of cryogenic liquid, a cryogenic liquid storage and filling unit, a reaction initiation stage, and a means for applying ligand solution to the sample and its holder as they are translating toward the cryogenic liquid surface.Sample support and wand

[0036] According to aspects of this disclosure, one or more crystals of a biological molecule, biomolecular complex, or a small molecule compound having a minimum dimension less than about 200 micrometers or, in some applications, less than about 20 micrometers, is held on a crystallography sample support.

[0037] According to aspects of this disclosure, the sample support may include a thin microfabricated film having a region configured to accept one or more crystals, where the film thickness in the sample-accepting region may be less than 25 micrometers or, in some applications, less than 10 micrometers or, in some applications, less than 5micrometers, a rod or frame to which the film is attached, and a goniometer base into which the rod or frame is inserted.

[0038] According to aspects of this disclosure, the region of the sample support film configured to accept one or more crystals has at least one through-hole to facilitate removal of excess liquid.

[0039] According to aspects of this disclosure, the region configured to accept one or more crystals may be plasma treated to render it more hydrophilic, while leaving the rest of the film in its native state or a state determined by a previous treatment (e.g., hydrophobic).

[0040] According to aspects of this disclosure, the sample support film is attached to a thin (25 to 200 pm) frame, which has an aperture or other opening exposing the sample-accepting portion of the sample support film.

[0041] According to aspects of this disclosure, the sample support film is alternatively attached to a curved support rod that imparts curvature to the film to rigidity it.

[0042] According to aspects of this disclosure, the system may include a wand that holds the crystallography sample support, such as a prior art wand used in cryocrystallography.

[0043] According to aspects of this disclosure, the wand may include a magnet at one end and may grab, hold, and release the goniometer base of a sample support during subsequent handling and processing.

[0044] According to aspects of this disclosure, the wand may be inserted into and removably held by the sample carriage attached to the sample vertical translation stage.

[0045] According to aspects of this disclosure, the wand may be inserted into a wand adapter that in turn can be inserted into and removably held by the sample carriage.

[0046] According to aspects of this disclosure, the crystallography sample support with its goniometer base may be magnetically held to the sample carriage, without use of a wand or wand adapter.

[0047] According to aspects of this disclosure, the sample support may be held via a sample carriage to a robotic arm, which executes sample vertical and horizontal motions.Sample vertical translation stage

[0048] According to aspects of this disclosure, motion of the sample carriage of the sample vertical translation stage may be driven by a motor (including a DC, servo, or stepper motor), by pneumatics, or electromagnetically.

[0049] According to aspects of this disclosure, motion of the sample carriage and sample may be driven by rotation of a lead screw driven by a motor.

[0050] According to aspects of this disclosure, motion of the sample and sample holder may be executed using a robot.

[0051] According to aspects of this disclosure, motion of the sample carriage and sample may be driven by gravity.

[0052] According to aspects of this disclosure, the speed of the sample carriage, sample wand, and sample when the sample enters the cryogenic liquid may be between 0.1 and 10 m / s and , in embodiments, between 1 and 4 m / s, to ensure rapid cooling without excessive splashing or excessive stopping distances within the cryogenic liquid.

[0053] According to aspects of this disclosure, the sample vertical translation stage may be configured so that side-to-side motion of the sample carriage and sample during a plunge and side-to-side irreproducibility of motion during a plunge of the sample is less than about 1 mm or, in some applications, less than about 0.1 mm.

[0054] According to aspects of this disclosure, the sample carriage may slide on double rails (one on each side of the carriage) to minimize side-to-side motion.

[0055] According to aspects of this disclosure, the sample vertical translation stage may allow the sample to be translated downward some distance, then held in place at that position for some time, and then plunged from that position at high speed into cryogenic liquid.

[0056] According to aspects of this disclosure, the sample vertical translation stage can be programmed to translate the sample between different heights above the cryogenic liquid surface, to stop and hold for a time interval at a location, to accelerate the sample to a given speed and to decelerate it to rest.

[0057] According to aspects of this disclosure, the sample carriage may be attached to a slider bushing / bearing / post and fall under the influence of gravity toward the cryogenic liquid, with its release to free fall controlled magnetically, electromagnetically, or mechanically.

[0058] According to aspects of this disclosure, the motor of the sample vertical translation stage may be configurable via software to drive the translation stage with different preset variable acceleration profiles, so that the vertical motion of the sample carriage along the plunge path can be varied.

[0059] According to aspects of this disclosure, the motor may be equipped with an encoder that allows the number of motor rotations and thus the sample position versus time to be monitored electronically.

[0060] According to aspects of this disclosure, the sample vertical translation stage may allow repeatable movement of the sample carriage along the plunge path such that the time to travel between any two positions is repeatable to within ±1 ms.Sample cryocooling and storage unit

[0061] According to aspects of this disclosure, the sample cryocooling and storage unit may be comprised of a cryocooling chamber, a gas exchange manifold, and a lid.

[0062] According to aspects of this disclosure, the cryocooling chamber may include structures to support the gas exchange manifold and cover, to hold a sample storage cassette, and to define a plunge channel below the gas exchange manifold containing a volume of cryogenic liquid.

[0063] According to aspects of this disclosure, the cryocooling chamber has insulation suitable for holding a cryogenic liquid.

[0064] According to aspects of this disclosure, the sample cryocooling chamber may be fabricated from a polymer foam (e.g., high density polyurethane foam) material.

[0065] According to aspects of this disclosure, the interior bottom of the cryocooling chamber may include a structure designed to accept a standard sample storage cassette and hold it in position (x and y within the plane and also rotationally), where the structure may be machined or otherwise fabricated into the interior bottom surface of the chamber, or where it may be a separately fabricated part that is fastened to the bottom interior surface of the chamber.

[0066] According to aspects of this disclosure, the structure designed to accept a standard sample storage cassette is located a horizontal distance d away from the sample plunge axis (the vertical axis on which the sample travels) where d is greater than the cassette radius + at least 2 cm to ensure that the cassette does not interfere with sampleplunging, and where d is greater than the cassette radius plus the length of the plunge channel from sample plunge axis to the channel’s end closest to cassette.

[0067] According to aspects of this disclosure a standard sample cryocooling cassette has a diameter between 6 and 12 cm, in some instances, about 6.7 cm.Gas exchange manifold

[0068] According to aspects of this disclosure, a gas exchange manifold including a plunge bore centered on the sample’s vertical plunge axis, and that is in contact with the cryogenic liquid within the cryocooling chamber, may be located above at least a portion of the cryocooling chamber.

[0069] According to aspects of this disclosure, the gas exchange manifold acts to remove cold gas that forms above the cryogenic liquid surface and replace it with ambient (or other) temperature dry gas, so that the sample experiences an abrupt transition from gas at ambient temperature to cryogenic liquid at cryogenic temperature when it is plunged.

[0070] According to aspects of this disclosure, the manifold has opposing channels intersecting the plunge bore through which suction can be applied to remove cold gas from the plunge bore and ambient temperature gas injected to replace the cold gas.

[0071] According to aspects of this disclosure, the gas exchange manifold further includes a heater that prevents frost formation on manifold surfaces exposed to moist air.

[0072] According to aspects of this disclosure, the gas exchange manifold rests with its plunge bore centered on the plunge channel in the cryocooling chamber, with the bottom edges of the plunge bore located within the plunge channel and below its top surface.

[0073] According to aspects of this disclosure, the gas exchange manifold is in contact with and rests on the cryocooling chamber adjacent to and outside the plunge channel, preventing cryogenic liquid from contacting and cooling the portions of the bottom surface of the gas exchange manifold that are remote from the plunge channel.

[0074] According to aspects of this disclosure, the lower portion of the gas exchange manifold adjacent to the plunge bore is partially submerged in cryogenic liquid.

[0075] According to aspects of this disclosure, the top surface of the gas exchange manifold coincides with the top surface of the cryocooling chamber.

[0076] According to aspects of this disclosure, the gas exchange manifold may have a sample exchange channel open at top and bottom extending from the plunge boretoward the main volume of the cryocooling chamber to allow transfer of a sample from the plunge bore and into the region of the main volume containing the sample storage cassette.

[0077] According to aspects of this disclosure, the walls defining this channel may contain opposing gas channels to remove cold gas from this channel and replace it with ambient temperature nitrogen gas to prevent cold gas from traveling into the plunge bore.

[0078] According to aspects of this disclosure, the side of the plunge bore perpendicular to the plunge channel in the cryocooling chamber, adjacent to the sample exchange channel, and nearest the sample storage cassette receptacle may have a vertically oriented swing arm door extending into the cryogenic liquid that is closed during plunging to isolate gas flows within the plunge bore from gas in the rest of the cryocooling chamber, and that swings open when a sample holding wand pushes against it, allowing the sample holding wand to be horizontally translated out of the plunge bore and plunge channel and into the main portion of the cryocooling chamber without being removed from the cryogenic liquid.

[0079] According to aspects of this disclosure, the door is held in the closed position using two magnets, one in the door and one opposite the magnet in the door.

[0080] According to aspects of this disclosure, the main body of the gas exchange manifold may be 3D printed from PLA, ABS, or a cryogenic-temperature compatible glass- stabilized resin.Lid for cryocooling chamber

[0081] According to aspects of this disclosure, the top surfaces of the cryocooling chamber and gas exchange manifold may be covered by a lid having an aperture over the plunge bore, that largely seals the cryocooling chamber from the outside air and minimizes contact of moist outside air with cold interior surfaces of the cryocooling chamber and gas exchange manifold.

[0082] According to aspects of this disclosure, the interior facing portion of the lid may be of a material with poor thermal conductivity such as a polymer or glass.

[0083] According to aspects of this disclosure, the top, outward facing surface of the lid may be of a thermally conducting material such as a metal.

[0084] According to aspects of this disclosure, the top portion of the lid may incorporate heaters to maintain the lid’s surface near room temperature and prevent frosting.

[0085] According to aspects of this disclosure, the lid may include a second circular aperture directly above the receptacle for a sample storage cassette in the cryocooling chamber that is large enough (about 7-12 cm in diameter for a 6.7 cm diameter cryocrystallography cassette) to allow insertion of a cassette into the cryocooling chamber and removal of the cassette.

[0086] According to aspects of this disclosure, the plunge bore aperture and the cassette aperture in the lid may be connected by a through-channel above the sample exchange channel in the gas exchange manifold large enough for a sample wand to be translated through.

[0087] According to aspects of this disclosure, a circular, optically clear disk of diameter matched to the inside diameter of the cassette aperture in the lid drops into that aperture and is supported by it, so that the disk may be rotated about a vertical axis.

[0088] According to aspects of this disclosure, the optically clear disk includes a through-channel projecting from the plunge bore to the lid and running from its outer edge inward toward its axis such that a sample held by a wand in the plunge bore can be translated through the channel in the lid and the channel in the clear disk, and then rotated with the disk until it is over a receptacle in a cassette into which the sample can be deposited, without the sample ever leaving the cryogenic liquid.

[0089] According to aspects of this disclosure, the optically clear disk may be of a glass or of an optically clear polymer.

[0090] According to aspects of this disclosure the channel in the main body of the lid and / or the channel in the clear disk lid may be partially sealed from outside air by brushes, membranes or other mechanisms that allow translation of the sample wand through them.Cryogenic liquid storage and filling unit

[0091] According to aspects of this disclosure, the cryogenic liquid level within the cryocooling chamber is set and maintained to reside within the height of the make-up gas and vacuum apertures in the gas exchange manifold where they intersect the plunge bore, to ensure a laminar flow of gas from the make-up gas aperture across the cryogenic liquid surface in the plunge bore to the vacuum aperture.

[0092] According to aspects of this disclosure, the cryogenic liquid level is held to within ±1 mm of the target value.

[0093] According to aspects of this disclosure, the cryogenic liquid level within the cryocooling chamber may be measured using heated RTDs, diodes, thermocouples, or using a laser level sensor.

[0094] According to aspects of this disclosure, the level sensors may be located in a portion of the cryocooling chamber that is isolated from waves on the surface of the cryogenic liquid or that includes feature that damp out waves.

[0095] According to aspects of this disclosure, a thermally insulated cryogenic liquid storage and filling unit may contain a second volume of the cryogenic liquid greater than the volume held in the cryocooling chamber.

[0096] According to aspects of this disclosure, the second volume of cryogenic liquid in the storage unit may be connected to the first volume of cryogenic liquid within the cryocooling chamber via a thermally insulated pipe, that includes a manually or electrically actuated valve.

