Patent Application
20250041871
This disclosure relates generally to the field of biotechnology. More particularly, aspects of this disclosure relate to the design of sample supports, sample cooling systems, and methods for handling samples for cryo-electron microscopy.
Single-particle cryo-electron microscopy (cryo-EM) is a powerful approach to obtaining near-atomic-resolution structures of large biomolecular complexes, membrane proteins, and other targets of major scientific, pharmaceutical, and biotechnological interest. Development of high efficiency, high frame rate direct electron detectors, algorithms for correcting acquired “movies” for electron-beam-induced motion, and computational tools for classifying and averaging 105-106 molecular images have dramatically increased achievable resolution and throughput. Enormous investments in new cryo-EM facilities and the development of easy-to-use software have greatly expanded access, especially to non-experts. Unlike X-ray crystallography, cryo-EM necessitates only a small amount of biomolecular sample dispersed in solution. It allows structural study of systems that have been intractable to crystallization, and is becoming a go-to method for initial attempts at structure determination.
As in X-ray cryocrystallography, key challenges in single-particle cryo-EM are associated with sample preparation and handling. Many basic principles and methods in current use were developed in the 1980s, and recent sample preparation technology development is firmly rooted in ideas and methods developed at that time. Cryoprotectant-free buffers containing a biomolecule of interest (e.g., about 0.3 milligrams per milliliter (mg/mL)) may be dispensed onto glow-discharge cleaned and charged (e.g., 10-50 nanometer (nm)) thick carbon or metal (e.g., gold) “foil” that is supported, for example, by a 200-400 mesh, 10-25 micrometer (μm) thick, 3.05 millimeter (mm) diameter metal (e.g., copper or gold) grid. Excess sample may be removed by blotting and evaporation, with a target thickness of several times the biomolecular diameter (e.g., about 10-50 nm) to maximize image quality while limiting the fraction of biomolecules preferentially oriented by interaction with interfaces. To vitrify the buffer for the best imaging, the sample-containing foil+grid is plunged, e.g., at about 1-2 meters per second (m/s) into liquid ethane at a temperature (T) of about 90 Kelvin (K) (e.g., produced by cooling gas in a liquid-nitrogen-cooled cup). The sample is transferred from ethane to liquid nitrogen (LN2), loaded into grid boxes, transferred to additional containers, and then into a storage Dewar. Samples are removed from the storage Dewar and grid boxes, and loaded into a cold microscope stage or else “clipped” and loaded into a cold sample cassette; the stage or cassette is then loaded in the microscope.
These complex procedures are fraught with difficulty. Grids and especially foils are routinely bent, torn, and otherwise damaged at each of the many manual handling steps. A new generation of sample preparation instruments integrates and automates sample deposition and blotting, sample plunging into liquid ethane, sample transfer into liquid nitrogen, and transfer into storage cassettes or containers. These instruments are complex and may be very expensive (e.g., costing in excess of $500,000 USD).
Systems and methods are currently available for cryocooling single-particle cryo-EM samples using only boiling liquid nitrogen as the coolant and obtaining high-quality images and molecular reconstructions. As a result, hazardous ethane, temperature-controlled stages to hold it, and robotics to transfer samples from ethane to liquid nitrogen for storage can be eliminated. This can substantially simplify designs for automated cryo-EM plunge coolers without compromises in performance.
The present disclosure relates to systems, methods, and devices using sample supports and sample cooling devices for cryo-electron microscopy.
Aspects of this disclosure include structures, apparatuses, and approaches to facilitate automated cryo-EM sample storage after cryocooling, particularly for systems that use only liquid nitrogen as the sample coolant, and to facilitate subsequent handling.
These structures and approaches are much simpler than robotics based approaches and can be implemented at much lower cost, without compromising performance.
Disclosed structures/apparatuses used for cryo-EM sample storage may be compatible with existing structures for both cryo-EM grid storage and for cryocrystallography sample storage. Since scientists generally use both cryo-EM and cryo-crystallography, structures that support sample handling in both approaches help to reduce complexity and cost for the end user.
