Cryo-electron microscopy sample preparation system, and modules and methods for operating same

Abstract

A system, modules and various methods are provided for cryo-EM grid preparation that can achieve a high throughput with parameterized multi-grid handling. The system can be configured to be scalable, updatable and future proof by using an open architecture and modular design. The system described herein can address problems with preferred orientation, timing, optimization, time-resolution experiments, particularly for new particles and techniques.

Description

CRYO-ELECTRON MICROSCOPY SAMPLE PREPARATION SYSTEM, AND MODULES AND METHODS FOR OPERATING SAMECROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 570,418 filed on March 27, 2024, the contents of which are incorporated herein by reference in their entirety.TECHNICAL FIELD

[0002] The following generally relates to sample preparation, in particular to cryoelectron microscopy (cryo-EM) sample preparation, including modules and methods for operating such a system.BACKGROUND

[0003] Since the development of electron microscopes (EMs), there has become a desire to examine biological specimens. Examining such specimens, however, was considered challenging due to difficulties in making the specimen thin enough to get an electron beam through it and preserving and contrasting the various structural elements of the material in the high vacuum of the microscope. Because of difficulties associated with maintaining hydration in a high vacuum environment, the early development of specimen preservation for biological EM focused on fixation, dehydration, embedding and sectioning combined with application of heavy metals to provide contrast in tissue samples.

[0004] More recent advances in cryo-EM such as access to better and faster microscopes, detectors, and data processing algorithms have accelerated data collection and processing speeds, thus driving down total costs per micrograph. Consequently, more single-particle structures can be determined routinely at subnanometer resolution and more biological objects may be imaged and identified in situ. For example, an increase in the number of cryo-EM entries in the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) has been observed.

[0005] Cryo-EM has become a powerful tool for imaging otherwise challenging samples such as large complexes and heterogeneous samples. One class of samples on which cryo-EM has had a large impact is membrane proteins. These proteins need to be embedded in a membrane mimetic to maintain their structuralintegrity, which makes them especially challenging for alternative structural techniques like X-ray crystallography.

[0006] Conversely, for cryo-EM, if the proteins can be extracted from the membrane, solubilized as individual particles, then vitrified in a thin layer on a grid, their structures can be solved with standard single-particle methods.

[0007] It is found that sample preparation and grid-making for cryo-EM can present significant challenges for many projects. Grid preparation for cryo-EM is used to suspend proteins in a well-vitrified layer of ice that is made to be as thin as possible, on a substrate supported by an EM grid. When done properly, the ice should be vitreous and uniform across the entire grid and just slightly thicker than the molecule of interest. The ice should also contain well-distributed particles at a sufficient concentration. The ability to reproducibly make good-quality grids means more high-quality images can be collected faster, making the entire process cheaper. Despite attempts at creating machines to automate and control the ice making process, challenges still remain, as well as the relatively high cost associated with these solutions.

[0008] Even with recent advances in robotic plunge freezers and the like, vitrification is a nontrivial optimization task that typically requires good fine motor skills while still being difficult to replicate from grid to grid.

[0009] To make a cryo-EM grid, a sample volume (e.g., -3 pL) is applied to a grid, then reduced to a thin film (e.g., -10-100 nm) by blotting with filter paper, followed by plunging the grid rapidly into a cryogen. In the process of making the thin film, the surface area to volume ratio of the sample increases dramatically, with the result that proteins collide with the air-water interface (AWI). These collisions with the AWI can cause a variety of problems. Although high-resolution electron cryo-EM of purified biological macromolecules has become a relatively more mature method, preparing the required thin, vitrified samples often remains a major challenge. Efforts have been under way for some time to improve the way in which samples are thinned before they are vitrified.

[0010] Cryo-sample preparation is found to be an important step in the process of obtaining high-resolution structures of macromolecules by using the single-particlecryo-EM method, however, cryo-sample preparation is commonly hampered by high uncertainty and low reproducibility.

[0011] Specifically, it is found that the reliability and repeatability of sample preparation can be a primary bottleneck with cryo-EM processes. For instance, the process is found to not be identical in execution, such that even “automated” machines rely on significant manual steps. Consequently, much of sample preparation remains trial and error.

[0012] Moreover, vitrification conditions may not be repeatable, the speed of the process and some environmental conditions can have major impacts on the quality of the results, and such results are often not recorded. Even with drastically high cost machines, some doubt may remain regarding the quality of samples produced.SUMMARY

[0013] In one aspect, there is provided an applicator module for a cryo-EM sample preparation system, the applicator module comprising: a holder for an applicator device, the applicator device configured to be actuated to express a determined portion of sample contained in the applicator device onto a grid; a carriage assembly supporting the applicator device, the carriage assembly being coupled to a withdrawal mechanism to enable the applicator device to be driven towards the grid to apply the sample and rapidly retract to provide space for a plunging module to further plunge the grid beyond the applicator module.

[0014] In another aspect, there is provided a blotting module for a cryo-EM sample preparation system, the blotting module comprising: at least one rotatable blotting arm to permit a blotting medium attached thereto to be presented towards and retracted from a grid having sample applied thereto; and a carriage assembly supporting the at least one blotting arm, the carriage assembly being coupled to a withdrawal mechanism to enable the blotting module to be driven towards the grid to blot the sample to keep the blotting medium from a humid environment in which the grid is being held.

[0015] In another aspect, there is provided an environmental module for a cryo- EM sample preparation system, the module comprising: a cooling system; a humidity system; and a chamber coupled to the cooling system via the humidity system torapidly cool a column of air defining a zone, the zone providing a cooled and humidified column of air into which a grid may be plunged by a plunging module positioned with the system above the chamber, the zone permitting access to a sample applicator to apply sample to a grid positioned in the chamber and a blotting module to blot the grid, the chamber being positioned above a cryo-module in the system and permitting the plunge module pass the grid through the chamber after preparation and be dropped into a crucible in the cryo-module.

[0016] In another aspect, there is provided a plunge system for a cryo-EM sample preparation system, the plunge system comprising: at least one drop rod operated by a high speed drive system, the drop rod being moveable vertically to plunge a grid held by the drop rod using a pair of tweezers at a bottom end thereof into an environmental chamber positioned beneath the plunge system; and the pair of tweezers coupled to the at least one drop rod, the pair of tweezers being actuatable to hold and release the grid.

[0017] In another aspect, there is provided a cryo-module for a cryo-EM sample preparation system, the cryo-module comprising: a crucible; a cup positioned within the crucible, the cup holding a plunge substance to be surrounded by liquid nitrogen contained around the cup within the crucible; and a control system to provide internal control of LN2 levels and cryogen levels.

[0018] In another aspect, there is provided a cryo-EM sample preparation system comprising: an applicator module according to the above; a blotting module according to the above; an environmental chamber according to the above; a plunge module according to the above; and a cryo-module according to the above.

[0019] Methods for operating the modules described above are also provided. Computer readable media storing computer executable instructions that when executed by a processor cause one or more of the above modules to perform such operations are also provided.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Embodiments will now be described with reference to the appended drawings wherein:

[0021] FIG. 1 is a modular block diagram of a cryo-EM sample preparation system.

[0022] FIG. 2 is a perspective view of a cryo-EM sample preparation system.

[0023] FIG. 3 is an elevation view of the system shown in FIG. 2 identifying the modular components of FIG. 1 .

[0024] FIGS. 4a and 4b together are a flow chart illustrating operations performed in a sample preparation processing using the system of FIG. 1.

[0025] FIGS. 5a, 5b, 5c, 5d, and 5e illustrate stages in a sample preparation process using the modules shown in FIG. 3.

[0026] FIGS. 6a and 6b illustrate further detail of an example of a plunge module.

[0027] FIGS. 7a and 7b illustrate further detail of an example of a sample applicator.

[0028] FIGS. 8a, 8b, 8c, and 8d illustrate further detail of an example of a blotting module.

[0029] FIGS. 9a and 9b illustrate further detail of an example of an environmental subsystem which includes an environmental chamber.

[0030] FIGS. 10a, 10b, 10c, 10d, 10e, 10f , and 10g illustrate further detail of an example of a cryo-module.

[0031] FIGS. 10h, 10i, 10j, and 10k illustrate an alternative configuration for a cryogen holding cup.

[0032] FIGS. 11a, 11 b, and 11 c together provide an example of a cryo-system piping diagram.

[0033] FIG. 12a is a block diagram of an example of a configuration for implementing a machine logging system coupled to the system of FIG. 1.

[0034] FIG. 12b is a message flow diagram for an MQTT system.

[0035] FIG. 12c is a block diagram illustrating a direct logging configuration.

[0036] FIG. 13 is a graph illustrating an example direct logging sample.

[0037] FIG. 14 is an example of a configuration for logging with an MQTT system.

[0038] FIG. 15 is a graph illustrating an example of a logging sample for sending messages to a local MQTT broker.

[0039] FIGS. 16a and 16b are graphs illustrating a logging sample post-MQTT without and with an outlier removed respectively.

[0040] FIG. 17 is a graph illustrating an example comparison of MQTT versus direct log samples.

[0041] FIG. 18 is a block diagram illustrating an example of a configuration for remote logging.

[0042] FIG. 19a is a graph illustrating a remote sending sample for sending to MQTT.

[0043] FIG. 19b is a graph illustrating remote logging sample post-MQTT.

