WO2024254310A2

WO2024254310A2 - Method and apparatus for cryogenic-electron microscopy sample preparation

Info

Publication number: WO2024254310A2
Application number: PCT/US2024/032811
Authority: WO
Inventors: Venkata P. DANDEY; Mario J. BORGNIA; Wyatt A. PEELE; Tony Jun Huang; Kaichun Yang
Original assignee: US Department of Health and Human Services
Current assignee: US Department of Health and Human Services
Priority date: 2023-06-09
Filing date: 2024-06-06
Publication date: 2024-12-12
Anticipated expiration: 2025-12-09
Also published as: EP4724786A2; WO2024254310A3

Abstract

An apparatus for preparing samples for cryogenic electron microscopy includes an acoustic transducer having a sample receiving area for receiving one or more liquid droplets. An acoustic signal generator is coupled to the acoustic transducer. Control circuitry is coupled to the acoustic signal generator, the control circuitry configured to cause the acoustic signal generator to generate a first acoustic signal at a first frequency for a first time period and subsequently to generate a second acoustic signal at a second frequency for a second time period.

Description

Method and Apparatus for Cryogenic-Electron Microscopy Sample Preparation

Field of Technology

[0001] The present disclosure is in the field of cryogenic electron microscopy, and more particularly, relates to acoustically mixing and dispensing liquid samples in preparation for scanning using cryogenic electron microscopy.

Background

[0002] Cryogenic electron microscopy (Cryo-EM) is a field that is rapidly advancing and is becoming popular due to a recent Nobel prize having been awarded in this field. There is a still a need for improved methods of preparing samples for Cryo-EM for more precise mixing and dispensing of the liquid sample onto the Cryo-EM grid.

Summary

[0003] According to one aspect, an apparatus for preparing samples for cryogenic electron microscopy is provided. The apparatus includes an acoustic transducer having a sample receiving area for receiving one or more liquid droplets. The apparatus also includes an acoustic signal generator coupled to the acoustic transducer. The apparatus further includes control circuitry coupled to the acoustic signal generator, the control circuitry configured to cause the acoustic signal generator to generate a first acoustic signal at a first frequency for a first time period and subsequently to generate a second acoustic signal at a second frequency for a second time period.

[0004] In some implementations of the apparatus for preparing samples, a computer apparatus comprising a user interface and at least one processor is further included. The apparatus for preparing samples further includes a gripper configured for gripping a cryogenic electron microscopy sample grid. In some implementations of the apparatus for preparing samples, a robotic arm configured to removably retain the gripper and to move the grid from a home position, and subsequently to a sample collection position facing the receiving area of the acoustic transducer, and subsequently to an ethane bath position; and subsequently to a storage position in response to computer executable instructions executable on the processor, is also included.

[0005] In some implementations of the apparatus for preparing samples, a mechanical actuator configured to move a piece of blotting paper from a retracted position to an extended position proximate the sample collection position of the grid in response to the computer executable instructions, is also included. The receiving area of the acoustic transducer of the apparatus for preparing samples may vibrate to mix the one or more liquid droplets in response to the first acoustic signal thereby generating a mixed liquid sample. The receiving area of the acoustic transducer of the apparatus for preparing samples may vibrate to dispense the mixed liquid sample in response to the second acoustic signal.

[0006] In some implementations of the apparatus for preparing samples, a camera configured to capture a video recording of the grid while the grid is moving from the sample collection to the ethane bath position, is included. A humidifier, wherein the humidifier provides a stream of humid air to the grid such that the relative humidity proximate to the grid is approximately 80%, may also be included. In some implementations, the low frequencies and high frequencies generated are in the 90 kHz to 110 kHz range, and the first time period and second time period are in the millisecond range.