Described herein are innovations for post-cooling sample storage of cryo-EM samples/grids in grid boxes.
According to aspects of this disclosure, the sample grid may be held using forceps that can be opened and closed robotically.
According to aspects of this disclosure, the forceps may be part of a sample wand that attaches to a vertical translation stage and that can be opened and closed via push-button actuation of the wand.
According to aspects of this disclosure, the sample grid may be translated vertically along a single axis from a start position in ambient or above-freezing temperature air or gas into liquid nitrogen for cooling, and then is translated along the same axis to a grid release point.
According to aspects of this disclosure, the grid may be released at the grid release point, by actuation of the forceps, into a funnel immersed in liquid nitrogen and positioned on the axis of vertical translation of the sample grid.
According to aspects of this disclosure, the funnel may have a tapering rectangular cross section ending in a narrow rectangular slot to precisely direct the grid.
According to aspects of this disclosure, the funnel opening at its top may have a width of about 2 mm to about 8 mm or, for at least some preferred configurations, about 4 mm, and a length of about 4 mm to about 10 mm or, for at least some preferred configurations, about 5 mm.
According to aspects of this disclosure, the funnel opening at its bottom may have a width of about 0.1 mm to about 1.5 mm or, for at least some preferred configurations, about 0.65 mm, and a length of about 3.2 mm to about 5 mm or, for at least some preferred configurations, about 3.5 mm.
According to aspects of this disclosure, a vertical distance between the top and bottom openings of the funnel may be about 5 mm to about 50 mm or, for at least some preferred configurations, about 16 mm.
According to aspects of this disclosure, one or more grid boxes may be held in a fixed position and orientation within a cryogenic-temperature-compatible grid box holder, that is in turn held on a stage. The grid box(es) and grid box holder may then be immersed in liquid nitrogen or other liquid cryogen. Each grid receptacle in each grid box can be positioned below the bottom opening of the funnel, allowing grids to be deposited in the grid box.
According to aspects of this disclosure, the grid box(es) and grid box holder may be replaced by a grid holder that directly has a pattern of receptacles for grids, as would be provided by an array of grid boxes.
According to aspects of this disclosure, the grid box holder may be attached to a stage that provides a single axis of rotation or a single axis of translation in a plane perpendicular to a plunge direction.
According to aspects of this disclosure, each grid box may have a grid holding receptacle layout that allows grid holding receptacles within each grid box to be positioned beneath the bottom outlet of the funnel. This may also allow grids to be deposited in the grid holding receptacles, one by one, via successive translations along a single axis or via successive rotations about a single axis of the stage and grid box holder.
According to aspects of this disclosure, the grid box holder may be attached to a stage that provides two axes of translational and/or rotational motion in a plane perpendicular to a plunge direction. This allows grid holding receptacles within grid boxes with arbitrary receptacle layouts to be positioned beneath the bottom outlet of the funnel, and may further allow grids to be deposited in the grid holding receptacles, one by one, via successive translations and/or rotations of the stage and grid box holder.
Also described herein are innovations for post-cooling sample handling to facilitate easy “clipping” of grids for loading into sample cassettes used in high-throughput cryo-TEMs.
According to aspects of this disclosure, a sample grid may be held using forceps that can be opened and closed robotically.
According to aspects of this disclosure, the sample grid may be translated vertically along a single axis from a start position in ambient or above-freezing temperature air or gas into liquid nitrogen or other liquid cryogen for cooling. Afterwards, the sample grid is then translated along the same single vertical axis to a grid release point.
According to aspects of this disclosure, the grid may be released by actuation of the forceps into a funnel immersed in the liquid nitrogen and whose top opening is positioned on the axis of vertical translation of the sample grid.
According to aspects of this disclosure, the funnel may have a tapering rectangular cross section.
According to aspects of this disclosure, the funnel may release grids into a grid clip ring holder, which has one or more receptacles for holding grid clip rings.