[0097] According to aspects of this disclosure, when the cryogenic liquid level in the cryocooling chamber drops below a target value, the valve connecting cryocooling and storage chamber may open, raising the cryogenic liquid level to its target value.Reaction initiation stage

[0098] According to aspects of this disclosure, the system for preparing samples for time-resolved crystallography experiments may be further comprised of a second vertical translation stage — the reaction initiation stage — adjacent to and with an axis parallel to the sample plunge axis, that allows the apparatus used to initiate the reaction in the sample to be translated up and down relative to the cryogenic liquid surface, allowing the distance above the cryogenic liquid surface at which the reaction is initiated and thus the time interval between reaction initiation and the start of cryocooling to be adjusted.

[0099] According to aspects of this disclosure, the reaction initiation stage may translate the reaction initiation apparatus up and down using a motor and lead screw, under either manual (switch) control or under computer control.

[0100] According to aspects of this disclosure, the reaction initiation stage may translate the reaction initiation apparatus up and down manually using a hand crank and screw or a slider with stops.

[0101] According to aspects of this disclosure the reaction initiation stage has a vertical position accuracy and reproducibility of at least 0.1 mm.

[0102] According to aspects of this disclosure, the motor driven reaction initiation stage has a maximum speed of 0.01 m / s or, in some instances, 0.2 m / s or, in some instances, 0.5 m / s.

[0103] According to aspects of this disclosure, the reaction initiation stage allows reaction initiation in the sample at a minimum sample height of 10 millimeters or, in some instances, of 5 mm or, in some instances, of 2 mm above a plane located at the vertical midpoint of openings of the dry gas and vacuum channels at the plunge bore, this plane in normal operation corresponding to the level of the cryogenic liquid.

[0104] According to aspects of this disclosure, the reaction initiation stage holding the reaction initiation apparatus can be moved from a position well above the cryogenic liquid surface, where the reaction initiation apparatus and any ligand solution contained by it is not appreciably cooled by proximity to the cryogenic liquid, to a position close to the cryogenic liquid surface prior to the sample plunge into the cryogenic liquid.

[0105] According to aspects of this disclosure, the time to translate the reaction initiation apparatus between an initial position equal to the lowest position at which any ligand solution contained therein does not cool more than 2 °C below ambient temperature (due to its proximity to the cryogenic liquid) when held there for at least 1 minute and a final position at which reaction initiation occurs and where such cooling may be significant is less than 2 seconds and, in embodiments, less than 0.5 s.

[0106] According to aspects of this disclosure, the lowest position at which the ligand solution within the reaction initiation apparatus does not cool more than 2 K below ambient temperature (when held for at least 1 minute) is between 2 and 6 cm above the cryogenic liquid.Loop-based reaction initiation apparatus

[0107] According to aspects of this disclosure, the reaction initiation stage accepts a reaction initiation apparatus that includes a break-way loop, a swing-arm door loop mechanism, a scissors loop mechanism, or another loop-based mechanism for depositing ligand solution on the sample, and positions the loop so that it is centered on the sample plunge axis at some vertical position between the sample’s initial position and the surface of the cryogenic liquid.

[0108] According to aspects of this disclosure, the reaction initiation mechanism may be comprised of a loop centered on the sample’s plunge axis that is spanned by a thin film of ligand solution. According to aspects of this disclosure, the sample is plungedY1through the ligand solution film spanning the loop, so that some of the solution within the loop is transferred onto the sample and its support.

[0109] According to aspects of this disclosure, the loop has a diameter between 1 and 10 mm and, in some instances, about 5 mm and, in some instances, about 3 mm, and in some instances about 2 mm, and in some instances, about 1 mm, and a thickness between 0.1 and 3 mm.

[0110] According to aspects of this disclosure, the plane of the loop is perpendicular to the sample plunge axis.

[0111] According to aspects of this disclosure, the loop is attached to the reaction initiation stage via one or more supporting members that insert into or attach to the stage.

[0112] According to aspects of this disclosure, the loop and supporting members are fabricated from a flexible material such as a polymer and may be 3D printed or injection molded.Breakaway loop

[0113] According to aspects of this disclosure, the loop is structured with a constriction or nick at a position around its circumference, and the supporting members are structured so as to easily bend downward, so that when a sample support including a goniometer base is plunged, the sample and supporting film, rod and / or frame passes through the loop, and then the goniometer base impacts the loop and the supporting member and the loop breaks open and the supporting member bends, allowing the goniometer base to pass on to the cryogenic liquid.

[0114] According to aspects of this disclosure, the supporting member holding the loop is structured with a constriction or nick at a position near the loop, so that when a sample support including a goniometer base is plunged, the sample and supporting film, rod and / or frame passes through the loop, and then the goniometer base impacts the loop and supporting member, the member breaks and releases the loop, and the loop initially continues downward with the base into the cryogenic liquid, eventually falling off the base and into the cryogenic liquid.

[0115] According to aspects of this disclosure, instead of a vertically translating reaction initiation stage, the ligand solution-containing loop can be held by a vertical member containing a series of holes arranged vertically between the initial (“home”) sample position and the cryogenic liquid surface that can each accept a supporting member of the loop and hold the loop centered on the plunge axis of the sample translation stage,allowing loops to be positioned at many different heights above the cryogenic liquid surface.Saloon door loop

[0116] According to aspects of this disclosure, the reaction initiation apparatus may comprise a “saloon door” loop mechanism attached to frame that attaches to the reaction initiation stage, where the ligand solution-holding loop may be comprised of two mating or overlapping semicircular halves, each attached to a separate arm or “door half”, configured so that after the sample passes through the loop, the doors and loop open and swing out of the way, allowing the rest of the sample support including the goniometer base and the sample carriage to travel downward toward the cryogenic liquid.

[0117] According to aspects of this disclosure, in a “load” position the two doors and / or the loop lie in a horizontal plane perpendicular to the sample plunge axis, allowing ligand solution to be deposited on and held by the loop.

[0118] According to aspects of this disclosure, each arm or door half is attached to a pivot or axle oriented perpendicular to the sample plunge axis and parallel to each other, and located on opposing sides of the sample plunge axis, such that the two door halves can swing downwards, separating the two halves of the loop and swinging them and the door halves out of the way of the sample support (including the goniometer base) and sample carriage as needed.

[0119] According to aspects of this disclosure, the two halves of the loop and / or the doors may be configured to interlock and stay held together within a horizontal plane when they are rotated upward to their ligand solution loading position.

[0120] According to aspects of this disclosure, the two doors may contain small opposing magnets that hold the doors in a horizontal position until the doors are pushed downward, separating the magnets and allowing the doors to swing downward.

[0121] According to aspects of this disclosure, the loop and doors may be fabricated from a rigid or semi-rigid material such as a polymer or a metal.

[0122] According to aspects of this disclosure, the loop and doors are opened, after the sample has passed through the loop, by physical contact with the goniometer base or with a member attached to the sample carriage as the base and carriage move toward the cryogenic liquid surface.

[0123] According to aspects of this disclosure, the loop and doors are structured to be robust against impacts of the goniometer base or sample carriage member, so that they withstand repeated impacts without failure.

[0124] According to aspects of this disclosure, the total mass and moment of the inertia of the doors may be minimized by fabricating the doors from a polymer, structuring the doors to provide rigidity and robustness while using as little polymer as possible, and by restricting the width and length of the doors perpendicular to the hinge to be comparable to the diameter of the goniometer base and between about 1 cm and 3 cm.

[0125] According to aspects of this disclosure, the doors may be angled downward when they are interlocked to form a horizontal loop at their bottom end, so that they receive a more glancing impact from the goniometer base or plunge stage that more gradually pushes them open and reduces wear and tear.Scissors loop

[0126] According to aspects of this disclosure, the reaction initiation apparatus may comprise a “scissors” loop mechanism attached to platform that attaches to the reaction initiation stage, where the ligand solution-holding loop may be comprised of two mating or overlapping semicircular halves, each attached to a separate arm that is attached to a common pivot or axle or to two independent pivots, oriented parallel to the sample plunge axis, allowing the arms and loop to be opened and closed in a plane perpendicular to the sample plunge axis in a scissor-like manner (in the case of a common pivot / axle) and in a tongs-like manner (in the case of two independent pivots.

[0127] According to aspects of this disclosure, the arms that hold the two halves of the loop may be pushed / pulled apart by one or more springs (e.g., a coil spring, two extension springs, and held together by a magnet located in one or both arms.

[0128] According to aspects of this disclosure, means are provided so that the loop opens after the sample has passed through it so that the sample support including the goniometer base and the sample carriage may continue travel toward the cryogenic liquid.

[0129] According to aspects of this disclosure, a portion of each arm extends upward from the loop at a radius larger than the loop but smaller than a radius of the goniometer base, and may be angled outward to end at radius that is larger than the radius of the goniometer base, so that the arms and loop are pushed open when the goniometer base passes through the upward extending portions of the arm, allowing the goniometer base to pass through the loop as the sample plunges toward the cryogenic liquid.Liquid capture

[0130] According to aspects of this disclosure, one or more small sheets of absorbent material may be held just below the plane of the loop or aperture, such that when ligand solution within the aperture is projected downward by contact with the sample and support, excess ligand solution is absorbed and prevented from falling into the cryogenic liquid.

[0131] According to aspects of this disclosure, one or more sheets of absorbent material may be held just above the gas exchange manifold near its bore to catch ligand solution drops that are projected downward from the loop.

[0132] According to aspects of this disclosure, absorbent sheets may be disposed on the plunge arms below the loop with their plane parallel to the plane of the loop, and forming an aperture on the sample plunge axis of 3 mm or 5 mm in diameter, such that when the sample passes through the ligand solution film, displacing and projecting the ligand solution downward, the sheets catch and absorb ligand solution that comes loose, preventing it from contacting the goniometer base or falling into the cryogenic liquid.

[0133] According to aspects of this disclosure, the two or more overlapping sheets have elongated partially overlapping apertures sized, shaped and oriented so as to form an opening 2-3 mm diameter, large enough to pass the sample and its holder but smaller than the goniometer base, and attached either to the bottom of the loop halves and doors or to a second set of doors located below the loop, so that they intercept ligand solution projected downward from the loop after sample contact that otherwise may be projected into the cryogenic liquid.Drop-on-demand based reaction initiation apparatus

[0134] According to aspects of this disclosure, the reaction initiation apparatus may include a drop-on-demand dispensing head with controller, with the head mounted on the reaction initiation stage to project drops in a largely horizontal direction.

[0135] According to aspects of this disclosure, the dispensing tip of the drop on demand dispensing head is positioned within 2 cm and, in some instances, within 0.5 cm of the sample plunge axis.

[0136] According to aspects of this disclosure, the drop dispensing head dispenses single drops with volumes between 0.05 and 100 nL and, in some instances, between 0.5 and 10 nL, and projects them horizontally at speeds between 0.5 and 20 m / s at the sample.

[0137] According to aspects of this disclosure, the drop dispensing head may dispense drops on the sample while the sample is stationary, or while the sample is plunging toward the cryogenic liquid surface at speeds up to the maximum speed of the sample vertical translation stage.

[0138] According to aspects of this disclosure, the gas exchange manifold above the cryogenic liquid includes a recessed portion adjacent to the plunge bore that allows the drop dispensing head to be positioned with its bottom surface near and below the level of cryogenic liquid in the bore, so that the drop dispensing tip is as little as 2 mm above the cryogenic liquid surface, allowing the tip to dispense drops within the plunge bore and onto the sample. If the lid covering the cryocooling chamber extends over the gas exchange manifold, the lid has an aperture that allows the dispensing head to access the recessed portion of the gas exchange manifold.

[0139] According to aspects of this disclosure, the bottom of the recessed portion of the gas exchange manifold may be between about 2 and 30 mm below the surface of the cryogenic liquid.

[0140] According to aspects of this disclosure, the reaction initiation stage and apparatus include a mechanism for precisely aiming the drop-on-demand dispenser tip so that dispensed drops hit the sample holding portion of the sample holder.

[0141] According to aspects of this disclosure, the system for preparing samples for time-resolved crystallography includes a mechanism for precisely timing the drop generation so that the drop strikes the sample even when the sample is moving at the maximum speed of the vertical translation stage.

[0142] According to aspects of this disclosure, this mechanism may include an optical, magnetic or other transducer mounted on the reaction initiation stage that senses when the sample is within a fixed vertical distance of the deposition tip and generates a delayed pulse to trigger drop generation at the appropriate time.

[0143] According to aspects of this disclosure, the timing signal from the transducer may be augmented with information from the motor controller regarding the motor’s speed and position to more precisely time the drop generation and account for variability in plunge stage motion.

[0144] According to aspects of this disclosure, ligand-containing liquid may be deposited onto the sample and sample-holding portion of the sample support while thesample and plunge stage are stationary using a piezoelectric drop / mist generator, using a venturi-type sprayer, or using continuous jet of ligand solution generated using a syringe.BRIEF DESCRIPTION OF THE FIGURES

[0145] FIG. 1A to ID shows examples of prior-art sample supports used to hold crystals for x-ray crystallography, suitable for holding crystals for time-resolved crystallography as implemented according to the present inventions.