[0044] FIGS. 20a through 20p together provide a flow chart illustrating user and machine control interface operations that may be implemented in operating the system of FIGS. 1-3.DETAILED DESCRIPTION

[0045] The following describes a system, modules and various methods for cryo- EM grid preparation that can achieve a high throughput with parameterized multi-grid handling. The system can be configured to be scalable, updatable and future proof by using an open architecture and modular design. The system described herein can address problems with preferred orientation, timing, optimization, time-resolution experiments, particularly for new particles and techniques.

[0046] The system described herein provides a robust implementation of a sample thinning method, along with a technique to enable fast, uniform application of very small sample volumes. The system can be operated to provide repeatable, reliable, thin ice production by automating operations from the cryogen controls to grid placement in a storage box. This may lead to a faster determination of ideal sample preparation parameters / conditions and a robust recall of previous parameters from a user-generated library.

[0047] The system provides a modular design with user-removable, replaceable modules for sample application, sample thinning, or other steps in the preparation workflow. The modularity allows for swappable components, e.g., to enable classification or custom / research modules to be used and tracked. Multiple modules may be installed simultaneously for sequential or parallel actions on the grid or sample. Distinct or duplicate modules may be used. The modules may be integrated into the system to provide full computer control of their operation as well as a hands- off grid preparation sequence, with implementations of the system allowing for multiple grids at a time as discussed further below. The system also permits containment and safety or protection mechanisms to be built into the overall unit to provide safety- and function-critical features.

[0048] The precision sample preparation provided by the system includes a computer controlled sample application from, for example, 0.3uL, with fast plunging times, fast sample cycling and precision control of parameter space for ice thickness optimization, while minimizing screening time, sample preparation cycle, and equipment time. With respect to grid optimization, in one preparation sequence and one screening session, one can determine optimum parameters.

[0049] The vitrification functionality described below can increase plunge times, while providing adjustability of same. This can create a nitrogen-free plunge path and allow for the vitrification of clipped and standard grids. The system may also be configured to allow for LN2 or hydrocarbon cryogen.

[0050] Grid handling may be fully automated from grid loading through the vitrified grids and in multi-grid implementations may provide capacity for preparing multiple (e.g., 12) grids with a single sample solution, automatically varying parameters for a single solution over up to the maximum number of grids it can take, or by having multiple completely unique grids (e.g., 12). The grid handling in this way may enable the performance of time series experiments and the box slot and box ID can be recorded for each sample. The grid handling described below also allows for automatic grid checking and characterization for alignment and insertion.

[0051] The cryo-module provided by the system can provide fully automatic, hands free, LN2 / cryogen filling and monitoring for an entire duty cycle. TheLN2 / cryogen transfer may be provided to a removable vessel at the session end for safe disposal in a fume hood. The cryo-module may also provide automatic identification of storage box serial number (e.g., via RFID) and can rapidly ramp up from start to a “ready to plunge” state. The cryo-module may use a mild negative pressure to prevent a disturbance of the sample chamber from cryo conditions and cold nitrogen intrusion.

[0052] The system may also be integrated with a user interface and provide network functionality. The user interface can provide adjustable access to features / modules based on user permissions (e.g., PI, supervisor, technician, researcher, intern user groups). The user interface may provide remote control / operation / monitoring of system as well as remote training and troubleshooting for systems and new capabilities.

[0053] The network functionality allows meta and sample data to be stored alongside physical information for each grid. Moreover, the date, time, user, sample info, preparation parameters (e.g., temp, RH, sequence times, blotting method, blot pressure, plunge speed, cryogen temp, etc.) may be stored, determined, controlled, changed, etc. This allows data to be stored locally and / or on a laboratory local area network (LAN) or a cloud-based or other centralized repository. The operational history and experimental history can be archived, and system performance data and predictive diagnostics / preventative maintenance and service data stored and analyzed, e.g., for multiple machines connected to a centralized server. Timings, currents, handshaking / synchronization, and performance may be recorded for each component on a “per prep” basis in addition to experiment data.

[0054] The modularity and integration of the modules enables the system to provide safety and reliability features. For example: a fully enclosed LN2 reservoir and filling system, hydrocarbon sensing, automated shutdown and “safe mode” functionalities, interlocks to prevent user contact with sensitive and hazardous systems and functions (e.g., module swap / installs), and unknown state exception detection at each degree of freedom.

[0055] Referring now to the figures, FIG. 1 illustrates a cryo-EM sample preparation system 10, referred to interchangeably herein as “the system 10” forbrevity. The system 10 provides a unit that may be used in an integrated workflow or separate and stand-alone portion of a workflow for electron microscopy of samples applied to a grid 14, which are prepared (by the system 10) for subsequent imaging.

[0056] The system 10 includes a plunge module 12, which lowers a grid 14 into an environmental chamber 16. The environmental chamber 16 provides a controlled environment in which to perform sample application using a sample applicator 18 and blotting using a blotting module 20. Once the sample has been applied to the grid 14 and the grid 14 blotted, the grid 14 is plunged into a cryo-module 22 to create the thin vitrified layer of ice before being stored for subsequent imaging.

[0057] It can be appreciated that the schematic diagram shown in FIG. 1 illustrates a base unit with at least one of each type of module. As described below and shown, for example, in FIG. 2, the system 10 may be configured to permit multiple modules of the same type or multiple subsystems of a particular portion of the process to permit higher throughput, sample variability, etc.

[0058] FIG. 2 illustrates an example of a multi-grid system 10 in which the plunge module 12 is integrated with a multi-grid carousel 24. The multi-grid carousel 24 enables multiple grids (not shown) to be loaded into the system 10 to increase the throughput. In this example configuration, the plunge module 12 is positioned above the carousel 24 such that grids may be rotated towards and beneath the plunge module 12 to be plunged downwardly and towards the environmental chamber 16.

[0059] Each slot 25 on the carousel 24 can be used to hold a mechanism for holding a grid 14 throughout the preparation cycle (e.g., as shown in FIG. 6, but without the plunge drive actuation). Specifically, each slot 25 can hold one drop-rod 78 and tweezer 60 combination (see also FIGS. 6a-6b) which hold the grids 14 to be prepared. By having multiple grids 14 held and ready when the system 10 goes through the preparation cycle, multiple grids 14 can be sequentially and rapidly prepared using pre-loaded samples, and with the same or different preparation steps. For example, several grids 14 can be rapidly prepared from a single syringe of sample using the same parameters or different variables (e.g., plunge speed, application volumes, blot times) can be parameterised and executed sequentiallythus providing a series of grids 14 in nearly the same time it would take to produce a single one in other machines.

[0060] The system 10 may be packaged with, within and on / around a frame 26 to provide a commercial device / machine that may be used to automatically prepare cryo-EM samples. Currently, it is found that much of the cryo-EM sample preparation is done manually. The preparation process requires high precision movements and timing to produce high quality samples, which are difficult for technicians to perform consistently. The system 10 is configured to replace multiple manual steps in sample preparation to reliably and repeatably prepare cryo-EM grid samples. The system 10 utilizes a modular design that can be customized and adapted to the specific installation requirements. Additionally, the system 10 can be upgraded if and when new preparation steps and techniques are developed. For example, multiple sample preparation modules can be installed by the user.

[0061] As shown in FIG. 2, the frame 26 permits the modules to be placed with and relative to each other, generally like the arrangement shown in FIG. 1 . That is, the plunge module 12 is held above the carousel 24 to obtain grids 14 to be plunged downwardly towards the environmental chamber 16 to position the grid 14 for sample application and blotting using the sample applicator 18 and blotting module 20 respectively. As described further below, the plunge module 12 further plunges the grid 14, post-blotting, downwardly into the cryo-module 22 to create the thin vitrified layer of ice required for subsequent imaging.

[0062] In the configuration shown in FIG. 2, the carousel 24 enables the system 10 to be loaded with multiple grids 14. This is achieved by using multiple drop rod units each having tweezers (see further details below). In the example shown, the carousel 24 accommodates up to twelve (12) drop rod / tweezer units, although more or fewer may be accommodated depending on the footprint utilized. The carousel 24 may then rotate about a central axis to position each unit in turn for plunging as described herein.

[0063] Since each tweezer holds one grid 14, this allows the user to pre-load up to N (e.g., 12) grids 14 and automatically prepare this many samples sequentially, with the grids 14 being prepared one at a time. At the front of the plunge module 12,a vertically mounted pulley and carriage (also referred to as a drop-rod) is able to engage with a selected tweezer to control the vertical translation of that tweezer. Tweezers are locked in a ‘home’ position when not engaged to the drop-rod. When the drop-rod engages a tweezer, a separate motor may simultaneously engage the tweezer to control its rotation about the vertical axis. The user may select the desired grid 14 for processing and the carousel 24 rotates to engage the appropriate tweezer to the carriage. The selected tweezer is then lowered from its home position such that the grid 14 enters the environmental chamber 16 as described more fully later.

[0064] FIG. 3 provides an elevation view of the system 10 to illustrate the positioning of the plunge module 12 above the environmental chamber 16 and cryomodule 22 to enable a staged plunging process from one stage to the next. From this view, and referring additionally to FIG. 2, it can be observed that the environmental chamber 16 is positioned directly below the grid’s ‘home’ position. This chamber 16 is where the sample is applied using the sample applicator 18 and processed using the blotting module 20, before freezing in the cryo-module 22. The environmental chamber 16 is temperature and humidity-controlled to minimize sample evaporation during processing. User-installed sample preparation modules (i.e. user-installed and inserted sample applicator(s) 18 and blotting module(s) 20) operate on the grid 14 in this chamber 16. In one example, the preparation involves two steps, namely application (apply sample to the grid 14), and blotting (i.e., remove excess sample to ensure even coating on the grid 14).