According to aspects of this disclosure, the grid clip ring may rest flat and not on edge on the bottom of the grid clip ring receptacle.
According to aspects of this disclosure, the bottom of the grid clip ring receptacle may be angled relative to the horizontal at an angle between 20 and 50 degrees.
According to aspects of this disclosure, the funnel's lower channel may curve so as to release the grid at an angle to the vertical, facilitating the grid drifting downward so that the grid lands with a specific side up within a grid clip ring.
According to aspects of this disclosure, the bottom end of the funnel may have features that facilitate release and subsequent motion of the grid at an angle to the vertical so that it lands with a preselected side up.
According to aspects of this disclosure, the bottom end of the funnel may have a cut-out on its lower side so that the grid is supported only on its edges via two ledges as it slides to the funnel's end. One ledge may end before the other so that the grid drifts sideways out of the funnel.
According to aspects of this disclosure, the ledges may end in a taper such that, when a portion of the grid edge closest to each sidewall of the funnel moves beyond and loses contact with the edge of the ledge, the trailing edge of the grid also moves beyond and loses contact with the edge of the ledge so that the grid experiences no net torque due to its interactions with gravity and the funnel.
According to aspects of this disclosure, a grid clip ring receptacle in the grid clip ring holder may be positioned so that its center is shifted relative to the center of the funnel outlet, in a plane perpendicular to the larger transverse dimension of the funnel, so as to facilitate transfer of grids through the funnel and into the grid clip ring with a desired orientation (foil or grid side up).
According to aspects of this disclosure, an upper portion of the grid clip ring receptacle may be configured to guide and correctly position a grid clipping tool so that grids within clip rings with the holder can easily be clipped by inserting a C-clip.
According to aspects of this disclosure, an array of funnels may be incorporated into a top portion of a holder of grid boxes, grids, or grid clip rings. These funnels may also removably attach to a bottom portion of the holder that has receptacles for grid boxes, grids, or grid clip rings, such that a grid released into each funnel in the top portion will be deposited into a unique grid box, grid receptacle, or grid clip ring receptacle in the bottom portion.
According to aspects of this disclosure, a series of grid clip rings may be held in a series of receptacles within a clip ring holder that is immersed in liquid nitrogen and that can be positioned just below the bottom outlet of the funnel. The clip ring holder has an array of receptacles that is sized and shaped to accept a clip ring and is compatible with grid clipping tools used to insert the C-clip spring and to clip grids. The walls of the receptacles may be angled outward or otherwise structured at the top to help guide the grid clipping tool to the bottom of each receptacle.
According to aspects of this disclosure, the grid clip ring holder may be attached to a stage that provides a single axis of rotation or a single axis of translation in a plane perpendicular to the grid plunge direction. This may allow grid holding receptacles containing grid clip rings within each grid clip ring holder to be positioned beneath the bottom outlet of the funnel, and may allow grids to be deposited in the grid clip rings within the clip ring receptacles, one by one, via successive translations or rotations of the stage and clip ring holder along a single translation or about a single rotation axis.
According to aspects of this disclosure, the grid clip ring holder may be attached to a stage that provides two axes of translational and/or rotational motion in a plane perpendicular to the plunge direction. This may allow grid clip rings within grid clip ring holding receptacles having arbitrary layouts within the grid clip ring holder to be positioned beneath the bottom outlet of the funnel. This may also allow grids to be deposited in the clip rings within the grid clip ring holding receptacles, one by one, via successive translations or rotations of the stage and clip ring holder.
Also described herein are innovations for post-cooling sample storage of cryo-EM samples/grids that are based on sample storage “pucks”-referred to as “UniPucks”—used in cryocrystallography.
According to aspects of this disclosure, a cryo-EM sample storage puck may consist essentially of a top portion and a bottom portion immersed in liquid nitrogen.
According to aspects of this disclosure, the cryo-EM sample storage puck may have a largely cylindrical shape that is sized and shaped to be compatible with hardware used for handling and storage of UniPucks.