[0146] FIG. 2 is a schematic illustration of a representative system for preparing samples for time-resolved crystallography and related measurements in accord with aspects of this disclosure.

[0147] FIGs. 3A-3C are schematic illustrations of a second representative system for preparing samples for time-resolved crystallography and related measurements in accord with aspects of this disclosure.

[0148] FIG. 4 is an isometric-view illustration of a representative system for preparing samples for time-resolved crystallography and related measurements in accord with aspects of this disclosure.

[0149] FIGs. 5A and 5B are front and right views of a representative sample vertical translation stage for use in the sample preparation system of FIG. 4 in accord with aspects of this disclosure.

[0150] FIGs. 6A to 6C are isometric-view illustrations of a magnetic sample wand, a magnetic sample wand adapter, and the wand / adapter assembly for use with the sample vertical translation stage of the sample preparation system of FIG. 4 in accord with aspects of this disclosure.

[0151] FIGs. 7A and 7B are top and isometric view illustrations of a representative sample cryocooling chamber.

[0152] FIGs. 8A and 8B are top and isometric view illustrations of a representative cryocrystallography sample storage cassette for use with the sample preparation system of FIG. 4 in accord with aspects of this disclosure.

[0153] FIGs. 9A and 9B are isometric-view illustrations and FIG. 9C and 9D are front and back views of a gas exchange manifold that is placed over a cryocooling chamber as in Fig. 8 for use with the sample preparation system of FIG. 4 in accord with aspects of this disclosure.

[0154] FIGs. 10A and 10B are isometric-view illustrations of a lid that is placed over a cryocooling chamber as in FIG. 7 and over a gas exchange manifold within the cryocooling chamber as in FIG. 8 for use with the sample preparation system of FIG. 4 in accord with aspects of this disclosure.

[0155] FIG. 11 is an isometric exploded view illustration of an assembly formed of a cryocooling chamber as in FIG. 7 with a gas exchange manifold as in FIGs. 9A-9D, lid as in FIG. 10, and crystallography cassette as in FIG. 8 for use with the sample preparation system of FIG. 4 in accord with aspects of this disclosure.

[0156] FIG. 12 is an isometric view illustration of a reaction initiation stage for use with the sample preparation system of FIG. 4 in accord with aspects of this disclosure.

[0157] FIGs. 13A-13C are isometric view illustrations of liquid transfer mechanisms comprised of breakaway and drop-in loops for use with the sample preparation system of FIG. 4 in accord with aspects of this disclosure.

[0158] FIGs. 14A-14C are isometric view illustrations of a saloon door loop liquid transfer mechanism for use with the sample preparation system of FIG. 4 in accord with aspects of this disclosure.

[0159] FIGs. 15A and 15B are isometric view illustrations of a scissors loop liquid transfer mechanism for use with the sample preparation system of FIG. 4 in accord with aspects of this disclosure.

[0160] FIG. 16 is an isometric view illustration of a sample preparation system as in FIG. 4 with a cryogenic liquid storage and filling in accord with aspects of this disclosure.

[0161] FIG. 17 is an isometric view illustration of an alternative loop liquid transfer mechanism allowing access to the earliest feasible time points for use with the sample preparation system of FIG. 4 in accord with aspects of this disclosure.

[0162] FIGs. 18A-18D is an isometric view illustration of the sequence of motion executed by the sample and the loop liquid transfer mechanism of FIG. 17 when a sample is plunged from an initial position, through the loop and liquid film it holds, and into liquid nitrogen.

[0163] FIG.

[0164] The present disclosure is amenable to various modifications and alternative forms, and some representative embodiments of the disclosure are shown by way of example in the drawings and will be described in detail herein. It should be understood, however, that the novel aspects of this disclosure are not limited to theparticular forms illustrated in the above-enumerated drawings. Rather, this disclosure covers all modifications, equivalents, combinations, permutations, groupings, and alternatives falling within the scope of this disclosure as encompassed, for example, by the appended claims.DETAILED DESCRIPTION OF THE EMBODIMENTS

[0165] This disclosure is susceptible of embodiment in many different forms. Representative embodiments of the disclosure are shown in the drawings and will herein be described in detail with the understanding that these embodiments are provided as an exemplification of the disclosed principles, not limitations of the broad aspects of this disclosure. To that extent, elements and limitations that are described, for example, in the Abstract, Introduction, Summary, Description of the Drawings, and Detailed Description sections, but not explicitly set forth in the claims, should not be incorporated into the claims, singly or collectively, by implication, inference or otherwise. Moreover, recitation of “first”, “second”, “third”, etc., in the specification or claims is not per se used to establish a serial or numerical limitation; unless specifically stated otherwise, these designations may be used for ease of reference to similar features in the specification and drawings and to demarcate between similar elements in the claims.

[0166] For purposes of this Detailed Description, unless specifically disclaimed: the singular includes the plural and vice versa (e.g., indefinite articles “a” and “an” are to be construed as meaning “one or more” unless expressly disclaimed); the words “and” and “or” shall be both conjunctive and disjunctive; the words “any” and “all” shall both mean “any and all”; and the words “including,” “containing,” “comprising,” “having,” and the like, shall each mean “including without limitation.” Moreover, words of approximation, such as “about,” “almost,” “substantially,” “generally,” “approximately,” and the like, may each be used herein to denote “at, near, or nearly at,” or “within 0-5% of,” or “within acceptable manufacturing tolerances,” or any logical combination thereof, for example.

[0167] Features of the present disclosure aim to address the design criteria and challenges listed below to optimize cryopreservation and recovery of small biological samples, including minimizing sample damage resulting from handling, osmotic shock, cryoprotectant toxicity, ice formation, fracturing, and other factors, to maximize survival and development of cellular systems. Additional information pertinent to this disclosure may be found, for example, in U.S. Patent Nos. 9,417,166 B2, to Thorne et al., 11,473,826 B2,to Closs et al., and 11,653,644 B2 to Thorne et al., all of which are incorporated herein by reference in their respective entireties and for all purposes.

[0168] Figs. 1A-1D shows examples of crystallography sample supports for holding protein and other biomolecular crystals in an x-ray beam. Herein, the term sample support and sample holder are synonymous. FIG. 1A depicts a sample holder 101, which includes a goniometer base 111, a steel rod 120 that is inserted (e.g., using tweezers) into the base, and a sample (crystal) holding portion 140, which may be formed of a polymer loop or polymer film. Base 111 may be a magnetic steel base that mates to goniometers used to rotate crystals during x-ray data collection

[0169] Fig. IB and 10 show examples 160 and 180 of sample holding thin films suitable for use with the present invention. Fig. ID shows sample holder 102, which includes a goniometer base 112, a thin frame 220 having an aperture that is spanned by a thin, x-ray transparent polymer film 240 on which crystals are deposited.

[0170] Fig. 2 shows a schematic illustration of a system for preparing samples for time-resolved crystallography in accord with aspects of the present invention. A sample (for example, a crystal) 260 on sample holder 101 base 111 is attached to a sample carriage 280, which is attached to and moved up and down by a sample vertical translation stage 203 that translates the sample from an initial position in air / gas to a final position within a cryogenic liquid 320.

[0171] A loop 340 containing solution 360 to be deposited on and soaked into the crystal 260 is located with its center on the path of the sample as it is translated toward the cryogenic liquid - the sample plunge axis. The cryogenic liquid is held within an insulated sample cryocooling chamber 270. At least a portion of the cryocooling chamber is covered by a gas exchange manifold 280. This manifold has a plunge bore 420 through which the sample travels. The gas exchange manifold has channels for dry gas 440 (e.g., dry N2 gas) and for applying suction / vacuum 201 that intersect the plunge bore 420 opposite each other, so that cold gas present within the plunge bore above the cryogenic liquid is sucked away and replaced by dry, ambient temperature gas, thereby creating an abrupt transition between room temperature gas and cold cryogenic liquid. This abrupt transition eliminates sample precooling in cold gas, resulting in larger average cooling rates.

[0172] In operation, the sample is plunged through the loop, causing solution to be transferred to the sample, and then on to the cryogenic liquid, where it may be released and stored. The plunge speed v and the height h of the loop above the cryogenic liquidsurface determine the time between ligand solution deposition and quenching of the reaction or conformation change by rapid cooling in the cryogenic liquid. Arbitrarily long times between ligand solution deposition and quenching can be achieved by plunging the sample through the loop, stopping and pausing, and then resuming the plunge to the cryogenic liquid after some time interval. Samples may also be translated slowly to increase the time between ligand solution deposition and quenching, but prolonged time within the loop may lead to excessive ligand solution transfer and crystals being displaced off the main sample-holding portion of the support.

[0173] Figs. 3A-3C show a schematic illustration of an example sample preparation system 300, which may be used for preparing samples for time-resolved crystallography. In Fig. 3A, a sample holder 302 is attached to sample carriage 280, which is translated up and down using the sample vertical translation stage 520. Examples of sample holder 302 include sample holders 101 and 102. In embodiments, sample holder 302 includes x-ray transparent film 240, which may hold a sample 260. Sample 260 may include one or more crystals. In operation of the sample preparation system 300, sample 260 is translated from an initial position above surface 549 of cryogenic liquid 540 within an insulated cryocooling chamber 370to a second position where the sample 260 is disposed within cryogenic liquid 540 (Fig. 3C). At least a portion of sample cryocooling chamber 370 is covered by a gas exchange manifold 380.

[0174] In system 300, a ligand solution may be deposited on sample 260 using a ligand solution deposition / liquid transfer system such as either a drop-on-demand dispenser 600 or a spray / jet generator 620 (e.g., an ultrasonic vaporizer, a piezo-actuated dispenser). Either ligand solution deposition / liquid transfer system may be mounted at a fixed height above a surface 549 of cryogenic liquid 540 (Fig. 3C) or on a second vertical translation stage, the reaction initiation stage 640, which translates reaction initiation apparatus up and down. In embodiments, system 300 may include both a fixed height solution depositor (e.g., for slow reactions or late time points) and an adjustable height solution depositor (for faster reactions and earlier time points). Ligand solution may be transferred to a stationary sample (sample 260), and the sample 260 may be then plunged to cryogenic liquid 540 after some time delay.

[0175] Alternatively, ligand solution may be deposited onto sample 260 as it travels toward the cryogenic liquid surface 549 (FIG. 3C). This latter approach may be used to achieve shorter time intervals between deposition and cryocooling, and thus to capture 1the earliest time points in, e.g., a reaction. For example, if the ligand solution is dispensed at a height of 5 mm above cryogenic liquid surface 549 and sample 260 plunges toward surface 549 at 2 m / s, the time interval will be 2.5 ms. The timing / sequencing of operations and other functions are controlled via a computer or microcontroller communicating through an interface to the various motor controllers, motors, switches, valves and drop dispenser controllers. Embodiments of sample preparation systems disclosed herein may include such a controller / computer, which may control one or both of a transfer mechanism or a translation stage of the preparation system.

[0176] A portion of system 300 above gas exchange manifold 380 and sample cryocooling chamber 370 that includes the sample plunge path may be contained within an environmentally controlled enclosure 660. Enclosure 660 may be supplied via a gas lines 681 by gas whose temperature and humidity are controlled by a warmer / chiller / humidifier (not shown). The temperature may be varied between about -20 °C (below which ligand solution on the surface of crystals and in the loop or liquid dispenser will tend to crystallize) and about 60-80 °C (above which proteins typically denature) and humidity between about 50% and 99% r.h. (to prevent crystal dehydration.)

[0177] In an example use scenario, dry gas (e.g., N2) is supplied from a source (e.g., a gas cylinder) to an input 682 to gas exchange manifold 380 on one side, and suction / vacuum generated by a pump or compressed gas vacuum generator is applied to the input 700 on the other side.

[0178] Fig. 3B is a cross-sectional view of manifold 380 in a plane A-A shown in Fig. 3A. Fig. 3C is a cross-sectional view of manifold 380 in a plane B-B shown in Fig. 3B. Gas exchange manifold 380 has a plunge bore 720, through which the sample travels on its path to the cryogenic liquid 540. The gas exchange manifold 380 may also have a recessed surface 382 defining a cavity 740 in its top that allows a reaction initiation stage (e.g., stage 640), drop dispenser 600 spray / generator 620 and / or loop (e.g., loop 340) to be placed so that ligand solution may be deposited on the sample very close to, e.g.., within 1-10 millimeters - of cryogenic liquid surface 549.