[0065] The user typically has at least a sample applicator 18 and a blotter module 20 installed in the system 10. The sample applicator 18 applies a small amount of sample, on the order of microliters, to the grid 14 using either direct deposition using a syringe or pipetter, or indirect deposition using a small piezoelectric transducer to vaporize the sample into a mist that adheres to the grid 14. The blotter module 20 then blots the grid 14 with a paper-like material to wick away excess sample, leaving an even coating on the grid 14. Once blotting is complete, the sample is immediately and rapidly lowered into the cryogen chamber contained in the cryo-module 22.

[0066] Although not shown in FIG. 3, an actuation barrier 28 is located between the environmental chamber 16 and the cryo-module 22 to act as a thermal isolatingbarrier and an actuator for opening and closing the tweezers in order to drop the grid 14, as described further below.

[0067] The cryo-module 22 may contain a crucible filled with LN2 that is mounted on an X-Y table, providing controlled motion in two axes. Within the crucible is a smaller container that is filled with cryogen. The cryogen is cooled and liquefied by the surrounding LN2.

[0068] Following preparation of a grid 14 in the environmental chamber 16, the grid 14 is rapidly lowered into the cryogen-module 22 causing the sample to vitrify on the grid 14.

[0069] A multi-slot grid holder 244 (see FIG. 10 described below) may be inserted into the cryogen container by the user before the start of the preparation cycle, into which vitrified grids are loaded by the system 10. When full, the grid holder 244 is removed from the system 10 by the user. As described further below, the grid holder 244 is a repository where the grids 14 are stored when the preparation cycle is complete and before the user has removed them. The grid holder 244 is immersed in the cryogen and the tweezers 60 lower the grids 14 into slots 246 in the grid holder 244.

[0070] The cryo-module 22 and user-installed sample preparation modules (i.e. , the sample applicator 18 and blotting module 20) may be mounted on a rotating platform 27 at the base of the frame 26, in order to facilitate access to the modules 18, 20, 22.

[0071] Referring now to FIG. 4a, a flow chart illustrating operations executed in utilizing the system 10 is provided once all required samples and materials are loaded into the system’s machine. At block 30, the sequence starts and the sample chamber environment, i.e., the conditions within the environmental chamber 16 are set at block 32. This may be done by an environmental system controller used to set the temperature and humidity of the sample environmental chamber 16 to the desired setpoints including humidity settings at block 34 and / or temperature settings at block 36.

[0072] At the same time, or sequentially, the cryogen system, e.g., settings for the cryo-module 22, are set at block 38. The cryogen system begins the fill cyclewhereby a crucible is chilled to cryogenic temperatures and filled with liquid nitrogen. Following this, a plunge cup is filled with a cryogen which may be some mixture of Pr and Et, or some other material to plunge the grid 14 into thereby vitrifying the sample.

[0073] The grid 14 may be prepared at block 42 after or while the cryogen system is set wherein the LN / cryogen fill cycle begins at block 40. The grid(s) 14 is / are prepared for the application of the sample, typically through glow discharge or other cleaning / preparation steps. The grid(s) 14 is / are then loaded into respective tweezers 60 and the tweezers 60 are loaded into the machine at block 44 and the application of the sample at block 46 as shown in FIG. 4b.

[0074] Referring to FIG. 4b, the application at block 46 may include selecting an automatic or manual option for certain filling settings at block 48. Sample thinning is then performed at block 50 using the blotting module 20, which may include selection of a blot type at block 52. Thinning may be accomplished by blotting the grid and excess sample or through other similar mechanisms. The thinned sample on the grid 14 is then plunged at block 54 into the cryo-module 20 to vitrify the sample, and the sequence ends at block 56. It can be appreciated that the plunge operation at block 54 may include actuation of the actuation barrier 28 to control access to the cryo-module 22 and actuation of the tweezers to drop the grid 14 into the cryogen. The grid 14 may then be placed in a receptacle (e.g., multi-slot storage device 244).

[0075] The process is also shown in the sequence of images in FIGS. 5a through 5e to illustrate automated operation of the modules 12, 18, and 20 relative to the chambers provided by modules 16, 22. Referring first to FIG. 5a, the grid 14 may be held by a set of tweezers 60, which is operated by the plunge module 12 in either a single or multi-unit configuration. The selected or loaded rod supporting the tweezers 60 is downwardly plunged into the environmental chamber 16 as can be seen in FIG. 5b. As detailed further below, the plunge module 12 may utilize a linear actuator and motor to drive the tweezers 60 and grid 14 through the stages shown herein.

[0076] Referring to FIG. 5c, while positioned in the environmental chamber 16, the sample preparation is performed with / on the grid 14. First, the sample applicator 18 operates to apply the sample to the grid 14 in a first preparation stage. Next, as shown in FIG. 5d, the blotting module 20 is operated to thin or “blot” the sample on the grid 14 to evenly distribute sample material on the grid 14.

[0077] Referring next to FIG. 5e, a further actuation of the plunge module 12 while operating the actuation barrier 28 enables the grid 14 to be further plunged downwardly towards the cryo-module 22 and the grid 14 released from the tweezers 60 into the cryo-chamber, to vitrify the sample as described above. Specifically, during the plunge part of the preparation cycle, the tweezers 60 containing a grid 14 is rapidly driven into the cryogen which is held at cryogenic temperatures. The thinned sample on the grid 14 is thus vitrified. Once the plunge step and vitrification are complete, the system 10 actuates to place and / or drop the grid 14 in a position such that it can be readily received by the user. This can, for example, be by accomplished placing into a grid-receiving space (the “drop slot”) in the plunge cup (see FIG. 10 below), or into a slot in the grid holder (also often called a “storage box”) which has many slots for holding multiple grids.

[0078] In the first example, the cryo-module 22 may open and allow the user to manually take the grid 14 out of the receiving space in the plunge cup, while remaining submerged in cryogen or LN2, and placing into a storage box. In the latter example, the system 10 may place the grid into the slot in the storage box automatically, At this point, the system 10 is ready to prepare another grid 14, or to have the grid(s) 14, either within the storage box or individually, removed to further steps in preparation for examination in the microscope.

[0079] As illustrated in FIGS. 5a-5e, the frame 26 can provide a structural support system and other covering and packaging for the system 10. The frame 26 may provide a support for the individual modules in both fixed and swappable / insertable / removable configurations. The module envelope is the outer structure that houses, and rigidly positions functional modules such as the sample applicator 18 and blotting module 20. These modules may thus be removable or permanent, although the description herein assumes hot-swappable / removable handling of the module envelope.

[0080] The module envelopes may be configured in a similar wedge shape to permit positioning radially about the environmental chamber 16 as illustrated in FIG. 2. Each module envelope may be given a unique serial on each part for version tracking. With respect to rigidity, the module envelope, due to the skeletal frame 24, can avoid bearing the weight of the overall superstructure (i.e., frame 24) of the system 10 and its removal should not reduce the integrity of the frame. This allows the individual modules to be physically independent from one another.

[0081] The module envelops may be made to be robust to frequent vibrations and external forces which may be caused by the operation of other modules. This may include an ability to prevent its displacement regardless of pulling, twisting, pushing, shaking, or lifting when a locking mechanism is engaged. The system 10 may be configured to indicate that a module is in the locked position or unlocked position (either, via mechanical position, LED, display message, etc.). Moreover, the system 10 may incorporate a mechanism to distinguish what serial module is connected and in what “locking bay” it sits in (positional data).

[0082] The module envelope should occupy only the spatial envelope for which it is allocated and should not impede on the adjacent module’s envelope. The module envelope may include a mechanism for handling the module that includes, for example, touch / grip contacts that are based on anthropometries. The envelope may also include a series of output ports and interactive components, such as LEDs, LCD displays as well as permit auditory alarms and cues, e.g., to indicate specific states.

[0083] To permit adjustability and / or calibration, the module envelope may be designed to have a mechanism for calibrating / intersecting the center line of the module (when locked in place) with the point of interest (position of the grid).

[0084] The module envelope may also include any one or more of the following features, namely: be robust to physical damage, be an integral part of the structure of the functional module it houses, be fully self-contained (house mechanical parts, electrical boards and wires and have no components sticking out of the physical perimeter of the module), include vibration isolation to prevent vibrations from transmitting between adjacent modules, block temperature and humidity controlled air entering the module, circulate temperature and humidity controlled air within themodule, and / or localize this circulation away from electric components and wires or protect these components.

[0085] FIGS. 6a and 6b provide an isolated elevation view of the plunge module 12. The plunge module 12 includes a housing or frame 70. The frame 70 supports an actuation motor 72, e.g., a linear drive motor or stepper motor. The motor 72 is coupled to a drive system 74, e.g., a belt drive, rack and pinion or other linear actuation-type drive. The drive system 74 is coupled to a plunge rod 78 via an attachment block 76. The attachment block 76 holds the plunge rod 78 during actuation of the plunge module 12 to plunge the rod 78 through an alignment block 80 at the bottom end of the frame 70. The rod 78 is coupled to a tweezer 60, which is used to grasp a grid 14. As shown in FIG. 6b, the plunge rod 78 may be downwardly translated or “plunged” in a controlled manner to align the grid 14 within the environmental chamber 16 then the cryo-module 22 as illustrated in FIGS. 5a-5e.

[0086] The plunge module 12 and its subsystems can fulfill certain important functionalities. First, the plunge module 12 is configured to hold and presents the grid 14 throughout the cycle to the user and other sub-modules wishing to interact with the grid 14. Second, the plunge module 12 accelerates the grid 14 towards the cryogenic bath / system in the cryo-module 22.