According to aspects of this disclosure, the puck may have a diameter of about 62 mm to about 72 mm or, for at least some preferred configurations, about 67 mm. The puck may have a height of about 30 mm to about 35 mm or, for at least some preferred configurations, about 32.4 mm. The top portion of the puck may have a height of about 12 mm to about 24 mm or, for at least some preferred configurations, about 18 mm.
According to aspects of this disclosure, the puck may have a central hole with a semi-cylindrical notch in its outer periphery that cooperatively facilitate puck handling using “cryotongs” used with UniPucks.
According to aspects of this disclosure, the bottom portion of the puck may have multiple recesses each shaped to accept and fix the position and orientation of a grid box. It may be desirable that the top surface of the grid box be approximately flush with a top surface of the bottom of the puck.
According to aspects of this disclosure, the top portion of the puck may include an array of rectangular cross-section funnels. In this instance, a bottom portion of the puck may be loaded with grid boxes and the puck assembled by inserting the top portion in the bottom portion. When the puck is fully immersed in liquid nitrogen, the funnels guide grids are released into an upper portion of the funnel, then downward and into grid receptacles within the grid boxes held within the lower portion.
According to aspects of this disclosure, the funnels may be arranged in a circle, the grid boxes may be arranged in a circle, and the slots within the grid boxes may be arranged so that deposition of the grids into successive funnels and into the receptacles within the grid boxes within the holder may necessitate only rotation of the puck about its central axis.
According to aspects of this disclosure, the funnels may be arranged in concentric circles and provide curved paths for the grids so as to guide them into grid boxes having receptacles within them in the different configurations/layouts of the grid boxes. In so doing, grid deposition within the grid boxes may necessitate two axes of motion of the puck.
According to aspects of this disclosure, in place of recesses for grid boxes, a bottom portion of the puck may have an arrangement of receptacles each sized and shaped to accept a single grid with its plane in a vertical or near vertical orientation. In this instance, a top portion of the puck may include an array of rectangular cross-section funnels. After the puck is assembled by inserting the top portion into the bottom portion, and the puck is substantially or fully immersed in liquid nitrogen, the funnels guide the grids, which were released into an upper portion of the funnel, downward and into grid receptacles within the lower portion of the puck.
Also described herein are innovations for post-cooling loading of cryo-EM sample grids into clip rings using sample storage “pucks” used in cryocrystallography.
According to aspects of this disclosure, a cryo-EM sample storage puck may consist essentially of a top portion and a bottom portion, immersed in liquid nitrogen.
According to aspects of this disclosure, the cryo-EM sample storage puck may have a substantially cylindrical shape.
According to aspects of this disclosure, a bottom portion of the puck may have multiple cylindrical recesses that are shaped to accept clip rings used in “clipping” grids for automated handling (e.g., in some THERMOFISHER™ cryo-transmission electron microscopes), and to accept the grid clipping tool used to insert a C-springs into a clip that holds the grid in a clip ring.
According to aspects of this disclosure, a top portion of the puck may include an array of rectangular cross-section funnels. In this instance, a bottom portion of the puck may be loaded with clip rings, the puck may be assembled by inserting the top portion in the bottom portion, and then the puck may be substantially or fully immersed in liquid nitrogen. The funnels may guide grids, which were released into an upper portion of the funnel, downward and into clip rings held within the lower portion of the puck such that the same side of the grid reliably ends up facing down into the clip ring.
According to aspects of this disclosure, a grid path through the funnels may be curved and a bottom end of the funnel may be shaped so as to orient the grid to help ensure that it lands with the correct orientation within the clip ring after falling through the liquid nitrogen.
According to aspects of this disclosure, the funnels may be arranged in a circle and the cylindrical receptacles holding clip rings may be arranged in a circle to help ensure that deposition of the grids into successive funnels and into the clip rings necessitates only rotation of the puck about its central axis.
According to an aspect of this disclosure, a bottom of the funnel structure may be configured to inhibit release of a grid and grid clip ring from the grid clip ring receptacles if the puck is tilted, inverted, or subject to acceleration.