[0007] According to another aspect, a method for preparing samples for cryogenic electron microscopy is provided. The method includes glow discharging a cryogenic electron microscopy grid to render the grid hydrophilic. The method includes gripping the grid with a tweezer. The method also includes mounting the tweezer to a robotic arm. The method includes robotically moving the tweezer from a home position to a sample collection position. The method further includes robotically move blotting paper to a position proximate the sample collection position such that the blotting paper touches the grid. The method includes depositing two or more microliter sized liquid samples side by side on a sample receiving area of an acoustic transducer, wherein the transducer is connected to a function generator. The method also includes activating the function generator to first generate low frequencies that premix the samples for a first time period and to subsequently generate high frequencies that spray the premixed samples onto the grid. The method further includes robotically plunging the grid into an ethane bath and subsequently robotically transferring the grid into a grid box submerged in nitrogen for long term storage.

[0008] In some implementations of the method, the method also includes triggering a camera to record a video of the grid while the grid is moving from the sample collection position to the ethane bath. In some implementations of the method, the method further includes robotically moving the blotting paper to a position proximate to the sample collection position via a mechanical actuator. In some implementations of the method, the robotic arm is configured to operate in response to computer executable instructions executable on a computer apparatus. In some implementations of the method, the computer apparatus includes a user interface and at least one processor.

[0009] In some implementations of the method, a receiving area of the acoustic transducer vibrates to mix the one or more liquid droplets in response to the first acoustic signal thereby generating a mixed liquid sample. The receiving area of the acoustic transducer may also vibrate to dispense the mixed liquid sample in response to the second acoustic signal, according to the method. In some implementations, the low frequencies and high frequencies are in the 90 kHz to 110 kHz range, and the first time period and second time period are in the millisecond range. The method may further include providing a stream of humid air to the grid such that the relative humidity proximate to the grid is approximately 80%.

[00010] According to another aspect, an apparatus for preparing samples for cryogenic electron microscopy is provided. The apparatus includes a first acoustic transducer having a sample receiving area for receiving one or more liquid droplets. The apparatus includes a second acoustic transducer coupled to a piece of blotting paper. The apparatus also includes an acoustic signal generator coupled to the second acoustic transducer. The apparatus further includes control circuitry coupled to the first acoustic signal generator and the second acoustic signal generator. Control circuitry coupled to both the first acoustic signal generator and the second acoustic signal generator is configured to cause the second acoustic signal generator to generate an acoustic signal at a frequency for a first time period. The apparatus further includes a computer apparatus comprising a user interface and at least one processor is further included. The apparatus for preparing samples further includes a gripper configured for gripping a cryogenic electron microscopy sample grid. A robotic arm configured to removably retain the gripper and to move the grid from a home position, and subsequently to a sample collection position facing the receiving area of the acoustic transducer, and subsequently to an ethane bath position; and subsequently to a storage position in response to computer executable instructions executable on the processor, is also included. A mechanical actuator configured to move the piece of blotting paper from a retracted position to an extended position proximate the sample collection position of the grid in response to the computer executable instructions, is also included. A camera configured to capture a video recording of the grid while the grid is moving from the sample collection to the ethane bath position, is included. A humidifier, wherein the humidifier provides a stream of humid air to the grid such that the relative humidity proximate to the grid is approximately 80%, is also included.

[00011] In some implementations of the apparatus for preparing samples, a receiving area of the first acoustic transducer vibrates to mix the one or more liquid droplets in response to the acoustic signal thereby generating a mixed liquid sample. The receiving area of the first acoustic transducer may also dispense the mixed liquid sample after the first time period.

[00012J According to another aspect, a method for preparing samples for cryogenic electron microscopy is provided. The method includes glow discharging a cryogenic electron microscopy grid to render the grid hydrophilic. The method includes gripping the grid with a tweezer. The method also includes mounting the tweezer to a robotic arm. The method includes robotically moving the tweezer from a home position to a sample collection position. The method further includes robotically move blotting paper to a position proximate the sample collection position such that the blotting paper touches the grid. The method includes depositing two or more microliter sized liquid samples side by side on a sample receiving area of an acoustic transducer, wherein the transducer is connected to a function generator. The method also includes activating the function generator to generate a frequency that mixes the samples on the grid. The method further includes robotically plunging the grid into an ethane bath and subsequently robotically transferring the grid into a grid box submerged in nitrogen for long term storage.