[0087] To achieve suitable functionality and reliability, the plunge module 12 may use impact absorbing materials built into the drive / or stroke to reduce component wear and isolate vibrations to other functional modules. In the configuration shown herein by way of example, the motor 72 and plunge drive 74 should be able to travel 40 mm (+ / - 1 mm) in less then 25 ms, have a positional accuracy of 0.5 mm at lower speeds (< 1 m / s), be able to hold a thin disk (e.g., 20-30 nm thick with a total diameter of 3.05 mm) while only contacting <5% of the total surface area of the disk, namely the grid 14.

[0088] The plunge module 12 is also configured to be able to physically mount the end effector to the plunge drive 74 with high positional accuracy at a fixed position (e.g., + / - 50 microns), withstand 4G acceleration without slipping at the attachment block 76 on the plunge drive, and withstand 4G acceleration without permitting the grid 14 to slip from the end effector, namely the tweezers 60.

[0089] The plunge module 12 may be configured to present the user with a space envelope with a minimum width for a user to reach a single hand to access the end effector (e.g., tweezers 60) between cycles at a designated location. The plunge module 12 may have the option for user-controlled speed adjustment, e.g., from a top speed to a very slow speed (e.g., to within 25% of top speed). The user may also be able to log performance data for every cycle as instructed by the user (e.g., current draw, top speeds, acceleration / deceleration rates, etc.)

[0090] The plunge module 12 may be adapted to have pinch point sign, warning signs, lockout systems for components in the plunge path that are not fully contained and isolated from the user. The plunge module 12 may also include an adjustment / calibration process for calibrating the position of the grid 14 as it relates to other physical / rigid components in the system 12.

[0091] Further details concerning the sample applicator 18 are shown in FIGS. 7a and 7b. The sample applicator 18 may be configured as a swappable userinsertable module that interacts with the grid 14 when placed within the environmental chamber 16. Referring first to FIG. 7a, the sample applicator 18 includes an injector body 90, which supports an actuator shaft 92 along which an actuation block 94 can travel. The actuation block 94 is aligned with an injector support 98, which receives and supports a syringe 96 or other applicator. The syringe 96 includes a needle 102 that extends towards an ultrasonic transducer 100. The ultrasonic transducer 100 may, optionally, be used to atomize the sample and spray a thin film onto the grid 14, which can be further thinned via blotting or plunged directly. It can be appreciated that while a syringe 96 is shown in FIG. 7, the sample can be held and applied by any device that can hold and express precise quantities of sample, such as a pipettor. Moreover, the ultrasonic transducer 100 is not necessary and the syringe needle 98 may be used to deposit the sample directly on the grid 14.

[0092] The body 90 is supported atop a base 104, which is connected to a carriage 106. The carriage 106 in this example is coupled to a spring-loaded drive 108 to permit the base 104 and body 90 to translate towards and away from the environmental chamber 16. The spring-loaded drive 108 may be provided by a trigger-sear release mechanism to drive the body 90 forward and back out of the wayof the plunge module 12. This movability allows the syringe 96 or other sample holder to be removed and reloaded in the module outside of the environmental chamber 16, simplifying such load / unload operations. The syringe, 96 being outside of the environmental chamber 16 also then does not disturb the conditions in the chamber 16 until it is needed for applying the sample to the grid 14. The needle 98 should also be able to move in to apply the sample then rapidly get out of the way of the plunging tweezer 60 and drop rod 78. The carriage 90, when moving into position may then trigger an opening in the environmental chamber 16 (through a door, for example) and provide a barrier for air movement and / or ambient conditions from disturbing the grid 14 and sample. There may also be a sensor which detects when the applicator 18 is in the correct position to apply the sample to the grid 14, whether directly by the syringe 96 or using the ultrasonic transducer 100.

[0093] As illustrated in FIG. 7b, the actuation block 94 translates along the actuator shaft 92 to engage a plunger of the syringe 96 to apply sample contained in the syringe 96 to the grid 14. In this way, the actuation speed and force may be controlled by the sample actuator 18 in an automated manner.

[0094] The sample applicator 18 and its subsystem provide a method to digitally control sample application onto a grid 14, using various methods determined by its end effector. The sample applicator 18 is designed to provide an accessible and maintenance-friendly method to replace individual components that have a shorter lifespan. That is, the sample applicator 18 may be configured to provide the following example delivery metrics: be able to store small volumes of liquids at a steady temperature (e.g., 4 degrees Celsius) for the entire duration the device is powered ON, if necessary, be able to express a volume of liquid directly on a grid 14, or onto an appropriate end effector without manual user interaction, have easy-to-clean, or disposable surfaces and components for all that which may come into contact with expressed liquids, be able to support the use of a standard syringe 96 (e.g., 700 series 5 microliter Hamilton™ syringe), be able to support the use of various types of micropipettes and pipette ends, be able to support various types of syringe needle ends, and permit the use of an end effector to modify the method of sample deposition onto a grid 14.

[0095] The sample applicator 18 may also be configured to prevent or inhibit the syringe needle or pipette ends coming into contact with parts (aside from an appropriate end effector, the ends an installed pair of tweezers 60, or a grid 14) between its installation and removal.

[0096] The sample applicator 18, in an example implementation, may be capable of expressing a volume of liquid in a controlled manner such that instances of air bubbles forming on the surface of a grid 14 or end effector are occurring in less than, for example, 20% of cycles, as well as express a volume of liquid at a small deviation (e.g., 2-15 degrees short of perpendicular) from any target surface (e.g., grid 14, or other end effector).

[0097] The sample applicator 18 may include an end effector that permits small volumes (e.g., 0.25-5 microliter) to be sprayed onto a grid 14. This may be done, in an example implementation, per the following: be able to spray a small volume of liquid onto a grid 14 from a short distance (e.g., 1-10 mm) away from the target surface; be able to pulse, or hold a spray for an arbitrary amount of time (volume permitting) without damage to the hardware; have parts and surfaces that come into contact with the expressed liquid be easy-to-clean or disposable to limit contamination between cycles; and have all parts and surfaces that come into contact with the aerosol liquid vapor be easy-to-clean or disposable to limit contamination between cycles.

[0098] The sample applicator 18 can permit a state to be held for an arbitrary amount of time (for example, an hour) without damage to the hardware, i.e., without reducing the lifespan of the unit.

[0099] In terms of workflow, the sample applicator 18 may be used as in the following example. First, completely evacuate from the trajectory of the plunge within a short timeframe (e.g., 15ms) of its execution at the grid 14. Next, the applicator 18 should be able enter and leave the work area (e.g., until it is within 25 mm radius of the target grid 14) without hindering or damaging any other module or component in that work area. In the event that it is within a 25 mm radius of the grid 14 in such an example, software controls and necessary feedback should be implemented to prevent any collision from occurring between independent moving parts.

[0100] The sample applicator 18 may be completely self-contained in its envelope when it is not within the work area and may be configured so as to not have its mobility impeded on by any wiring by routing the wiring accordingly. This may be done to ensure there is a space envelope with a minimum width (e.g., 9.6 cm - 95th percentile hand breath of a 40-Year-Old American Man) for a user to reach a single hand to access and replace the syringe 96, pipette or other end effector or end point akin to the needle 98 or sprayer 100, in between cycles at a designated location. The sample applicator 18 may then be positioned such that the end of the syringe 96 (or pipette) is relatively near the target surface (e.g., 0.5-2 mm away from the target surface) such as the grid 14 as shown in FIG. 7b.

[0101] The sample applicator 18 provides both adjustability and repeatability. For instance, the device may be configured to express a volume of liquid concentric to a grid 14 (e.g., with a maximum offset of 0.9 mm), or on an end effector (e.g., with a maximum offset = 20% of the diameter or largest geometric dimension of the target surface). The applicator 18 may permit small volumes to be expressed with a maximum (e.g., +10%) error, which may consider device hardware limitations, and any workflow-specific directives that result in sample loss.

[0102] To provide such adjustability, the sample applicator 18 may permit mechanical adjustment to the concentricity of a syringe end, a pipette end or an end effector relative to a grid 14. It may also permit mechanical adjustment of the angle between the axis that runs through the barrel of the syringe 96 (or pipette) and the plane of the grid 14 and permit mechanical adjustment of the time it takes for evacuating the blotting media from the trajectory of the plunge (e.g., + / - 2 ms).

[0103] The sample applicator 18 may also be configured to permit mechanical adjustment of the distance between the end of a syringe 96 (or pipette) and the target surface (e.g., 0.1 -1.5 mm) at the point of execution.

[0104] The exact “appropriate” distance may be experimentally derived through visual examination or through resulting EM graphs to have subsequent calibration be consistent (e.g., up to + / - 0.1 mm) and repeatable.

[0105] With respect to sensing, the sample applicator 18 may be configured such that it is able to report the environmental conditions in the module, namely at thelocation where the sample is stored. The sample applicator 18 may also be configured to dispose of used pipette ends within the envelope of the module, include a fail-safe against failure modes that result in complete, or substantial sample loss; detect an installed syringe 96 (or pipette); detect the specific model of syringe 96 (or pipette) installed, prevent the environmental conditions within the work area from flowing into the module when net working in the environmental chamber 16, and / or to circulate the environmental conditions within the work area within the module.