The above Summary does not represent every embodiment or every aspect of the present disclosure. Rather, the foregoing synopsis merely provides an exemplification of some of the novel concepts and features set forth herein. The above features and advantages, and other features and attendant advantages of this disclosure, will be readily apparent from the following detailed description of illustrated examples and representative modes for carrying out the present disclosure when taken in connection with the accompanying drawings and the appended claims. Moreover, this disclosure expressly includes any and all combinations and subcombinations of the elements and features presented above and below
The present disclosure is amenable to various modifications and alternative forms, and some representative embodiments 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 the particular forms illustrated in the above-enumerated drawings. Rather, this disclosure covers all modifications, equivalents, combinations, subcombinations, permutations, groupings, and alternatives falling within the scope of this disclosure as encompassed, for example, by the appended claims
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 the disclosure. To that extent, elements and limitations that are described, for example, in the Abstract, Technical Filed, Background, Summary, 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.
The present disclosure describes devices and methods by which cryo-EM grids that are plunge cooled in a liquid cryogen may be automatically stored after cooling. In particular, described herein are devices and methods that allow grids that are translated along a single vertical axis during plunging to be translated and released on that same axis and then stored in grid boxes or loaded into clip rings for subsequent handling. The disclosure provides a dramatically simplified and highly convenient approach to automated post-cooling grid handling and storage.
With the arrangement of clip ring receptacles along an arc as in
Extensive experimentation with prototypes of the grid box system in
Nearly all researchers who use single-particle cryo-electron microscopy to determine the structures of biomolecules also use X-ray cryocrystallography. As a result, common methods and hardware used to store and transport cryo-EM samples are based on existing “standardized” hardware for cryocrystallography, including sample pucks as in
The top portion of the puck includes a series of funnels 440 with rectangular cross-sections that have a wide end on the top surface of the top portion and a narrow tapered end at the bottom surface of the top portion. The bottom portion of the puck 410 has multiple receptacles each shaped and sized to accept and fix the position and orientation of a “standard-format” grid box 450. When the top and bottom portions are assembled together, each funnel in the top portion has its bottom end aligned and in near contact with a grid receptacle in a grid box held in the bottom portion, so that when grids are released into each of the funnels, they fall and are guided into each of the grid receptacles in each of the grid boxes. In the example shown, the grid boxes are arranged in a circle, each has grid receptacles that lie on a circle, and the funnels are arranged in a circle and guide the grids straight down into each receptacle. This design allows all grid box receptacles to be loaded using a single axis of puck rotation.
To prevent grids from falling out if the puck is inverted, the top portion can be covered with a cap; the cap could be of magnetic steel and the top portion may include one or more magnets; the cap may insert and be captured by (e.g., using twist and lock) the central hole of the top portion; or the bottom portion may rotate slightly after grid loading so that the funnels no longer align with the grid box receptacles and so that the each grid box receptacle is covered by a solid region of the top portion.
Aspects of the present disclosure have been described in detail with reference to the illustrated embodiments; those skilled in the art will recognize, however, that many modifications may be made thereto without departing from the scope of the present disclosure. The present disclosure is not limited to the precise construction and compositions disclosed herein; any and all modifications, changes, and variations apparent from the foregoing descriptions are within the scope of the disclosure as defined, for example, by the appended claims. Moreover, the present concepts expressly include any and all combinations and subcombinations of the preceding elements and features. Additional features may be reflected in the following clauses:
This application is a continuation of U.S. patent application Ser. No. 18/852,838, which was filed on Sep. 30, 2024, is now pending, and is a U.S. National Phase Entry of International Patent App. No. PCT/US2023/017096, which was filed on Mar. 31, 2023, is now pending, and claims the benefit of priority to U.S. Provisional Patent App. No. 63/326,473, which was filed on Apr. 1, 2022, and is now lapsed. All of the foregoing patent applications are incorporated herein by reference in their respective entireties and for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 63326473 | Apr 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 18852838 | Jan 0001 | US |
| Child | 18919875 | US |