[0179] Embodiments of system 300 include a wall 760 between cavity 740 and plunge bore 720. Wall 760 includes one or more vertical slits 762 that terminate above surface 549 of cryogenic liquid 540 that allow passage of liquid, e.g., dispensed drops, between, e.g., a drop dispenser 600 in cavity 740 and bore 720. Accordingly, a portion of reaction initiation stage 640 or dispenser 600 may be located below the surface 549 ofcryogenic liquid 540. Wall 760 may be part of manifold 380, e.g., attached to or integrally formed with manifold 580. Part of surface 382 may be a surface of wall 760.

[0180] Dry gas (e.g., N2) supplied from a source to the input 682 flows through a channel in the gas exchange manifold (not shown) that intersects the plunge bore 720 on one side. Suction / vacuum is applied to the input 700 to a channel intersecting the plunge bore 720 on the opposite side. This acts to remove cold gas present above the surface of the cryogenic liquid within the plunge bore and replace it with room temperature gas, thereby eliminating sample precooling.

[0181] Fig. 4 shows an isometric illustration of a representative system 400 for preparing samples for time-resolved crystallography and related measurements in accord with aspects of this disclosure. The major system components are a high-speed sample vertical translation stage 840, a sample wand / adapter assembly 860 which holds a sample holder 880, a reaction initiation vertical translation stage 900, a reaction initiation / liquid transfer apparatus 920, the sample cryocooling and cryostorage assembly 940, and a receiving area (supporting platform) 946 for assembly 940. System 400 may also include sample holder 880.

[0182] A total volume of cryostorage assembly 940 available for holding a cryogenic liquid may range from IL to 40 L, where example volumes include, but are not limited to: 2L, 4L, 6L, 10L, 15L, 20L, 30L, 35L.

[0183] Figs. 5A and 5B show a respective side view and a front view of a highspeed sample vertical translation stage 500, which is an example of translation stages 840 and 520. Vertical motion is driven by a motor 960 (e.g., de, servo, stepper), which turns a lead screw 980. The motion can be stopped once the sample has entered the cryogenic liquid either via programmed motor deceleration or using an electromagnetically actuated brake 1000. The brake gives the shortest stopping distance, allowing the maximum plunge speed (and cooling rate) to be maintained for a longer time while minimizing the required cryogenic liquid depth and volume.

[0184] The sample carriage 1020 includes a block 1040 with lead screw threads and bearings that mate with the lead screw and convert the rotational motion of the lead screw into vertical motion of the sample carriage. This block is attached to a rigid plate 1060. The sample carriage includes a member 1080 that accepts sample wand assembly 860 and holds it tightly (e.g., using magnets and tight machining) with very little play.

[0185] To reduce wobble of the sample carriage during high-speed translation, blocks 1100 attached to the plate 1060 slide via roller bearings on a double rail 1120, and can generate a sample plunge path that is reproducible to within ± 1 mm and even ± 0.1 mm along the entirety of the plunge path. The motor and lead screw are selected to allow acceleration to speeds between 1 and 4 m / s and in some cases, 1 m / s and, in some cases, 2 m / s and, in some cases, 3 m / s over the distance between the upper “home” position and the position where the sample holder 880 enters the cryogenic liquid.

[0186] The combination of the motor and brake allow the sample carriage and sample to be decelerated to rest over the distance between the top surface of the cryogenic liquid to the sample’s final position within the cryogenic liquid. Speeds up to 10 m / s are feasible, but require large stopping distances to prevent sample loss and create substantial splashing of the cryogenic liquid. Speeds of 0.1 m / s and lower are readily produced using the motor and lead screw approach.

[0187] Fig. 6A shows an isometric view illustration of a sample wand 1200 used in cryocrystallography to manipulate samples. Wand 1200 has a magnet at its sampleaccepting end 1220 that holds a magnetic steel goniometer base to the end. A push-button 1240 connected to a rod running along the axis of the wand pushes the goniometer base away from the sample accepting end to release it. Fig. 6B is an isometric view illustration of an adapter 1260 that holds the wand 1200 securely and that either is of magnetic steel or includes magnets that allow it to be securely gripped when placed in member 1080 of the sample carriage 1020. Fig. 6C is an isometric-view illustration of sample wand / adapter assembly 860 comprised of the sample wand and adapter.

[0188] FIG. 7A and 7B are top and isometric view illustrations of a representative cryocooling chamber 1300, which is part of the sample cryocooling and cryostorage assembly 940. Chamber 1300 is an example of chamber 370. Chamber 1300 holds the volume of cryogenic liquid that is used to cryocool the sample as well as a crystallography sample storage cassette into which the sample may be inserted following cryocooling. The cryogenic liquid capacity of the chamber for a laboratory device may be between 1 and 10 liters. Cryocooling chambers holding more than one sample storage cassette may have capacities up to 40 liters. Chamber 1300 is of a thermally insulating material such as a polymer foam (e.g., machined high density polyurethane foam) to minimize heat transfer to and evaporation of the cryogenic liquid. Chamber 1300 may include a recess 1320 and an alignment pin 1340 in its interior bottom that allows precise placement of acrystallography cassette in a well-defined orientation. Alternatively, a separate receptacle for holding the crystallography cassette may be attached to the chamber’s interior bottom. Chamber 1300 has a feedthrough in its side wall (not shown) for filling with cryogenic liquid from a cryogenic liquid storage and filling unit.

[0189] Cryocooling chamber 1300 may have raised portions 1360, whose tops are above the level of the cryogenic liquid, beneath the gas exchange manifold to support the manifold. These raised portions define a plunge channel into which the sample is plunged and through which it is translated before insertion into the sample cassette. They reduce the total volume of cryogenic liquid required to fill the chamber. Chamber 1300 may also have a cut-out 1380 for the gas exchange manifold.

[0190] Fig. 8A and 8B are top and isometric view illustrations of a representative crystallography sample storage cassette 1400, that can be inserted into the chamber of Fig. 7. Several cassette designs are in routine use, with the UniPuck being the most common, which has a diameter of 6.7 cm. The cassette includes sixteen receptacles 1420 for sample supports mounted in goniometer bases, where each receptacle has a magnet to hold the base in place. The cassette has an alignment hole 1440 and an alignment notch 1460 that can be used to orient the cassette and to identify the different receptacle locations.

[0191] FIG. 9A is an isometric view illustration of a gas exchange manifold 1500 that is a component of the sample cryocooling and cryostorage assembly 940. Manifold 1500 is an example of manifold 380. Gas exchange manifold 1500 may rest on the raised portions 1360, the cutout 1380 and the top 1381 of the cryocooling chamber 1300. Gas exchange manifold 1500 has multiple functions. (1) It defines a plunge bore 1520 through which the sample is plunged into the cryogenic liquid. (2) It isolates the cryogenic liquid surface within the plunge bore from the surrounding liquid surface, reducing waves and helping to create a flat interface. (3) It removes cold gas present immediately above the cryogenic liquid and replaces it with dry ambient temperature gas, eliminating precooling of the sample in cold gas before it enters the cryogenic liquid. (4) It prevents contact of warm moist air with cold surfaces including that of the cryogenic liquid. (5) It prevents frost from forming on cold surfaces. (6) It facilitates transfer of cold samples held by a wand out of the plunge bore and into a sample storage cassette while the sample remains in cryogenic liquid. Many components can be 3D printed using a glass-embedded resin with reduced thermal expansion, suitable for cryogenic temperature use.

[0192] Fig. 9B is a sectioned isometric view of gas exchange manifold 1500 showing the plunge bore 1520, and Figs. 9C and 9D are front and rear views of the gas exchange manifold, respectively. Gas exchange manifold 1500 has horizontal bores 1540 and 1560 with connectors (not shown) for connection to a supply of dry room temperature N2 gas (from an N2 cylinder or house supply) and to a source of suction or vacuum (e.g., a vacuum pump or venturi vacuum generator), respectively. These bores connect to channels 1580 that slope downward toward the plunge bore and that form rectangular openings where the channels intersect the plunge bore. A metal (e.g., aluminum) block 1590 with one or more heaters within it inserts into the top of the gas exchange manifold. It defines the top surfaces of the vacuum and make-up channels and maintains constant temperature of the portion of the bore wall above the cryogenic liquid.

[0193] Gas exchange manifold 1500 has a through-slot 1600, the sample exchange channel, connecting the bore to the portion of the cryocooling chamber holding the sample storage cassette. This allows the sample on the wand to be translated through the cryogenic liquid from the bore and into the cassette without leaving the cryogenic liquid. Gas exchange channels 1620 on opposing sides of the slot prevent cold gas from the main volume of the cryogenic liquid container from entering the bore during gas exchange within the bore and minimize frosting along the slot. The bottom of gas exchange manifold 1500 has a downward projection 1530 that helps to isolate the cryogenic liquid within the bore from surrounding cryogenic liquid. This projection may extend along the bottom of the through-slot 1600.

[0194] During operation, the cryogenic liquid level is maintained at a height within the height of the vacuum and make-up gas channels where they intersect the plunge bore, and , in embodiments, near the vertical midpoint of those channels. Valves (not shown) on the vacuum and make-up gas lines open, so that the cold gas present within the plunge bore along the plunge path is completely removed and replaced with dry ambient temperature gas. This eliminates precooling of the sample prior to entering the cryogenic liquid and ensures that the reaction proceeds at room temperature until the sample enters the cryogenic liquid.

[0195] Figs. 10A and 10B are isometric views of a lid assembly 1660, which may rest on top of cryocooling chamber 1300 (FIG. 7A) and gas exchange manifold 1400 (Figs. 8A, 8B) and forms part of the sample cryocooling and storage assembly 940. The main body of the gas exchange manifold 1400 may be fastened to the underside of the lid, as in Fig. 4,or the gas exchange manifold may replace the portion of the lid within its footprint. Lid assembly 1660 helps prevent moist ambient air from going inside the cryocooling chamber and provides insulation that reduces evaporation of the cryogenic liquid. The main portion 1680 of lid assembly 1660 includes heaters (not shown) that prevent moisture condensation and frost formation on the lid. The heaters may be cartridge heaters embedded in lid assembly 1660, or resistive sheet heaters located on a lid surface. The heaters may be PID-controlled and may maintain its surface at 25 °C.

[0196] Lid assembly 1660 has an opening 1004 for the plunge bore that connects via a slot 1720 over the sample exchange channel to a larger circular opening 1740 through which a crystallography cassette (as in Fig. 8) is loaded and removed. The cassette aperture may have a diameter between about 7 and 12 cm, larger than the 6.7 cm diameter of a standard cassette. The cassette aperture is covered by an optically clear, slotted, freely rotatable window 1760, which may be of a polymer (e.g., an acrylic) or glass. After each sample is plunged through the plunge bore, the sample wand assembly is removed from the sample vertical translation stage’s sample carriage, translated into slot 1720 of the lid and into a slotted portion 1762 of the window 1760, and rotated (with the window) into position above an open cassette receptacle. The sample is then released from the wand and into the cassette. This configuration allows all post-plunge sample motions to be completed with the sample remaining in cryogenic liquid, minimizing the risk of inadvertent sample warming, and helps minimize exposure of the cryogenic liquid to moist ambient air. Exposure to ambient air can be further minimized by lining slot 1720 of the lid and of the window 1760 with brushes, membranes or other mechanisms that allow translation of the sample wand through them.

[0197] Fig. 11 is an exploded isometric view of the sample cryocooling and cryostorage assembly 940, illustrating the relationship between the lid assembly 1660, the gas exchange manifold 1500, cryocooling chamber 1300, and sample storage cassette 1400.

[0198] Fig. 12 is an isometric view of reaction initiation stage 900 introduced in FIG. 4. The basic design may be similar to that of the high-speed sample vertical translation stage 840, except that only relatively low speed motions are required. A stepper motor 1800 may drive a lead screw 1820. A reaction initiation carriage 1840 has a threaded element through which the lead screw passes, converting the rotational motion of the lead screw into translational motion of the carriage. Two rods 1860 pass through roller bearings within the reaction initiation carriage and on either side of it, largely eliminating its side-to-side wobble motion. The reaction initiation carriage 1840 may hold a variety of different systems for depositing ligand solution on the sample, including a drop-on-demand dispenser, a sprayer, or a liquid film holding device. The stage allows the height h of ligand solution deposition above the cryogenic liquid surface and thus the time Ar(rave;between ligand solution transfer / reaction initiation and entrance into cryogenic liquid to be varied under computer control. Precise, highly repeatable motion of the reaction initiation carriage is essential when drop-on-demand dispensers are used for reaction initiation. Motion should be repeatable to within ±0.1 mm or better, in x, y and z.

[0199] For reaction time points of ~5 ms and shorter, the ligand solution may be dispensed within millimeters of the cryogenic liquid surface, within the plunge bore of the gas exchange manifold. Even with cold gas removed, at this distance radiative cooling will eventually lower the ligand solution temperature. The reaction initiation carriage 1840 can initially be held at a position well above the cryogenic liquid surface, and then lowered (by the stepper motor 1800) into position immediately before the sample plunge, minimizing the time for ligand solution cooling via its proximity to the cryogenic liquid.