[0106] The blotting module 20 is shown in greater detail in FIGS. 8a through 8d. Referring first to FIG. 8a, the blotting module 20 includes a base 110 which supports a track 112. The track 112 supports a pair of rotatable blotting arms coupled to an end wall 114 towards a front end of the module 20. Each rotatable blotting arm includes a blotting hand 118 that is coupled to a rotatable shaft 122 via a connection point 116. The blotting hand 118 is configured to support a piece of blotting paper 120. As shown in FIG. 8a, the blotting hands 118 of the opposing arms are positioned such that the pieces of blotting paper 120 face each other. The shafts 122 are coupled to a drive block 124 supporting a drive motor 126 for each arm. At least one of the blotting arms may include a magnetic clutch or similar mechanism to rapidly disengage from the grid to permit high speed plunging of the grid. Various types of magnet clutches may be used, for example, ones that use off-axis magnets and plates as shown in FIG. 8a, which can hold or release one another to release the arm from the motor. In another example, radial in-line clutches may be used, with a clutch plate in line with the drive shaft, which his magnetically drawn to a friction plate to hold, then released to spin freely and allow the blotting arms to release from the motor. An alignment motor 128 may also be provided as shown to permit movement of the pair of rotatable blotting arms along the track 112 toward and away from the environmental chamber 16.

[0107] Referring to the side view shown in FIG. 8b, it can be seen that the alignment motor 128 may be actuated to advance the blotting hands 118 towards a grid 14 that is being used to prepare a sample. Referring next to FIGS. 8c and 8d, with the hands 118 aligned on either side of a grid 14, the motors 126 may rotate the shafts 122 in opposite directions to bring the pieces of blotting paper 120 towardsand into contact with the opposite sides of the grid 14 in a controlled and automated manner to blot the sample that was applied using the sample applicator 18.

[0108] The blotting module 20 and its subsystems provide a device to implement a method for digitally controlling the sample thinning process of a grid 14 by way of controlling contact with a porous blotting material, e.g., the pieces of blotting paper 120 shown in FIG. 8. The blotting module 120 may be configured to accommodate standard or otherwise commercially available blotting media, which may have a thickness of, for example, 0.18 mm in one case, or 0.4-1.27 mm in another case.

[0109] The subsystems shown in FIGS. 8a-8d should provide a non-porous and easy to clean surface for all blotting material to interface with the grid 14. In the event that a porous material is backing the surface (thin film), that porous material should be completely sealed to the environment such that humidity or nearby aerosols cannot be absorbed; or is a consumable that may be replaced between cycles to avoid contaminants.

[0110] In an example implementation, the blotting module 20 may incorporate the following features and capabilities: provide a minimum area (e.g., 38.5 mm2) of unused blotting paper 120 to blot each respective grid 14, wick away some volume of liquid from the surface of a grid 14, such that, e.g., 40% of the vitrified area contains visible particles, and / or not permit installed media to come into contact with other parts, aside from, for example, unused blotting material, the ends an installed pair of tweezers 60, or a grid 14 between its installation and removal.

[0111] The blotting module 20 may be configured to permit a static state to be held for a minimum amount of time (e.g., one (1) hour) without damage to the hardware and may: permit installed blotting paper 120 to make contact with a single side / surface of a grid 14, permit installed blotting paper 120 to make contact with both surfaces of a grid 14 (e.g., simultaneously (within 250 ms of one another)), and / or permit adjustment in the force (e.g., + / - 2 newtons) the blotting paper 120 exerts on the grid 14 without damage to the hardware that would decrease its lifespan.

[0112] In operation, the blotting module 20 may be completely evacuated from the trajectory of the plunge within a short amount of time (e.g., 15 ms) of itsexecution at the grid 14 and be able to enter and leave the work area at a speed of, for example, 2cm / s (e.g., until it is within 25 mm radius of the target grid 14) without hindering or damaging any other module or component in that work area. In the event that the blotting module 20 is within a 25 mm radius of the grid 14 in such an example, software controls and necessary feedback may be implemented to prevent any collision from occurring between independent moving parts.

[0113] The blotting module 20 can be configured to be completely self-contained in its envelope when it is not within the work area and to not have any of its mobility impeded by any wiring. As with the sample applicator 18, the module envelope may be sized and configured to ensure there is a space envelope with a minimum width (e.g., of 9.6 cm (95th percentile hand breath of a 40-Year-Old American Man)) for a user to reach a single hand to access the blotting paper 120 in between cycles at a designated location.

[0114] The blotting module 20 may present blotting paper 120 parallel to the grid 14 within a determined degree of error, e.g., 0.25 degrees. The blotting module 20 may also permit any mechanical adjustment to the angle of the blotting paper 120 relative to the grid 14 to hold for a portion (e.g., ! ) of the product lifetime without slipping and permit the preset force exerted on a grid 14 to be repeatable, e.g., up to + / - 0.25 Newtons.

[0115] The blotting module 20 may permit the preset blot time holding blotting paper 120 against a grid 14 to be repeatable, e.g., up to + / - 50 ms and permit the surface (e.g., hand 118) presenting the blotting paper 120 to be angularly adjustable (roll axis, yaw axis). The blotting module 20 may also allow for mechanical adjustment of the time it takes for evacuating the blotting paper 120 from the trajectory of the plunge (e.g., + / - 2 ms). The blotting module 20 may also have the following static states return a known feedback: home position, minimum position, maximum position, blotting position, clear-of-plunge position, clear-of-work-area position (either with the use of an optical sensor, or encoder feedback) and have the time it takes to evacuate the plunge be directly sensed and measurable.

[0116] Additional features of the blotting module 20 that may be implemented include, without limitation: be able to support multiple blots (e.g., minimum 3) with theinstalled blotting paper 120, possess an automated method for presenting unused blotting paper 120 to a grid 14 between cycles without manual replacement of the paper 120, possess a method for disposing used blotting paper 120 within the envelope of the module 20, prevent or inhibit the environmental conditions within the work area from flowing into the module 20 when not working in the chamber 16, and circulate the environmental conditions within the work area within the module 20.

[0117] An environmental subsystem 160 that includes the environmental chamber 16 is shown in greater detail in FIGS. 9a and 9b. Referring first to FIG. 9a, the subsystem 160 includes a cooler 142 coupled to the environmental chamber 16 via a humidity module 144 and a coupler 146. The chamber 16 includes an aperture 150 in its top surface or lid to permit the tweezers 60 to plunge a grid 14 thereinto. The upper surface of the chamber 16 includes a wedge shaped bracket 152 for holding any actuation or support mechanisms needed to open and close the aperture and the top of the chamber 16..

[0118] FIG. 9b provides a cross-section of the subsystem 160. Referring first to the cooler 142, this device includes an airflow inlet 154 that introduces the air flow into a hollow section 156 that includes fins 160 of one or more heatsinks 158, one on either side of the hollow section 156. The base of the heatsinks 160 are affixed, thermally, to thermoelectric or “Peltier” modules in the top and bottom surfaces of the cooler 142. The Peltier modules may be used to cool the heatsinks 158, the fins 160 of which, extending into the hollow section 156, cools the air as it passes over them. In this way, as the air traverses the length of the cooler 142 it becomes progressively colder through exposure to the cold heat sink fins 160. The air that is cooled as it travels through the hollow section 156 of the cooler 142 is directed from the cooler 142 into an interior 162 of the humidity module 144 on one side of a wet sponge 164. The air passes through the wet sponge 164 to pick up moisture and increase its humidity as it enters a humid zone 166 on the other side of the sponge 164. The humid air then passes through the coupler 146 to an interior zone 168 of the chamber 16. The chamber 16 includes an openable window 170 to enable a user to see the operation of the system 10, to access the environmental chamber 16 (e.g., to clean the inside), or to manually apply sample to the grid 14. The subsystem 140 enables the system 10 to control the environmental conditions within theenvironmental chamber 16 using the cooler 142 and humidity module 144 to control temperature and humidity. This allows the sample to be applied to the grid 14 and the grid 14 to be blotted at optimal environmental conditions as needed for the particular sample being prepared.

[0119] That is, the environmental subsystem 140 maintains a controlled environment at a pre-set temperature and relative humidity for the grid 14, in order to preserve the native state of applied biological samples and affect evaporation rates.

[0120] The subsystem 140 may be configured to be able to dry, drain, or replace surfaces that hold moisture for moderately long periods (e.g., > 12 hours), wherein replacement is defined as a method of discarding surfaces as a “consumable” and installing a new surface in its place. Surfaces that hold moisture for long periods (e.g., > 24 hours) should be replaced after a single use. Other surfaces should be replaced periodically, e.g., every 25 000 cycles.

[0121] The cooler 142 should be able to heat or cool a volume of air to any preset temperature value (e.g., between 2-30 degrees Celsius + / - 2%), provide a method of sensing temperature levels at multiple positions in the volume of cooled or heated air, and be able to heat / cool a volume of air to the maximum operating differential (e.g., a total of 40 degrees Celsius or higher) within a certain period of time (e.g., 25 minutes), given the system 10 is operating in ambient temperatures.

[0122] For the humidity module 144, it should envelope the grid 14 with a pre-set humidity in a x radius bubble, be able to humidify a volume of air to any pre-set relative humidity (e.g., between 95-100%), and provide a method of sensing humidity levels at one or more positions in the volume of humid air generated.

[0123] In operation, the subsystem 140 may be configured to enable user-set inputs for temperature control, and humidity control and provide sensor functionality to report to the host-parent system and / or the user, data such as: outlet humidity, outlet temperature, and water reservoir level. The subsystem 140 can log performance data for every cycle as instructed by a user (e.g., water reservoir level, temperature and humidity readings, airflow, air speed, etc.) The subsystem 140 may also have the capability of user controlled airflow, airspeed or rate of cooling, heating, or humidifying.

[0124] The actuation barrier 28, located between the environmental chamber 16 and the cryo-system 22 is shown in FIG. 9c. The barrier 28 acts as both a thermal isolating barrier between the two environments and an actuator for opening and closing the tweezers 60 in order to drop the grid 14. The barrier 28 includes an annular flange 180 with a central aperture 182 that permits the tweezers 60 to position the grid 14 about a retractable window 184. The retractable window 184 in this example mimics an optical iris to radially open and close. This can be operated to facilitate opening the tweezer 60 no matter the rotational position of the grid 14. The modularity built-in to the barrier 28 means that the grid 14 may face various modules at any position preceding the plunge and the retractable window 184 and its iris shape allows one to plunge with minimal or without any delay. That is, in order to open the tweezers 60 to drop the grid 14 into a designated receptacle or slot, the tweezers 60 are squeezed (i.e. operated in reverse). In the configuration described herein, the tweezers 60 can be facing in any direction so that any or multiple of the modules can work on it before plunge. Because the idea is to plunge as rapidly as possible after the last module has worked on the grid 14, the tweezer 60 may not have time to rotate to a specific angle. Hence, the circular iris-like window 184 is used to squeeze the tweezers 60 so that they can be opened no matter what is the current angle.

[0125] Moreover, when dropping the grid 14 directly into a grid storage box 244 (which is circular and has radially arranged slots 246 in it for the grids - see FIG. 10 described below), the grid / tweezers 14 / 60 may need to be at any arbitrary angle, hence leveraging the configuration of a circular actuator (the iris) to open the tweezer 60 and let go of the grid 14.

[0126] The cryo-module 22 is shown in greater detail in FIGS. 10a through 10g. Referring first to FIG. 10a, the module 22 includes a base 202 that may be secured to the turntable 27 to allow the module 22 to be rotated about the central axis of the turntable 27 as module envelopes are loaded and unloaded from the system 10. The base 202 supports a crucible 200 via a set of support legs 204. The crucible 200 includes an aperture 206 in its upper surface or lid 210. The crucible 200 is fed by a manifold 208 that is used to supply LN2 into an inner containment vessel 222. The manifold 208 may be coupled to a tube through which the LN2 flows.

[0127] The crucible 200 is shown in elevation in FIG. 10b and in cross-section in FIG. 10c. The crucible 200 includes an inlet tube 212, which provides a port used to draw off dry nitrogen gas that is used to clear fog away from the surface of the cryogen for visibility and to keep ambient air away from the surface of the cryogen, e.g., to reduce ice crystal formation and contamination. Referring to FIG. 10c, the crucible 200 includes an interior zone 220 that contains the vessel 222 and provides a structural wall to provide precise mounting functionality to the crucible 200. The vessel 222 contains the LN2 and the plunge cup 226. The vessel 222 is supported by a set of pegs 224 to hold / support the containment vessel 222 and maintain it at the appropriate height within the crucible 200.. The vessel 222 holds liquid nitrogen 230 such that the liquid nitrogen 230 surrounds and cools the inner cup 226 in communication with the aperture 206 to permit a grid 14 to be dropped into propane / ethane 232 contained in the cup 226 to be cooled by the liquid nitrogen 230 indirectly. It may be noted that the space between the containment vessel 222 and the structural wall of the crucible (i.e., the zone 220) is used to thermally insulate the containment vessel 222 (at cryogenic temps) from the structural wall of the crucible 200 (the outside of which is near room temp). That space defining the zone 220 can be filled with a highly insulating substance such as expanding urethane foam. This also helps make the system water-tight (or LN2-tight).

[0128] The vessel 222 is where the vitrification process of sample on the grid 14 occurs. The vessel 222 should be able to contain the cryogen in its liquid phase and maintain the cryogen within the specified temperature range to enable vitrification of the sample for the minimum time required for a sample prep session. The cryomodule 22 may provide a mechanism to cool and liquify the cryogen from a gas to liquid phase using LN2 and provide a mechanism to drain any vessels containing LN2 or cryogen to a separate vessel for disposal external to the system 10.

[0129] The cryo-module 22 provides a path for the grid 14 to travel immersed in cryogen during the plunge process and / until a user removes the grid 14 and provides a location for the grid 14 to be released post-plunge and readily accessible for a user (e.g., if not in the storage location or drop slot). For example, if the grid 14 is not dropped into a drop slot 246, or placed in a storage box, it is possible that a user could remove it directly from the tweezers 60. As such, after the grid 14 is immersedand the system 10 translates the grid 14 relative to the storage box 244, the drop slot 246, or this intermediate location to remove it by hand, the grid 14 remains submerged in cryogen.

[0130] Referring to FIG. 10d, a plan view of the lid 210 is shown. The lid 210 exposes the cup 226 via aperture 206. The cup 226 includes a deep portion referred to herein as a dive pool 240 which is adjacent to a drop slot 242 which is between the dive pool 240 and a storage box 244. Referring also to FIG. 10e, the storage box 244 in this example includes a set of radially spaced slots 246 each providing a drop point for a grid 14. The cup 226 is shown in isolation in FIGS. 10f and 10g. The cup 226 includes a relatively deeper portion for the dive pool 140, which is supported above a flange 250, which may be secured to the interior of the vessel 222. It can be seen from FIG. 10g that the cryogen level may submerge the storage box 244 to maintain the grid 14 in the cryogen until the storage box 244 may be removed.

[0131] An alternative cryo-cup 1226 is shown in FIGS. 10h, 10i, 10j and 10k. In the alternative configuration 1226, the cryogen into which the grid 14 is plunged is maintained in a separate chamber 1240 from the storage area 1244 (see FIG. 10k) and storage box 244. In this way, the storage box 244 is not in the non-LN2 cryogen, as some of the cryogen may still freeze in the very small volumes in the slots 246 in the storage box 244, which may freeze the grid 14 in the slots 246. In the configuration shown in FIGS. 10h-10k, post application, blotting etc., the system 10 plunges down into the first chamber 1240 containing cryogen which vitrifies the sample in a thin layer on the grid 14. The system 10 then lifts the grid 14 out of the cryogen briefly and as close to the surface of the cryogen as possible (so as not to warm enough to de-vitrify), the crucible then moves so that grid 14 is now over the section containing the drop slot 1242 (see FIG. 10k) and the storage box 244, and the grid 14 is lowered into LN2. The rest of the process may be similar to that described above. The cup 1226 can be designed with radiating fins 1228 etc., so that even as the LN2 drops over time in the rest of the crucible, it stays in the vessel with the storage box 244, even when the LN2 in the rest of the crucible is much lower than that part of the vessel. In this configuration, the vessel is designed to hold the LN2 higher than the main reservoir.

[0132] The cryo-module 22 may provide a temporary storage location where the grid 14 can be placed by a user, and which contains and protects the grid 14 (e.g., commercial grid storage box) and may include a mechanism to reduce visibilityhampering fog during handling. The cryo-module 22 may be adapted to include a mechanism to reduce water ice building (frost) from ambient humidity and may provide the user with ready access to the vitrified grid 14 and any temporary storage for it (e.g., eject the crucible 222 so that the user can access and remove the grid 14).

[0133] The cryo-module 22 may provide a mechanism to remove any residual water in the cryogen system. The cryo-module 22 is installed below the environmental chamber 16 as discussed above and may include various features, such as the following, without limitation: provide a mechanism for the automated filling of LN2, provide a mechanism for the automated filling of cryogen, provide a mechanism for the manual filling of cryogen prepared external to the system 10, provide sensor functionality to report to the host-parent system and / or the user. The sensor data may include, for example: cryogen level, cryogen temperature, coolant level, coolant availability, and cryogen availability.

[0134] FIGS. 11 a, 11 b, and 11c together provide an example of a cryo-system piping diagram, e.g., to implement the cryo-module 22 described herein. When referring to FIGS. 11 a-11c, reference numerals will be omitted and labels instead referred to preserve readability of the figures. Referring to FIG. 11a, room temperature propane and ethane may be supplied by respective tanks via respective manual shut off valves and respective regulators to a Pr / Et mixer to prepare a room temperature, high pressure Pr / Et mixture. This mixture may be monitored by a pressure sensor and feeds the cryo-chamber 200 via a cryogen input valve as shown in FIG. 10c.

[0135] Referring back to FIG. 10b, air at room temperature and at a low pressure is supplied by a LN2 pump to an LN2 tank with a pressure relief valve provided to maintain the low pressure. The pressure into the tank forces LN2 out, which is at a low temperature (e.g., -190c as shown) and at a low pressure and feeds the cryochamber 200 via an LN2 inlet as shown in FIG. 10c. The cryogen inlet and LN2 inletfeed the crucible 222 and inner chamber 226 as shown, cryogen and LN2 outlets may fee respective dump valves for disposing of the cryogen and LN2 after use.

[0136] The system 10 may be configured to permit logging features to obtain and store data acquired from use of the system 10. Such data may be used in machine learning or other analytics processes to improve usage, track errors and record operations.

[0137] Referring now to FIG. 12a a machine logging system is shown. In this example, a user’s browser 300 connects to the machine, namely the system 10, via a web socket and webserver 302. The web socket and webserver 302 allow for bidirectional, event-driven communication between the user’s browser 300 and a backend. The webserver 302 may initiate activity in the backend, such as logging data or sending / receiving messages to other machines. The system 10 may be configured to include a database 304, an MQTT broker 306 and a MQTT client 308. The webserver 302 connects to a cloud server 310, specifically a server-side MWTT broker 312. The MWTT broker 312 is connected to a MQTT client 308, which has a database 316 for storing data in the cloud server 310. The MQTT system allows for bidirectional communication between the system 10 (i.e. the machines) and the cloud server(s) 310, both locally and remotely. The database(s) 304, 316 store data received from the MQTT clients 308, 314 and / or the webserver 302.