[0200] A variety of different reaction initiation apparatus / liquid transfer mechanisms (920 in Fig. 4) for applying ligand solution to the sample - while it is stationary or while it is plunging toward the cryogenic liquid surface - to initiate the reaction or structural change within the sample can be mounted on the reaction initiation carriage 1840. If ligand solution is dispensed while the sample is stationary, a commercial drop-on-demand dispenser can be used to dispense one or many drops. For example, a Microdrop Technologies MD-K-130 could be used to dispense 50 to 150 pl drops onto the sample support at a maximum rate of about 1 kHz, so that dispensing volumes of a few nanoliters sufficient to cover crystals on a roughly 200 micrometer diameter support requires 10-50 drops and a time between 10 and 50 ms. Ligand solution can be sprayed onto the sample using a small nozzle (e.g., with a venturi mechanism) or using an ultrasonic mist generator. However, if ligand solution is to be dispensed when the sample is traveling at high speed, as is necessary to achieve millisecond time resolution, the options are more constrained.

[0201] For commercial drop generators, drop generation frequencies and the size of the sample target (typically from 50 to 200 pm) are such that only a single drop can be dispensed onto a sample moving at 1-2 m / s. That drop must have sufficient volume to fully cover the sample, the timing of its dispensing must be precisely coordinated with themotion of the sample plunge stage, and the drop trajectory from dispense tip to sample must be adequately precise and reproducible. Suitable commercial drop dispensers cost roughly $20,000. Nozzles generating short continuous streams / jets require large flow rates to adequately cover a rapidly moving sample and can be wasteful of the dispensed ligand solution.

[0202] A simpler and less costly approach is to plunge the crystals on their support through a thin film containing the ligands / reactants / solutes required to trigger the reaction or conformational change. A loop can be positioned centered on the sample plunge axis at some height h above the cryogenic liquid. Ligand solution can be loaded onto the loop, and the sample plunged through the ligand solution film spanning the loop and into the cryogenic liquid. The time between entry into the film - the start of diffusion and reaction - and quenching is then determined by the travel time Attravei from the loop position to the LN2 + the time tcooito cool below 200 K or 150 K.

[0203] Figs. 13A-13C shows examples of much simpler, loop-based apparatus (that may be part of reaction initiation / liquid transfer apparatus 920 in Fig. 4) for transferring ligand solution to samples for reaction initiation during high-speed plunges. In all cases, a ligand solution film is created on a loop, the sample is translated through the loop, and some of the ligand solution initially spanning the loop is transferred to the sample. The loop-based dispenser may be made of a compliant, breakable material such as polymer. As the sample continues its plunge, the goniometer base impacts the loop, displacing and / or breaking it, so that the sample can continue its journey to the cryogenic liquid surface.

[0204] Fig. 13A shows a very simple break-away loop-based ligand solution dispenser 1900. A loop 1920 - larger than the transverse size of the sample support loop / film (about 0.8-1.5 mm] and, if present, copper post (2.7 mm, present in some goniometer base designs) but smaller than the maximum diameter of the goniometer base (about 1 cm) - is attached to a support post 1940 that can be held by the reaction initiation stage (for the example shown in Fig. 13A, by insertion of the end opposite the loop into a hole in the stage) or by a vertical plate with a regular array of holes. The inner diameter of the loop may be between ~1 mm and 1 cm and, in some instances, about 2-5 mm, and the thickness of the loop may be between 50 micrometers and 1 mm.

[0205] The loop dispenser 1900 is made of a compliant, bendable and breakable material such as a polymer and may be 3D printed. A taper at the point where the loop1920 connects with the support post 1940 provides a weak link that breaks upon contact with the larger diameter portion of the goniometer base, after the sample has traversed the loop and passed through its ligand solution film, and impact of the base with the support post 1940 bends the post downward and out of the way. This allows the sample and support to continue moving along the plunge path toward the cryogenic liquid after ligand solution deposition. The weak, breakable link may also be placed along the circumference of the loop itself, or somewhere along the support post. In embodiments, the loop breaks during each sample plunge, and a new loop is used for each sample.

[0206] Fig. 13B shows a loop-based ligand solution dispenser 1960 designed to place the loop 1980 as close as possible to the cryogenic liquid surface, as required for the shortest time points. In the design shown, pads 2000 rests on top of the gas exchange manifold and plunge chamber cover and vertical arms 2020 project down into the plunge bore of the gas exchange manifold and hold the loop below within the bore and just above the cryogenic liquid surface. Alternatively, the pads 2000 may rest on a horizontal member that attaches to the reaction initiation carriage 1840 and that has an aperture through which the arms 2020 may project down into the plunge bore. This allows the loop to be loaded well above the plunge bore and then translated into position within it. As with the design in Fig. 13A, solution dispenser 1960 has features 2040 that form weak points, so that when loop 1980 is impacted by the goniometer base the dispenser breaks at those locations, allowing the sample and support to continue into the cryogenic liquid. In embodiments, a new loop dispenser is used for each sample. In a current design, loop 1980 may be placed within 5 mm of the cryogenic liquid surface. With a plunge speed of 2 m / s, this corresponds to a 2.5 ms time interval between ligand solution deposition and entry into the cryogenic liquid.

[0207] Fig. 13C shows an alternative loop-based ligand solution dispenser 2060. In this design, a supporting ring 2080 rests on the edge of a circular aperture in a horizontal member that attaches to the reaction initiation stage (not shown). The members 2100 project downward at an angle and form part of a conical surface. Members 2100 attach to loop 2120, e.g., with a raised, sharp inner lip (not shown), that is split in half at its midpoint between members 2100. During a sample plunge, the sample first passes through the ligand solution spanning loop 2120. When the goniometer base begins to impact the members 2100, members 2100 gradually separate, opening loop 2120 so that the basepasses through it to the cryogenic liquid. In its open state, loop 2120 may include two complementary sections, e.g., arc lengths that when joined form a closed loop.

[0208] Fig 14 shows a “saloon door” loop-based liquid transfer apparatus 2140, a specific realization of reaction initiation apparatus 920 in Fig. 4, that is fully reusable. The apparatus 2140 may be attached to the reaction initiation stage (e.g., 640, 900) via screws that insert into the slots 2160, allowing fine adjustment of the loop position. Two “doors” 2180 that swing downward (as in Fig. 14C) are attached to the main frame 2200 of the dispenser via horizontal screws 2220. Magnets 2240 are inserted into receptacles 2260 in each door that, when the doors are “closed”, are directly opposite each other and in close proximity. The magnets then act to hold the doors in the closed, horizontal position. The loop is formed from two semi-circular parts 2280, one on each door, that may butt against each other when closed or that may be shaped to overlap in forming a continuous loop when closed. The loop may have raised, sharp edges, e.g., at its inner periphery (not shown) that help to pin the ligand solution contact line and stabilize the loop-spanning ligand solution film. The loop forming portion of each door may be removable and replaceable, which may be desirable when using multiple distinct ligand solutions.

[0209] During a sample plunge, after the sample passes through the film, the impact of the goniometer base with the doors 2180 forces them to swing downward and out of the way. The doors may be 3D printed and very light weight, so that their inertia and the forces of impact may be minimized. Experiments show that 3D printed doors of a not- particularly-tough polymer survive hundreds of high-speed impacts with goniometer bases without breakage or noticeable degradation.

[0210] Loop-based reaction initiation apparatus 2140 may be lowered by the reaction initiation stage into contact with the top of the cryocooling chamber lid and / or gas exchange manifold. In an embodiment, this places the loop 40 mm above the cryogenic liquid surface, giving a minimum time point of about 20 ms. The stage can be raised to obtain longer times between ligand solution deposition and cryocooling. The gas exchange manifold and lid can be modified, as manifold 380, to allow the reaction initiation stage to be lowered closer to or below the cryogenic liquid surface to allow dispensing with a few millimeters of the cryogenic liquid surface and so to achieve earlier time points.

[0211] Fig. 15 shows a “scissors” loop-based ligand solution / liquid transfer mechanism 2300, which may be part of reaction initiation apparatus 920 in Fig. 4. In this design, the two halves of a loop 2320 overlap when the scissors are closed, forming acontinuous loop 2329 (Fig. 15B). The loop halves are each attached to an arm 2340 that can rotate about a shaft 2360. The arms 2340 cross over at the shaft, as with scissors. A spring (not shown) with its straight portions (for a torsion spring) or ends (for a regular spring) attached to the ends of the arms forces the two arms apart. The ends of the arms opposite the loop ends may have receptacles 2380 in which a two-pronged clip 2400 is inserted, holding the two arms together so that the loop is closed. The clip 2400 can be attached to a solenoid, so that when the solenoid is energized, the clip is pulled up, releasing the arms so that the loop springs open. An optical or magnetic gate / sensor that senses the position of the sample and support can be used to generate a trigger pulse, and electronics used to generate an appropriately delayed pulse to activate the solenoid after the sample has passed through the loop and before the goniometer base strikes the loop. Prototypes were 3D printed to make very light and rigid arms and are found to open in less than 1 millisecond, sufficiently fast to get out of the way of the goniometer base.

[0212] Alternatively, the opening of the loop may be triggered by direct physical contact with the goniometer base or with a suitable member projecting down from the reaction initiation stage. For example, the two-pronged clip could be attached to a horizontal shaft perpendicular to the horizontal line bisecting the jaws, and the clip could have a tab extending from the shaft to the rear of the jaws that is horizontal when the clip is inserted into the jaw receptacles 2380. Impact of a member projecting downward from the sample carriage could push the tab down, lifting the clip up and releasing the jaws to open. Alternatively, conical structure (or simply two or more angled arms) like that in Fig. 13C could project upward from the arms at the loop end, such that as the goniometer base contacts the arms the loop is forced apart. Alternatively, a member projecting down from the reaction initiation stage could force apart the jaws in the region between the loop and the shaft 2360.

[0213] An alternative configuration is to use two arms each attached to a separate vertical shaft, where the arms form a loop when brought into contact. The arms can be held in contact using, e.g., magnets or a clip, and pushed apart using one or two springs.

[0214] Films of ligand-containing solution with a wide range of viscosities, including pure water, can be stably suspended across the loops in Figs. 13, 14 and 15 for more than a minute, which is significantly longer than the few seconds required to load the film and initialize plunging. Solutions with low viscosity - e.g., protein in pure water tend totransfer less well than higher-viscosity solutions containing, e.g., ethylene glycol, DMSO, or PEG. These compounds may be added to inhibit crystalline ice formation in transferred ligand solution. Experiments suggest that transfers of a few nL - sufficient to cover the active area of a 200 micrometer diameter crystal support film to a depth of about 25 micrometers - are reliably obtained by this method.

[0215] Ligand solution may be loaded into the loop using a pipette or syringe. Alternatively, a tool including a pipette tip with a largely circular ball bearing or bead placed near its open end to form a structure similar to that of a ball point pen may be used, and this tool has been found to reproducibility produce stable thin films across the loop.

[0216] Fig. 16 is an isometric illustration of a sample preparation system for time-resolved crystallography as in Fig. 4 that implements automated control of the cryogenic liquid level within the sample cryocooling chamber. Control of this level is important for effective removal of cold gas above the cryogenic liquid surface by the gas exchange manifold, as the liquid level must lie within and ideally near the middle of the gas and vacuum channels where they intersect the plunge bore. In one current implementation, this requires that the cryogenic liquid level be maintained to within ±4 mm. Level control could be achieved using a commercial pressurized cryogenic liquid storage dewar with a flow valve. The cryogenic liquid level can be sensed using resistors, diodes, heated RTDs, or a laser level sensor attached to the interior of the cryocooling chamber or to the gas exchange manifold, and a feedback controller used to open the flow valve to maintain the level.

[0217] Fig. 16 shows a simpler and less costly approach that relies on gravity rather than pressure to feed the cryogenic liquid. Cryogenic liquid is stored within an insulated container 2420 that is elevated above a cryocooling chamber 1640 using a support 2440 (here shown as a table.) Liquid cryogen flows out of the container 2420 and through a thermally insulated cryogenic-compatible line 2460 to a cryogenic compatible valve 2480, and then through additional insulated line 2500 and into the sample cryocooling chamber. The cryogenic liquid line preferably enters the cryocooling chamber at a level below the top surface of the cryogenic liquid to minimize splashing and generation of waves. Baffles within the chamber and near the cryogen line’s outlet can reduce waves generated during filling with cryogenic liquid.

[0218] In a realization of the system shown in Fig. 16, all system functions - plunging, reaction initiation stage positioning, heater power and P1D control, gas flow valveoperation - except for braking and motor encoder data acquisition and processing were controlled using custom scripts in Python and communicating with the instrument components through a National Instruments data acquisition (Nl-DAQ) system including a terminal block (N1 SCB-68A) for distributing signal input / output and multifunction I / O board (N1 PCle-6361) for signal processing.

[0219] Figs. 17 and 18 show a loop-based reaction initiation apparatus 1700, which is based on the “saloon door” design in Fig. 14. Apparatus 1700 allows placement of the ligand solution film within roughly 2 mm of the liquid nitrogen surface. When a sample is plunged at high speed through the loop and into the liquid nitrogen, the short travel distance between the loop and liquid nitrogen is traversed in a very short time, allowing access to the earliest possible time points in a reaction. For example, with an example plunge speed of 2 m / s, the time to traverse 2 mm is 1 millisecond.

[0220] As shown in Fig. 17, sample 260 on a sample holder 880 is attached to a sample wand / adapter assembly 860 (see, e.g., Fig. 4 and Fig. 5A), which in turn is held by the sample vertical translation stage (not shown). The two loop-forming “doors” 2180 in the design of Fig. 14 are replaced with two loop-forming arms 2620 that project downward. The horizontal separation of the hinged portion of the arms is comparable to or smaller than the corresponding width of the bore through the gas exchange manifold 1500 (if present). The downward projection of the arms is sufficient to allow the loop to be placed a minimum desired distance (for example, 2 millimeters in a current realization) above surface 549 of cryogenic liquid 540, and may be determined by the distance between the top of the gas exchange manifold and the liquid nitrogen surface.

[0221] The loop 2660 is formed from distal portions of the loop-forming arms 2620, such as, for example, two halves (e.g., two semicircular portions) that project approximately horizontally from the lower end of the arms 2620. In some aspects, the loopforming arms 2620 may define an opening to hold a film that is not circular in shape (e.g., an ovoid shape, a square shape, a rectangular shape, etc.).

[0222] The diameter of the loop 2660 may be as small as is feasible to minimize consumption of the ligand-containing solution required to form a film. In embodiments, the inside diameter is limited by at least one of (a) the size of the sample and the portion of the sample holder immediately adjacent to the sample that passes through the loop and (b) the amount of the sample’s side-to-side motion and variability in that motion at the position of the loop during sample plunges through the loop. In some aspects, the arms may be heldtogether to form a loop using one or more magnets 2680 (e.g., one magnet 2680 on each arm 2620) or by one or more mechanical fasteners (e.g., latch(es), clip(s), etc.). In some examples, the arms 2620 are attached to the frame 2700 of the reaction initiation apparatus, which in turn may be attached to a reaction initiation stage 900.

[0223] Figs. 18A-18D shows an example mode of operation of reaction initiation / liquid transfer apparatus 1700. In this example, the sample 260 reaches a downward speed of about 2 m / s and the loop holding the liquid film is placed about 4 mm above liquid nitrogen surface 549. The time interval between liquid transfer (to, e.g., initiate a reaction in the sample) and the start of sample cooling in liquid nitrogen is then 4 mm / (2 m / s) = 2 milliseconds. First, in Fig. 18A, arms 2620 are brought together so that the distal ends of each arm form a loop 2660, and a ligand-containing solution is dispensed into the loop to form a liquid film bearing the ligand that spans the opening defined by the loop-forming arms 2620. Next, as shown in Fig. 18B, sample 260 is translated downward, passing through both loop 2660 and the liquid film spanning loop 2660. toward cryogenic liquid 540.

[0224] As the sample progresses downward, eventually the larger diameter portion of the sample holder 880 comes into contact with the two arms 2620, forcing them apart (Fig. 18C). This allows the sample and holder to continue to the cryogenic liquid unimpeded by the loop or loop-forming arms 2620, as shown in Fig. 18D, in which magnets 2680 are visible. The downward angle of the arms leads to a glancing impact of the sample holder 880 with the arms 2620, reducing the force of impact and ensuring a smooth motion of the arms. In some aspects, lower surfaces of the sample holder 880 that would come into contact with arms 2620 may be chamfered or otherwise angled or configured to increase and / or tailor a contact area between the sample holder 880 and arms 2620. As a result of the downward angling of the arms when they contact to form a loop, the motion of the arms when they separate after contact with the sample holder is largely horizontal, keeping the loop from entering into the liquid nitrogen.

[0225] When the liquid film and loop are brought in close proximity to the liquid nitrogen surface, they will tend to cool due to cold gas present above the liquid nitrogen surface and also due to radiative cooling. In embodiments, the liquid film’s temperature is maintained at the same temperature as the sample at its initial position. In some examples, cooling of the film can be minimized by, first, holding it at an initial position well above surface 549 where cooling is negligible, and then rapidly translating to its final positionimmediately prior to (for example, within 1 or 2 seconds before) sample passage through the loop. In some examples either in isolation or in combination with the above-noted translation of the film immediately prior to sample passage through the loop, cooling of the liquid film can also be minimized by removing cold gas above the liquid nitrogen surface using the gas exchange manifold.

[0226] An example method of using an example system in accord with aspects of the disclosed concepts follows. In a system initialization, a cryocooling chamber and any storage chamber to be used with the system is filled with a cryocoolant, such as liquid nitrogen. In some aspects, control software is initialized. The sample translation stage and the sample deposition stage are registered or homed to specified positions or a predetermined setpoints or positions (e.g., selected from a plurality of available setpoints or positions) and a sample support is attached to a wand and adapter. In some aspects, arms of the reaction initiation apparatus / liquid transfer mechanism are closed so as to form or define an opening capable of supporting a thin liquid film (e.g., a loop). Herein, examples of reaction initiation apparatus / liquid transfer mechanisms 920 and their key components include loop 340 (Fig. 2); loop-based liquid dispenser 1900, 1960, and 2060 (Figs. 13A-13C), reaction initiation apparatus 2140 in Figs. 14A-14C; loop-based liquid dispenser 2300 (Fig. 15); reaction initiation apparatus 1700 of Fig. 17. Herein, a transfer mechanism may include a solution holder (such as loops and solution dispensers disclosed herein), and may also include a translation stage, such as translation stage 900.

[0227] In some aspects of a sample and liquid loading process, the opening (e.g., loop) is loaded with solution to be deposited onto the sample. In some examples, the loading may be performed using, for instance, a pipette or other fluid delivery device (e.g., syringe pump, burette, microfluidic device, automated liquid handling system, etc.), to form a thin unsupported film in the opening. A test sample, such as a biological sample, is mounted on a sample support held by the wand and adapter, following which any excess liquid is removed from the surface of the sample and support, and the sample support, wand and adapter are attached to the sample translation stage.

[0228] In some aspects of the plunging, liquid transfer, and cryocooling process using an example system in accord with aspects of the disclosed concepts, following the acts of sample and liquid loading, a command is issuing a command to the controller (e.g., control software, etc.) to cause the sample to plunge through the loop and unsupported liquid film and into the cryocoolant (e.g., liquid nitrogen). In some aspects, the controllertranslates the transfer mechanism and its opening bearing the thin film to a selected vertical position, set by a desired time point to be captured. Where a gas exchange manifold is advantageously provided, vacuum and make-up gas flows to the gas exchange manifold are activated to remove cold gas from above the liquid nitrogen surface. The sample stage and test sample borne thereby are then translated to a position where the sample contacts the liquid film spanning the opening (e.g., loop), resulting in the transfer of at least a portion of the liquid volume of the liquid film onto the test sample, following which the translation of the sample stage and test sample to a position where at least the test sample is disposed in the cryocoolant (e.g., liquid nitrogen). In other aspects, the sample stage and test sample may be translated to (e.g., under control of a drive system controlled by a controller) or allowed to translate to (e.g., under gravity, with movement limited by mechanical stops) to a position where the test sample and a portion of, or an entirety of, the wand and / or adapter are disposed in the cryocoolant (e.g., liquid nitrogen).

[0229] Following the plunge, in some aspects, the test sample, sample support, wand and adapter are removed from the sample translation stage and the test sample is inserted into and released in a sample cassette or puck, or the like, disposed in the cryocoolant (e.g., liquid nitrogen) within the cryocooling chamber. The cassette or puck, or the like, containing the test sample is then removed and maintained or stored at an appropriate cryogenic temperature until testing of the test sample can be performed. For instance, the cassette or puck, or the like, may be transported to an X-ray source where X- ray data can be collected from the test sample while it is maintained at an appropriate cryogenic temperature.

[0230] Although the present disclosure has been described with respect to one or more particular example(s), it will be understood that other example(s) of the present disclosure may be made without departing from the scope of the present disclosure.

[0231] In a first example, a system for preparing samples for time resolved studies includes a sample support to receive a biological sample, an at least substantially vertical sample translation stage or robot, a means for removably attaching the sample support to the vertical translation stage or robot, an insulated chamber configured to bear a cryogenic liquid, a drop-on-demand liquid drop dispenser head with a drop dispensing tip, and a manifold extending from the top surface of the insulated chamber downward into the chamber to a position below the surface of the cryogenic liquid, where the manifold seals the chamber from the air above except within a through-hole defining a plunge borealigned with the axis of vertical motion of the sample support, and where the manifold has a recessed region adjacent to the plunge bore extending from the top of the manifold to a distance below the surface of the cryogenic liquid within the plunge bore sufficient to accommodate the vertical height of drop dispenser so that the dispensing tip is positioned within 2 mm of the cryogenic liquid surf ace within the plunge bore. The system includes a mechanism for adjusting the height of the drop dispenser above the surface of the cryogenic liquid that allows the dispensing tip to be positioned within 2 mm of the level of the cryogenic liquid within the plunge bore of the manifold and a mechanism for triggering the dispensing of a drop based on the sample's vertical position, where the drop dispensing tip is adjusted to a desired height, the sample is translated from an initial position into the cryogenic liquid, and a drop is dispensed onto the sample as the sample is translating toward the cryogenic liquid in response to a trigger.

[0232] In some aspects of the first example, the biological sample comprises a protein crystal, a thin layer of biomolecule-containing solution, a thin layer containing cells, and / or a thin slice of tissue.

[0233] In some aspects of the first example, the vertical sample translation stage or robot is configured to accelerate the sample to a speed between 0.1 and 10 m / s and , in embodiments, between 1 and 4 m / s over the distance between an initial sample position and a position within the insulated chamber where the sample enters the cryogenic liquid.

[0234] In some aspects of the first example, the drop on demand dispenser produces single drops of volume between 50 pL and 100 nL and, in some instances, between 0.5 and 10 nL, and dispenses the drop with a velocity of between 0.5 and 20 m / s.

[0235] In some aspects of the first example, the drop on demand dispenser head has minimum vertical dimension perpendicular to the drop dispensing axis of about 10 to 20 mm.

[0236] In some aspects of the first example, the recessed portion of the manifold extends between 5 and 30 mm below the level of the cryogenic liquid in the plunge bore.

[0237] In some aspects of the first example, the mechanism for adjusting the height of the drop dispensing head is a motor-driven vertical translation stage.

[0238] In some aspects of the first example, the dispensing of a drop is triggered by electronically counting the number of rotations of the motor driving the vertical translation stage.

[0239] In some aspects of the first example, the dispensing of a drop is triggered using an optical gate, a capacitive sensor, or a magnetic sensor that detects the passing of the sample support or the passing of a portion of the vertical translation stage.

[0240] In some aspects of the first example, the manifold includes gas channels and connections to allow exchange of cold gas present within the plunge bore with dry ambient temperature gas.

[0241] In some aspects of the first example, the sample support includes a magnetic steel goniometer base as used in cryocrystallography, and where the means for removably attaching the sample support to the vertical sample translation stage.

[0242] In a second example, a system for preparing samples for time resolved studies includes a sample support to receive a biological sample, a sample translation stage or robot, a means for removably attaching the sample support to the translation stage or robot, an insulated chamber configured to bear a cryogenic liquid, a drop-on-demand liquid drop dispenser head with a drop dispensing tip, and a manifold extending from the top surface of the insulated chamber downward into the chamber to a position below the surface of the cryogenic liquid, where the manifold seals the chamber from the air above except within a through-hole defining a plunge bore aligned with the axis of vertical motion of the sample support, and where the manifold has a recessed region adjacent to the plunge bore extending from the top of the manifold to a distance below the surface of the cryogenic liquid within the plunge bore sufficient to accommodate the vertical height of drop dispenser so that the dispensing tip is positioned within 2 mm of the cryogenic liquid surf ace within the plunge bore. The system also includes a mechanism for adjusting the height of the drop dispenser above the surface of the cryogenic liquid that allows the dispensing tip to be positioned within 2 mm of the level of the cryogenic liquid within the plunge bore of the manifold, and a mechanism for triggering the dispensing of a drop based on the sample's vertical position. In this system, where the drop dispensing tip is adjusted to a desired height, the sample is translated from an initial position into the cryogenic liquid, and a drop is dispensed onto the sample as the sample is translating toward the cryogenic liquid in response to a trigger.