[0138] FIG. 12b illustrates an example of an MQTT configuration. In this example, a temperature sensor 320 is shown, which publishes a temperature reading to a topic “temp”. The MQTT broker 306, 312 publishes this reading and a computer 322 that subscribes to the topic “temp” will obtain the reading. As shown, a mobile device 324 may also subscribe to the topic “temp” and receive the reading published by the MQTT broker 306, 312. A MQTT network normally includes at least one broker 306, 312 and several devices 322, 324, known as clients. The MQTT network utilizes a publish / subscribe messaging protocol, wherein a broker receives all messages and distributes to clients based on their subscriptions, and clients can send and receive messages to / from a broker. Publishing thus sends a message to one or more topics while subscriptions receive all messages from the topics the client is subscribed to.

[0139] Local logging may be used when the machine (e.g., system 10) wants to log to its own database(s). Typically, this approach does not have network security as a major concern, as the machine communicates with itself and does not involve any users or machines outside of its internal (i.e. within the structure of the machine) network. With this system, there are two possibilities for local logging: a) directly log into the database (browser communication and logging process embodied in a simple process), b) send a message through the local MQTT network to the database (browser and MQTT communication decoupled from logging process, i.e. two separate processes).

[0140] The components from FIG. 12a that may be adapted for use in a direct logging scenario are shown in FIG. 12c. FIG. 13 illustrates a graph illustrating local direct logging timestamps showing a temperature reading.

[0141] FIG. 14 illustrates a local logging configuration using MQTT, with the components from FIG. 12a described above and need not be reiterated. FIG. 15 provides a graph illustrating local logging using MQTT (t2) and the relatively higher sampling rate that can be achieved. The data t3 being saved to the database 304 (see also FIG. 14) is illustrated in a local logging post MQTT in FIG. 16a and the same graph with an outlier removed in FIG. 16b. A comparison of all three graphs is shown in FIG. 17.

[0142] In terms of logging speed, direct logging may be favorable compared to the logging speed of the decoupled process method. However, on the user end, the decoupled process method may allow for faster and more reliable collection of data (e.g., 0-1 ms). In this case, the decoupled process may be the chosen method of local logging, since : receiving data from user and sending data to the MQTT broker may have a consistent and fast range, may allow for more detailed logs, may give user confidence in the r reliability and speed of logs. Here, the data sending / receiving communication process is separated from the logging process.

[0143] A remote logging configuration is shown in FIG. 18, wherein the MQTT broker 312 and MQTT client 314 are located solely at the cloud server 310. Remote logging may be chosen when the machine (i.e., system 10) wants to communicate with other machines, regardless of whether the other machine is on the samenetwork or not. The process is similar to local logging, except the destination is not itself. For security reasons, there may be no direct logging to remote devices. FIGS. 19a and 19b illustrate remote logging send to MQTT and logging post-MQTT operations respectively.

[0144] Turning now to FIGS. 20a through 20p, an end-to-end example of operations performed in using the system 10 is shown. Referring first to FIG. 20a, the system 10 may be packaged as a contained machine as illustrated in FIGS. 1-3 and is powered on at 800. The system 10 is booted and diagnostics and status checks may be performed at 802. The system 10 may then connect to the network at 804, if configured to do so, and determine if network settings should be changed at 806 and the settings may be displayed at 808 if they are to be changed. Moving now to FIG. 20b, an account login screen may be displayed at 810. In this example, an engineer may login and be able to see user controls as well as technical details of the machine at 812. A visitor may login to see limited information with some inputs and parameters locked at 818. An admin may login to display an admin home screen, which may list all users of the system 10 at 814. A user who plans to operate the system 10 may login at 816 to display a home screen with an overview of parameters and the environment.

[0145] Moving to FIG. 20c, the user may change the sample chamber environment at 820. If so, control moves to what is shown in FIG. 20d. As illustrated in FIG. 20d, at 826, the parameters and settings may be displayed. The user may confirm the correct parameters at 828. If not, the user may set temperature and humidity parameters at 830. Once the parameters are set, the user may be prompted to begin cooling the environmental chamber 16 using subsystem 140. This process may include determining if the water reservoir is low at 836 and displaying a warning at 838 if so. If not, the system may determine at 840 if the humidity and temperature settings have been reached. Operations 834, 836, and 838 may be repeated until the settings have been reached. At that time, the system 10 may stop the fan circulation to maintain the settings at 842 and then continue to the plunge screen at 844.

[0146] Referring again to FIG. 20c, if the chamber environment does not need to be changed and / or the changes shown in FIG. 20d are made, the system 10 maydetermine at 822 if the plunge speed is to be changed. If so, control moves to what is shown in FIG. 20e. As illustrated in FIG. 20e, the system 10 may display a grid preparation screen with the plunge speed setting at 846 and determine at 848 if the parameters are correct. If not, the user may set the desired plunge speed at 850. Once correct, the system 10 may prompt the user to load the tweezers 60 with a grid 14 at 852. The system 10 may determine when a user button input has been received at 854. If not, the system 10 waits at 856. Once the user button input is received, the system 10 may continue to an application screen at 858.

[0147] Referring again to FIG. 20c, once the plunge speed is set or does not need to be changed, the user may determine at 824 if the application settings need to be changed. If so, control moves to what is shown in FIGS. 20f and 20g. Referring next to FIG. 20f, the application screen may be displayed at 860 and the system 10 may determine if an automatic or manual operation is selected at 862. If automatic, the application volume and apply-to-blot time delay may be set at 864 and the system 10 may determine at 866 whether the user wants or needs to refill the syringe 96. If so, the syringe 96 may be removed at 868 and the system 10 waits for the user to load the syringe 96 at 870. This may require a user input button, which the system 10 waits for at 872. Once the syringe 96 is refilled or if not required, or if the manual operation is selected at 862, control moves to what is shown in FIG. 20g.

[0148] Referring now to FIG. 20g, it can be seen that for manual operation, the system 10 may continue to the blotting screen at 890. If automatic, the system 10 determines at 874 if the syringe levels are low or empty. If so, a warning may be displayed at 876. The system 10 may then determine if the air circulation fan shown be turned on at 880. If so, the auto fan cycle may begin at 882. The system 10 may then determine if the piezo spray is to be used, at 884. If so, the system 10 may wait for the user to install the piezo at 886 and the user input is received at 888. The system 10 may then continue to the blotting screen at 890.

[0149] Referring to FIGS. 20c and 20h once the application settings are changed or changes are not needed, control may proceed to what is shown in FIG. 20h. Referring now to FIG. 20h, the system 10 determines at 892 if the user will change the blot settings. If not, the system 10 may start the fill process at 894. If blot settings are to be changed, the system 10 displays the sample thinning screen at896. The system 10 then determines if the blot time is correct at 898. If not, the blot time may be adjusted by the user at 900. If the blot time is correct or after changing same, operation may proceed to what is shown in FIG. 20i. Referring to FIG. 20i, the system 10 determines if the number of blots is correct at 902. If not, this may be adjusted at 904. Once adjusted or if correct, the system 10 determines if the number of blots set exceeds the number of blots remaining at 906. If not, a warning may be displayed to the user at 908 and the user is prompted to clear or change the blot material (e.g., blot paper 120) at 910. The system 10 may then determine at 912 if the blot type is correct. If not, the blot type can be adjusted at 914.

[0150] Operations may then proceed as shown in FIG. 20j. Referring now to FIG. 20j, the system 10 may determine if the blot-to-plunge delay is correct at 916. If not, this may be adjusted at 918. The system 10 may then determine if the blot force is correct at 920. If not, this may be adjusted at 922. The system 10 may then continue to the fill settings at 924.

[0151] Returning to FIG. 20h, the fill process starts at 894 and operation may move to what is shown in FIG. 20k. Referring now to FIG. 20k, the system 10 displays the LN / cryogen levels and readings at 926 and determines if the user has selected an automatic or manual fill at 928. If automatic, the LN / cryogen levels are set at 930 and the system 10 determines if these levels are correct at 934. If not they may be adjusted by repeating 930. If the levels are correct, the system 10 begins or continues the automatic fill cycle for LN at 936 until the system determines at 938 that the LN level has been reached. Operation may then continue to what is shown in FIG. 20m. Referring now to FIG. 20m, the system 10 may display a completion message to the user at 940 and begin or continue the auto-fill cycle for cryogen at 942. The system 10 determines at 944 if the cryogen level has been reached. Once reached, the system displays a completion message to the user at 946.

[0152] Referring again to FIG. 20k, if the manual process is selected at 928, the system 10 may start or continue filling LN after the user presses a button at 948. The system 10 determines whether the LN is full at 950 and displays a warning if so at 952. If the LN is not full, the system 10 may determine at 954 whether the LN level is correct. If so, the system 10 stops filling by receiving an input from the user at 956. Operation may then proceed to what is shown in FIG. 20m. Referring again to FIG.20m, an input button may be used to begin filling cryogen at 958. The system 10 may determine at 960 whether the cryogen is full. If so, a warning is displayed at 962. If not, the system 10 determines if the LN levels are low and a warning is displayed at 968. The system 10 may then determine if the correct cryogen level has been achieved at 970. If not, the process may repeat at 958. Once the correct cryogen level has been achieved, the system 10 stops filling cryogen based on a user input at 972. The system 10 then continues to the sequence at 974 after either the automatic or manual fill process is completed.