[0243] In aspects of the second example, the sample translation stage is configured to translate the sample support at an angle of between about 60 degrees to about 90 degrees relative to the insulated chamber, and wherein the sample translation stage is motor-driven, gravity- driven, actuator-driven, or pneumatic-driven.

[0244] In aspects of the second example, the sample translation stage is configured to translate the sample support at an angle of about 90 degrees relative to the insulated chamber.

[0245] In a third example, a system for cryocooling and storing a sample m a cryogenic liquid includes a sample support configured to hold a sample, a wand configured to hold the sample support, a mechanism for translating the sample support along an at least substantially vertical direction into the cryogenic liquid, a means for removably attaching the sample support and wand to the sample translation mechanism, a thermally insulated chamber configured to hold a volume of cryogenic liquid, a gas exchange manifold defining a sample plunge bore and sample exchange channel, the manifold being located over a first portion of the thermally insulated chamber with a first portion of the manifold extending downward from the top surface of the thermally insulated chamber and into the thermally insulated chamber to place the gas exchange manifold in fluid communication with a cryogenic liquid in the thermally insulated chamber. The system also includes a receptacle on the inside bottom surface of a second portion of the thermally insulated chamber that accepts a sample holding storage cassette, a lid extending over the top surface of the thermally insulated chamber, the lid including an aperture for the plunge bore, an aperture sized to allow passage of a sample holding storage cassette, and a channel connecting these two apertures and extending over the sample exchange channel. The system includes a substantially flat plate sized to fit and freely rotate within the aperture of the lid or sized to cover the aperture of the lid and to be freely translated relative to the lid, having a channel extending inward from its outer edge toward its center, where a wand with attached sample support is plunged by the vertical translation mechanism through the gas exchange manifold bore and into the cryogenic liquid, horizontally translated through the sample exchange channel and plunge channel and into the channel of the substantially flat plate, rotated and / or translated into position above a sample receptacle where the wand releases the sample holder into the receptacle, and where the sample remains immersed in the cryogenic liquid during transfer from the plunge bore to the sample receptacle.

[0246] In aspects of the third example, the mechanism for vertically translating the sample and sample support comprises a robot, a motor driven translation stage, a gravity driven translation stage, a pneumatic-driven translation stage, or an actuator- driven translation stage.

[0247] In aspects of the third example, the vertical translation mechanism is configured to accelerate the sample to a speed between 0.1 and 10 m / s and , in embodiments, between 1 and 4 m / s at the point where it enters the cryogenic liquid.

[0248] In aspects of the third example, the system also includes a first fluid source comprising a cryogenic liquid disposed in the thermally insulated chamber and a second fluid source configured to deliver dry ambient temperature gas to a second portion of the gas exchange manifold, wherein the gas exchange manifold is configured to, or configurable to, allow cold gas from the plunge bore to be replaced by dry ambient temperature gas.

[0249] In aspects of the third example, the lid is of a thermally insulating material.

[0250] In aspects of the third example, the lid is comprised of a thermally insulating layer on its bottom side, facing the cryogenic liquid, and a thermally conducting layer on its other, top side.

[0251] In aspects of the third example, the lid comprises one or more heating elements to minimize frosting and ice accumulation on at least a portion of a top surface of the lid or an entirety of a top surface of the lid.

[0252] In aspects of the third example, the substantially flat plate is optically transparent, and made of a material including a polymer and a glass.

[0253] In aspects of the third example, the channel in the substantially flat plate and the channel in the lid may be partially sealed from outside air by brushes, membranes or other mechanisms that allow translation of the sample wand through them.

[0254] In aspects of the third example, the gas exchange manifold optionally includes a recess in its top surface that allows a stage to be lowered into the recess to a level below the level of the cryogenic liquid within the plunge bore.

[0255] In a fourth example, the present concepts include a film suspension device comprising a frame and one or more arms movably disposed relative to the frame, a proximal end of the one or more arms being movably attached to the frame and a distal end of the one or more arms defining or comprising a holding portion, having one or more parts, defining a film retaining opening having a longest dimension along any axis of between 1-10 mm and , in embodiments, between 3-5 mm, the film retaining opening being configured to hold a freely suspended liquid film having a longest dimension along any axis of between 1-10 mm and , in embodiments, between 3-5 mm, the one or morearms being configured to move from a first position to a second position with a vertical displacement of less than 2 mm and with a total lateral displacement of between about 1 cm and 3 cm.

[0256] In aspects of the fourth example, the film suspension device comprises a plurality of arms, each of the plurality of arms having a proximal end movably attached to the frame and a distal end comprising a holding portion, wherein the holding portions of the plurality of arms collectively defining the film retaining opening when the plurality of arms are each disposed in the first position. In aspects of the fourth example, each of the plurality of arms downwardly depends from the frame.

[0257] In aspects of the fourth example, one or more of the plurality of arms comprises one or more positioning devices to maintain the each of the plurality of arms in the first position to collectively define the film retaining opening. In some aspects, the one or more positioning devices comprise one or more mechanical, electromechanical, or magnetic members. In some aspects, a first arm comprises a first latch member and a second arm comprises a second latch member, wherein the first latch and the second latch cooperatively engage one another to maintain the first arm and the second arm in the first position to collectively define the film retaining opening. In some aspects, the plurality of arms each comprise an actuator to bias the respective arm away from the arms’ first position toward the arms’ second position, wherein the one or more mechanical, electromechanical, or magnetic members (e.g., latch(es)) are configured to counter the bias of the arm actuators. In some aspects, the actuator comprises one or more springs.

[0258] In a fifth example, a system for preparing samples for time-resolved studies includes a support for a biological sample (optionally including a biological sample on a sample support), a thermally insulated chamber configured to bear a cryogenic liquid, a sample translation stage configured to move a sample between a first position at some height above the thermally insulated chamber and a second position within the thermally insulated chamber and below the surface of the cryogenic liquid, a means for removably attaching the sample support to the sample translation stage, and a mechanism for holding a freely suspended liquid film of a diameter between 1 mm and 10 mm and , in embodiments, between 3 and 5 mm, and positioning this film along the sample’s vertical translation axis between an initial sample position above the thermally insulated chamber and the surface of the cryogenic liquid, such that, after a biological sample has passedthrough the liquid film, the film-holding mechanism moves out of the path of the sample support, so that the sample and its support may pass unimpeded into the cryogenic liquid.

[0259] In at least some aspects of the fifth example, the biological sample comprises a biomolecular crystal, a thin film containing biomolecules, a thin film containing cells, and / or a thin section of a biological sample such as a tissue sample.

[0260] In at least some aspects of the fifth example, the sample translation stage is at least substantially vertical, and wherein the sample translation stage is motor-driven, gravity- driven, actuator-driven, or pneumatic-driven.

[0261] In at least some aspects of the fifth example, the sample translation stage is configured to accelerate the sample to a speed between 0.1 and 10 m / s and, in some instances, between 1 and 4 m / s before it enters the cryogenic liquid.

[0262] In at least some aspects of the fifth example, the sample support includes a magnetic steel goniometer base as used in cryocrystallography, and where the means for removably attaching the sample support to the sample translation stage includes a magnetic wand as used in cryocrystallography.

[0263] In at least some aspects of the fifth example, the mechanism for creating and holding the liquid film comprises two separately hinged elements attached to a frame, where the axes of rotation of the hinged elements are parallel and in the plane of the frame, and where the hinged elements each includes a portion of a loop such that when the hinged elements are rotated into contact, a complete loop capable of supporting a liquid film is formed.

[0264] In at least some aspects of the fifth example, the two hinged elements are held in contact using magnets or a clip, so that when the hinged elements are pushed downward and out of contact, they continue rotating downward under the influence of gravity or a spring until they are out of the path of the sample holder.

[0265] In at least some aspects of the fifth example, the hinged elements are pushed downward by contact with the sample holder or with a member attached to the sample translation stage.

[0266] In at least some aspects of the fifth example, the mechanism for creating and holding the thin film is comprised of two members that rotate on a common vertically oriented axle as in the manner of scissors, each member including a portion of a loop at the same distance from the axle, such that when the two members are in contact they form a complete loop capable of supporting a liquid film.

[0267] In at least some aspects of the fifth example, each member is attached to a biasing member, such as a spring, that acts to separate the members.

[0268] In at least some aspects of the fifth example, each member includes a magnet or a clip that holds them in a configuration in which the complete loop is formed.

[0269] In at least some aspects of the fifth example, opening of the loop may be triggered by contact of the members or of a clip with a downward moving member attached to the sample translation stage.

[0270] In at least some aspects of the fifth example, opening of the loop may be triggered by a sensor that detects the position of the sample and / or of the sample translation stage, and where the opening may be electrically or electromechanically actuated.

[0271] In at least some aspects of the fifth example, the mechanism for creating and holding the thin film comprises of two members that rotate on separate vertically oriented axles, each member including a portion of a loop, such that the two members can be rotated into contact to form a complete loop capable of holding a liquid film.

[0272] In at least some aspects of the fifth example, each member is attached to a spring that acts to separate the members so as to open the loop.

[0273] In at least some aspects of the fifth example, each member includes a magnet or a clip that holds them in a configuration in which the complete loop is formed.

[0274] In at least some aspects of the fifth example, the system further comprises a mechanism for adjusting the height of the suspended liquid film above the surface of the cryogenic liquid.

[0275] In at least some aspects of the fifth example, the mechanism for adjusting the height of the liquid film above the surface of the cryogenic liquid comprises a second motor-driven or manually-driven vertical translation stage, to which the mechanism for holding the liquid film is attached.

[0276] In at least some aspects of the fifth example, the portion of the sample support in contact with and adjacent to the sample where liquid is to be deposited is treated to produce a hydrophilic surface and / or an adjacent portion of the sample support is treated to produce a hydrophobic surface.

[0277] In at least some aspects of the fifth example, the portion of the mechanism bearing the liquid film is disposed at a height of 2 mm above the surface of the cryogenic liquid.

[0278] FIG. 19 is a flowchart illustrating a sample preparation method 1900, which includes at least one of steps 1910, 1920, 1930, 1940, and 1950.

[0279] Step 1910 includes translating a sample support from a first position toward a second position. The first position is at a first height above a thermally insulated chamber disposed in the receiving area. In the second position, a test sample borne by the sample support is disposed below a surface of a cryogenic liquid within the thermally insulated chamber. In step 1910, said translating may be along a translation axis, which may be vertically oriented.

[0280] Step 1920 includes moving the test sample into contact with and through a freely suspended liquid film in an opening of a transfer mechanism located between the first position of the sample and a surface of a cryogenic liquid. The transfer mechanism defines, in a first state, an opening to form and hold the freely suspended liquid film. At least part of the moving of step 1920 may occur during the translating of step 1910.

[0281] Step 1930 includes transferring at least a part of the freely suspended film to the test sample. At least part of the transferring of step 1930 may occur during and / or after step 1920.

[0282] Step 1940 includes changing the transfer mechanism from the first state to a second state, wherein the film-holding opening is disrupted or moved to facilitate continued movement of the sample support and test sample along the translation axis as the sample moves toward its second position within the cryogenic liquid. Step 1940 may occur after step 190, e.g., after the sample has moved into contact with and through the freely suspended film.

[0283] Step 1950 includes continuing the motion of step 1910 until the sample reaches a second position within the cryogenic liquid.Combinations of Features

[0284] Features described above, as well as those claimed below, may be combined in various ways without departing from the scope hereof. The following enumerated examples illustrate some possible, non-limiting combinations.

[0285] Embodiment 1. A sample preparation system comprising: a sample support configured to bear a test sample; a receiving area configured to receive a thermally insulated chamber bearing a cryogenic liquid; a sample translation stage configured totranslate the sample support along a translation axis between a first position at a first height above a thermally insulated chamber disposed in the receiving area and a second position where a test sample borne by the sample support would be disposed below a surface of a cryogenic liquid provided within a thermally insulated chamber disposed in the receiving area; and a transfer mechanism defining, in a first state, an opening to form and hold a freely suspended liquid film; wherein, the transfer mechanism opening is configured to be disposed along the translation axis of the sample support in the first state with a liquid film in the opening of the transfer mechanism and, following movement of a test sample borne by the sample support into contact with and through a film in the opening of the transfer mechanism, the transfer mechanism is configured to assume a second state, in which the opening defined by the transfer mechanism is disrupted or moved to facilitate continued movement of the sample support and test sample along the translation axis toward the second position of the sample translation stage, wherein a portion of a film in the opening of the transfer mechanism is transferred to the test sample following contact of the test sample with the film. The translation axis may be vertically oriented.