[0153] Referring now to FIG. 20n, a sequence includes load tweezers 60 at 976 and the sequence begins at 978, followed by grid removal at 980. Operation may then continue with what is shown in FIG. 20p. Referring now to FIG. 20p, the system 10 determines if the user wishes to continue with a new process at 982. If so, the user is able to continue with the screens as described above by returning to what is shown in FIG. 20c. If not, the user logs out at 988.

[0154] The system 10 determines at 988 if a new login is attempted. If so, the process repeats from login as shown in FIG. 20b. If a new login is not attempted, the system 10 determines at 992 if there is a change to the network or machine settings. If so, the current screen may remain at 994. If not, the system 10 determines at 996 if it is to be powered down. If not, the current screen may remain at 998. If so, the machine is powered down at 1000.

[0155] For simplicity and clarity of illustration, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements. In addition, numerous specific details are set forth in order to provide a thorough understanding of the examples described herein. However, it will be understood by those of ordinary skill in the art that the examples described herein may be practiced without these specific details. In other instances, well-known methods, procedures and components have not been described in detail so as not to obscure the examples described herein. Also, the description is not to be considered as limiting the scope of the examples described herein.

[0156] It will be appreciated that the examples and corresponding diagrams used herein are for illustrative purposes only. Different configurations and terminology canbe used without departing from the principles expressed herein. For instance, components and modules can be added, deleted, modified, or arranged with differing connections without departing from these principles.

[0157] It will also be appreciated that any module or component exemplified herein that executes instructions may include or otherwise have access to computer readable media such as transitory or non-transitory storage media, computer storage media, or data storage devices (removable and / or non-removable) such as, for example, magnetic disks, optical disks, or tape. Computer storage media may include volatile and non-volatile, removable and non-removable media implemented in any method or technology for storage of information, such as computer readable instructions, data structures, program modules, or other data. Examples of computer storage media include RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other non-transitory computer readable medium which can be used to store the desired information and which can be accessed by an application, module, or both. Any such computer storage media may be part of the system 10, any component or module, sub-system of or related thereto, etc., or accessible or connectable thereto. Any application or module herein described may be implemented using computer readable / executable instructions that may be stored or otherwise held by such computer readable media.

[0158] The steps or operations in the flow charts and diagrams described herein are provided by way of example. There may be many variations to these steps or operations without departing from the principles discussed above. For instance, the steps may be performed in a differing order, or steps may be added, deleted, or modified.

[0159] Although the above principles have been described with reference to certain specific examples, various modifications thereof will be apparent to those skilled in the art as having regard to the appended claims in view of the specification as a whole.

Claims

Claims:1 . An applicator module for a cryo-EM sample preparation system, the applicator module comprising: a holder for an applicator device, the applicator device configured to be actuated to express a determined portion of sample contained in the applicator device onto a grid; a carriage assembly supporting the applicator device, the carriage assembly being coupled to a withdrawal mechanism to enable the applicator device to be driven towards the grid to apply the sample and rapidly retract to provide space for a plunging module to further plunge the grid beyond the applicator module.

2. The applicator module of claim 1 , wherein the withdrawal mechanism is spring loaded.

3. The applicator module of claim 2, wherein the withdrawal mechanism comprises a trigger-sear release mechanism.

4. The applicator module of claim 1 , wherein the applicator module further comprises an ultrasonic transducer supported at a distal end of an applicator to spray the expressed sample onto the grid.

5. The applicator module of claim 1 , wherein the applicator device comprises a syringe or pipettor.

6. The applicator module of claim 1 , further comprising a module envelope surrounding the holder and carriage assembly, the module envelope being releasably attachable and removeable from the sample preparation system about an environmental chamber in which the grid is prepared.

7. The applicator module of claim 6, wherein the module envelope is wedge shaped to permit a plurality of wedge shaped modules to be loaded around the environmental chamber.

8. A blotting module for a cryo-EM sample preparation system, the blotting module comprising: at least one moveable blotting arm to permit a blotting medium attached thereto to be presented towards and retracted from a grid having sample applied thereto; and a carriage assembly supporting the at least one blotting arm, the carriage assembly being coupled to a withdrawal mechanism to enable the blotting module to be driven towards the grid to blot the sample to keep the blotting medium from a humid environment in which the grid is being held.

9. The blotting module of claim 8, comprising a pair of blotting arms that move towards each other to enable both sides of the grid to be blotted.

10. The blotting module of claim 9, configured to selectively blot either side or both sides of the grid.11 . The blotting module of claim 8, wherein the at least one blotting arm comprises a magnetic clutch to rapidly disengage from the grid to permit high speed plunging of the grid.

12. The blotting module of claim 8, further comprising a module envelope surrounding the at least one arm and carriage assembly, the module envelope being releasably attachable and removeable from the sample preparation system about an environmental chamber in which the grid is prepared.

13. The blotting module of claim 12, wherein the module envelope is wedge shaped to permit a plurality of wedge shaped modules to be loaded around the environmental chamber.

14. An environmental module for a cryo-EM sample preparation system, the module comprising: a cooling system; a humidity system; and a chamber coupled to the cooling system via the humidity system to rapidly cool a column of air defining a zone, the zone providing a cooled and humidified column of air into which a grid may be plunged by a plunging module positioned with the system above the chamber, the zone permitting access to a sample applicator to apply sample to a grid positioned in the chamber and a blotting module to blot the grid, the chamber being positioned above a cryo-module in the system and permitting the plunge module pass the grid through the chamber after preparation and be dropped into a crucible in the cryo-module.

15. The environmental module of claim 14, wherein the cooling system comprises a plurality of heat sink fins for cooling a source of air as the air flows towards the humidity system.

16. The environmental module of claim 15, wherein the heat sinks are cooled by an external cooler.

17. The environmental module of claim 14, wherein the humidity system comprises a wet sponge to humidify the air flowing from the cooling system towards the chamber.

18. The environmental module of claim 14, wherein the chamber comprises an openable viewing window to permit visibility into the chamber and permit access to the grid and sample applied thereto.

19. The environmental module of claim 14, further comprising a circular closeable window beneath the chamber, the window closing an aperture through which the gridmay be dropped and closing towards a center thereof to operate on a pair of tweezers holding the grid.

20. A plunge system for a cryo-EM sample preparation system, the plunge system comprising: at least one drop rod operated by a high speed drive system, the drop rod being moveable vertically to plunge a grid held by the drop rod using a pair of tweezers at a bottom end thereof into an environmental chamber positioned beneath the plunge system; and the pair of tweezers coupled to the at least one drop rod, the pair of tweezers being actuatable to hold and release the grid.21 . The plunge system of claim 20, comprising a plurality of drop rods, each drop rod being selectively coupled to the high speed drive system.

22. The plunge system of claim 21 , each of the plurality of drop rods comprising a respective pair of tweezers for holding a respective grid.

23. The plunge system of claim 21 , comprising a rotatable carousel for holding the plurality of drop rods, the carousel being indexable relative to a holder coupled to the high speed drive system to select a next grid to be processed.

24. The plunge system of claim 20, wherein the pair of tweezers is actuatable by squeezing same.

25. The plunge system of claim 20, wherein the drop rod is actuatable by the drive system into at least two positions, a first position to place the grid within the environmental chamber and a second position beneath the environmental chamber to drop the grid into a cryo-chamber to vitrify the sample on the grid.

26. A cryo-module for a cryo-EM sample preparation system, the cryo-module comprising: a crucible; a cup positioned within the crucible, the cup holding a plunge substance to be surrounded by liquid nitrogen contained around the cup within the crucible; and a control system to provide internal control of LN2 levels and cryogen levels.

27. The cryo-module of claim 26, wherein the control system comprises a set of room temperature valves and feeder lines for both hydrocarbon gases and LN2.

28. The cryo-module of claim 26, comprising a gas mixer and heat exchanger / condenser to automatically liquify the cryogen.

29. The cryo-module of claim 26, comprising compressed air to drive the LN2 while enabling pressure relief and over pressure protection via 3-way valves.

30. A cryo-EM sample preparation system comprising: an applicator module according to any one of claims 1 to 7; a blotting module according to any one of claims 8 to 13; an environmental chamber according to any one of claims 14 to 19; a plunge module according to any one of claims 20 to 25; and a cryo-module according to any one of claims 26 to 29.31 . A method of high speed retraction of an applicator in a cryo-EM sample preparation system, the method comprising: enabling the applicator to be driven towards a grid to apply a sample to the grid; and rapidly retract the applicator to provide space for a plunging module to further plunge the grid beyond the applicator module into a cryochamber.

32. The method of claim 31 , comprising: expressing the sample onto the grid.

33. The method of claim 31 , comprising: providing at least one moveable blotting arm; having a blotting medium attached thereto be presented towards and retracted from the grid having the sample applied thereto.

34. The method of claim 31 , comprising: providing at least one drop rod operated by a high speed drive system, the drop rod being moveable vertically to plunge the grid held by the drop rod using a pair of tweezers at a bottom end thereof into an environmental chamber positioned beneath the plunge system; and actuating the pair of tweezers, coupled to the at least one drop rod, to hold and release the grid.

35. The method of claim 31 , comprising: positioning an environmental chamber above a cryo-module; permitting a plunge module to pass the grid through the chamber after preparation and be dropped into a crucible in the cryo-module; and rapidly cool a column of air defining a zone, the zone providing a cooled and humidified column of air into which a grid may be plunged by the plunging module.

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