[0286] Embodiment 2. The system of embodiment 1, wherein the sample support is configured to bear a test sample comprising a biomolecular crystal, a thin film containing biomolecules, a thin film containing cells, and / or a biological sample.

[0287] Embodiment 3. The system of either one of embodiments 1 and 2, wherein the sample support is removably attached to the sample translation stage via one or more magnetic connectors or mechanical connectors or wherein a sample holding portion of the sample support is removably attached to the sample support via one or more magnetic connectors or mechanical connectors.

[0288] Embodiment 4. The system of any one of embodiments 1-3, wherein the sample translation stage is at least substantially vertical, wherein the sample translation stage is motor-driven, gravity- driven, actuator-driven, or pneumatic-driven, and wherein the sample translation stage is configured to accelerate the test sample to a speed between 0.1 and 10 m / s and, in some instances, between 1 and 4 m / s before the test sample enters the cryogenic liquid.

[0289] Embodiment 5. The system of any one of embodiments 1-4, wherein a portion of the transfer mechanism defining the opening comprises a frangible or breakable material that is displaced or broken by the sample support or a member attached to thesample support following movement of the test sample borne by the sample support through a film in the opening of the transfer mechanism.

[0290] Embodiment 6. The system of any one of embodiments 1-5, where the transfer mechanism comprises two separately hinged elements attached to a frame at a proximal end of the hinged element, where axes of rotation of the hinged elements are parallel and are spaced apart from the translation axis, and wherein the hinged elements each define a portion of the opening such that when distal ends of the hinged elements are rotated into the first position of the transfer mechanism, the opening capable of supporting a liquid film is formed.

[0291] Embodiment 7. The system of embodiment 6, where the two hinged elements are held in the first position by one or more magnets or one or more mechanical latches, so that when the hinged elements are biased away from the first position, they rotate toward the second position due to gravity, responsive to a resilient element, or responsive to an actuator, until they are out of a path of the sample support.

[0292] Embodiment 8. The system of either one of embodiments 6 or 7, where the hinged elements are biased away from the first position by contact of a portion of the sample support, a member attached to the sample support, sample translation stage, or a member attached to the sample translation stage, with the transfer mechanism.

[0293] Embodiment 9. The system of any one of embodiments 1-8, wherein the transfer mechanism comprises a first member and a second member configured to rotate on a common vertically oriented axle, the first member defining a first portion of the opening at a predetermined distance from the axle, the second member defining a second portion of the opening at the predetermined distance from the axle, such that when the first portion of the first member and the second portion of the second member are disposed in engagement, the first portion and the second portion are adjacently positioned to together define the opening capable of supporting a liquid film.

[0294] Embodiment 10. The system of embodiment 9, where the first member and the second member are each attached to a biasing member biasing the first member and the second member out of engagement.

[0295] Embodiment 11. The system of either one of embodiments 9 or 10, wherein the first member, the second member, or both the first member and the second member includes a magnet or a latch to hold the first member and the second member in engagement.

[0296] Embodiment 12. The system of any one embodiments 9-11, wherein the first member and the second member of the transfer mechanism are caused to disengage from one another responsive to contact of a portion of the sample support, a member attached to the sample support, a portion of the sample translation stage, or a member attached to the sample translation stage, with at least one of the first member or the second member, or a latching member holding the first member and the second member in engagement, during movement of the sample translation stage.

[0297] Embodiment 13. The system of any one embodiments 9-12, wherein an actuator or switch is configured to cause disengagement of the transfer mechanism first member and second member responsive to a signal from a sensor configured to detect a position of the sample and / or a position of the sample translation stage.

[0298] Embodiment 14. The system of any one of embodiments 1-13, where the transfer mechanism comprises two members that rotate on a separate vertically oriented axles, each member defining a portion of the opening, such that the two members can be rotated into a first position wherein the respective portions of the opening are adjacent to form the opening capable of holding a liquid film.

[0299] Embodiment 15. The system of embodiment 14, where each of the two members is attached to a spring that acts to bias the two members away from the first position and away from one another to a second position.

[0300] Embodiment 16. The system of either one of embodiment 14 or 15, further comprising one or more magnets or latches configured to hold each of the two members in the first position.

[0301] Embodiment 17. The system of any one of embodiments 1-16, further comprising a mechanism for adjusting a height of the transfer mechanism or a height of the opening of the transfer mechanism relative to a surface of a cryogenic liquid disposed in a thermally insulated chamber in the receiving area.

[0302] Embodiment 18. The system of any one of embodiments 1-17, where the mechanism for adjusting the height of the transfer mechanism or a height of the opening of the transfer mechanism relative to a surface of a cryogenic liquid disposed in a thermally insulated chamber in the receiving area comprises a motor-driven or a manually-driven driven translation stage attached to the transfer mechanism.

[0303] Embodiment 19. The system of any one of embodiments 1-18, wherein a first portion of the sample support in contact with and adjacent to a test sample comprisesa hydrophilic surface and wherein a second portion of the sample support adjacent the first portion comprises a hydrophobic surface.

[0304] Embodiment 20. The system of any one of embodiments 1-19, wherein a height of the transfer mechanism opening is configured to be positionable at a height of 2 mm or greater above a surface of a cryogenic liquid disposed in a thermally insulated chamber in the receiving area.

[0305] Embodiment 21. The system of any one of embodiments 1-20, wherein the sample translation stage is configured to translate the sample support at an angle of between 60 degrees to about 90 degrees relative to the receiving area or a thermally insulated chamber disposed in the receiving area, and wherein the sample translation stage is motor-driven, gravity-driven, actuator-driven, or pneumatic-driven.

[0306] Embodiment 21. The system of any one of embodiments 1-21, further comprising: a test sample borne in the sample support; and a thermally insulated chamber, bearing a cryogenic liquid, disposed in the receiving area.

[0307] Changes may be made in the above methods and systems without departing from the scope of the present embodiments. It should thus be noted that the matter contained in the above description or shown in the accompanying drawings should be interpreted as illustrative and notin a limiting sense. Herein, and unless otherwise indicated the phrase “in embodiments” is equivalent to the phrase “in certain embodiments,” and does not refer to all embodiments.

[0308] As used in this specification, any appendices thereto, and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. Regarding instances of the terms “and / or” and “at least one of,” for example, in the cases of “A and / or B,” “at least one of A and B,” and “at least one of A or B,” such phrasing encompasses the selection of (i) A only, or (ii) B only, or (hi) both A and B. In the cases of “A, B, and / or C, ” “at least one of A, B, and C,” and “at least one of A, B, or C,” such phrasing encompasses the selection of (i) A only, or (ii) B only, or (iii) C only, or (iv) A and B only, or (v) A and C only, or (vi) B and C only, or (vii) each of A and B and C. This may be extended for as many items as are listed.

[0309] The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the present method and system, which, as a matter of language, might be said to fall therebetween.

Claims

CLAIMSWe claim:

1. A sample preparation system comprising: a sample support configured to bear a test sample; a receiving area configured to receive a thermally insulated chamber bearing a cryogenic liquid; a sample translation stage configured to translate the sample support along a vertical translation axis between a first position at a first height above a thermally insulated chamber disposed in the receiving area and a second position where a test sample borne by the sample support would be disposed below a surface of a cryogenic liquid provided within a thermally insulated chamber disposed in the receiving area; and a transfer mechanism defining, in a first state, an opening to form and hold a freely suspended liquid film; wherein, the transfer mechanism opening is configured to be disposed along the vertical translation axis of the sample support in the first state with a liquid film in the opening of the transfer mechanism and, following movement of a test sample borne by the sample support into contact with and through a film in the opening of the transfer mechanism, the transfer mechanism is configured to assume a second state, in which the opening defined by the transfer mechanism is disrupted or moved to facilitate continued movement of the sample support and test sample along the vertical translation axis toward the second position of the sample translation stage, wherein a portion of a film in the opening of the transfer mechanism is transferred to the test sample following contact of the test sample with the film.

2. The system of claim 1, wherein the sample support is configured to bear a test sample comprising a biomolecular crystal, a thin film containing biomolecules, a thin film containing cells, and / or a biological sample.

3. The system of claim 1, wherein the sample support is removably attached to the sample translation stage via one or more magnetic connectors or mechanical connectors or wherein a sample holding portion of the sample support is removably attached to the sample support via one or more magnetic connectors or mechanical connectors.

4. The system of claim 1, wherein the sample translation stage is at least substantially vertical, wherein the sample translation stage is motor-driven, gravity- driven, actuator-driven, or pneumatic-driven, and wherein the sample translation stage is configured to accelerate the test sample to a speed between 0.1 and 10 m / s and, in some instances, between 1 and 4 m / s before the test sample enters the cryogenic liquid.

5. The system of claim 1, wherein a portion of the transfer mechanism defining the opening comprises a frangible or breakable material that is displaced or broken by the sample support or a member attached to the sample support following movement of the test sample borne by the sample support through a film in the opening of the transfer mechanism.

6. The system of claim 1, where the transfer mechanism comprises two separately hinged elements attached to a frame at a proximal end of the hinged element, where axes of rotation of the hinged elements are parallel and are spaced apart from the vertical translation axis, and wherein the hinged elements each define a portion of the opening such that when distal ends of the hinged elements are rotated into the first position of the transfer mechanism, the opening capable of supporting a liquid film is formed.

7. The system of claim 6, where the two hinged elements are held in the first position by one or more magnets or one or more mechanical latches, so that when the hinged elements are biased away from the first position, they rotate toward the second position due to gravity, responsive to a resilient element, or responsive to an actuator, until they are out of a path of the sample support.

8. The system of claim 6, where the hinged elements are biased away from the first position by contact of a portion of the sample support, a member attached to the sample support, sample translation stage, or a member attached to the sample translation stage, with the transfer mechanism.

9. The system of claim 1, wherein the transfer mechanism comprises a first member and a second member configured to rotate on a common vertically oriented axle, the first member defining a first portion of the opening at a predetermined distance from the axle, the second member defining a second portion of the opening at the predetermined distance from the axle, such that when the first portion of the first member and the second portion of the second member are disposed in engagement, the first portion and the second portion are adjacently positioned to together define the opening capable of supporting a liquid film.

10. The system of claim 9, where the first member and the second member are each attached to a biasing member biasing the first member and the second member out of engagement.

11. The system of claim 9, wherein the first member, the second member, or both the first member and the second member includes a magnet or a latch to hold the first member and the second member in engagement.

12. The system of claim 9, wherein the first member and the second member of the transfer mechanism are caused to disengage from one another responsive to contact of a portion of the sample support, a member attached to the sample support, a portion of the sample translation stage, or a member attached to the sample translation stage, with at least one of the first member or the second member, or a latching member holding the first member and the second member in engagement, during movement of the sample translation stage.

13. The system claim 9, wherein an actuator or switch is configured to cause disengagement of the transfer mechanism first member and second member responsive to a signal from a sensor configured to detect a position of the sample and / or a position of the sample translation stage.

14. The system of claim 1, where the transfer mechanism comprises two members that rotate on a separate vertically oriented axles, each member defining a portion of the opening, such that the two members can be rotated into a first position wherein the respective portions of the opening are adjacent to form the opening capable of holding a liquid film.

15. The system of claim 14, where each of the two members is attached to a spring that acts to bias the two members away from the first position and away from one another to a second position.

16. The system of claim 14, further comprising one or more magnets or latches configured to hold each of the two members in the first position.

17. The system of claim 1, further comprising a mechanism for adjusting a height of the transfer mechanism or a height of the opening of the transfer mechanism relative to a surface of a cryogenic liquid disposed in a thermally insulated chamber in the receiving area.

18. The system of claim 1, where the mechanism for adjusting the height of the transfer mechanism or a height of the opening of the transfer mechanism relative to a surface of a cryogenic liquid disposed in a thermally insulated chamber in the receiving area comprises a motor-driven or a manually-driven driven vertical translation stage attached to the transfer mechanism.

19. The system of claim 1, wherein a first portion of the sample support in contact with and adjacent to a test sample comprises a hydrophilic surface and wherein a second portion of the sample support adjacent the first portion comprises a hydrophobic surface.

20. The system of claim 1, wherein a height of the transfer mechanism opening is configured to be positionable at a height of 2 mm or greater above a surface of a cryogenic liquid disposed in a thermally insulated chamber in the receiving area.

21. The system of claim 1, wherein the sample translation stage is configured to translate the sample support at an angle of between 60 degrees to about 90 degrees relative to the receiving area or a thermally insulated chamber disposed in the receiving area, and wherein the sample translation stage is motor-driven, gravity- driven, actuator-driven, or pneumatic-driven.

22. The system of claim 1, further comprising: a test sample borne in the sample support; and a thermally insulated chamber, bearing a cryogenic liquid, disposed in the receiving area.

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