Brief Description of the Drawings

[00013] FIG. 1 is a schematic of an exemplary apparatus for preparing samples for cryogenic electron microscopy, according to an aspect of the present disclosure;

[00014] FIG. 2A is a schematic view of an exemplary method for preparing samples for cryogenic electron microscopy, according to an aspect of the present disclosure;

[00015] FIG. 2B is a process flow of the exemplary method of FIG. 2A for preparing samples for cryogenic electron microscopy;

[00016] FIG. 3 A is a photographic view of an exemplary robotic system setup for cryogenic electron microscopy, according to an aspect of the present disclosure;

[00017] FIG. 3B is a photographic view of the exemplary robotic system setup for cryogenic electron microscopy of FIG. 3 A, illustrating a shroud setup;

[00018] FIG. 3C is a photographic view of the exemplary robotic system setup for cryogenic electron microscopy of FIG. 3B, illustrating a sample being sprayed by an acoustic transducer to a grid;

[00019] FIG. 4A is a photographic view of an exemplary graphical user interface relating to the exemplary robotic system setup for cryogenic electron microscopy of the present disclosure; [00020] FIG. 4B is a schematic of an exemplary acousto-fluidics setup for cryogenic electron microscopy, according to an aspect of the present disclosure;

[00021] FIG. 5 includes photographic views of an exemplary acoustic transducer in the process of mixing and spraying a sample, according to an aspect of the present disclosure;

[00022] FIG. 6A is a Cryo-EM photographic view of exemplary preliminary testing results for an Apoferritin sample on Cryo-EM grid was obtained by simple blot and plunge method, according to an aspect of the present disclosure;

[00023] FIG. 6B is a Cryo-EM photographic view of exemplary preliminary testing results for an Apoferritin and dGTPase sample on the same grid mixed and dispensed using an acoustic transducer, according to an aspect of the present disclosure;

[00024] FIG. 7 is an exemplary 3D rendering of Apoferritin, illustrating the efficacy of the disclosed systems and methods via successful determination of Apoferritin by manual application of the sample, blot and plunge method;

[00025] FIG. 8A is a schematic of an exemplary implementation of an alternative embodiment of the present disclosure, in which a robotic arm may be used to sequentially present a grid to multiple transducers that acoustically dispense, and optionally mix, additional liquid sample component onto a grid;

[00026] FIG. 8B is a photographic view of the exemplary schematic of FIG. 8A;

[00027] FIGs. 9A and 9B are concentrated images of an exemplary implementation of an alternative embodiment of the present disclosure; [00028] FIG. lOA is Cryo-EM photographic view of exemplary preliminary testing results for mixing an ACE2 sample with a spike protein sample on an Cryo-EM grid, according to an aspect of the present disclosure; and

[00029J FIG. 10B includes images of 2D class averages, showing the SARSCOV2 spike interacting with ACE2 to provide improved results, according to an aspect of the present disclosure.

[00030] Like reference numerals in different Figures indicate like elements.

Detailed Description

[00031] This description of the preferred embodiments of the invention is intended to be read in connection with the accompanying drawings, which are to be considered part of the entire written description of this invention. The drawings are not necessarily to scale, and certain features of the invention may be shown exaggerated in scale or in somewhat schematic form in the interest of clarity and conciseness.

[00032] Aspects of the present disclosure include apparatus and methods for time-resolved preparation of liquid samples for cryogenic electron microscopy (Cryo-EM) experiments. In particular, the present disclosure describes a system and method for mixing and dispensation of liquid samples using specific electrical signals that can be transduced into specific acoustic frequencies. In response to the specific electrical signals, the transducer mixes the liquid samples (low frequency) and then dispenses the mixture (high frequency) in small (nanoliter) volumes onto a Cryo-EM grid, in some embodiments. The disclosed apparatus and methods may thereby provide more precise control over liquid sample mixing and dispensing, and improved dispensation of the mixture onto the Cryo-EM grid.

[00033J The disclosed apparatus and methods also provide improved quality of captured images of homogenous macromolecular structures is achieved due to a more uniform mixing and dispensed sample on the EM grid. This allows electrons to be transmitted through very thin liquid film in the holes of the Cryo-EM grid and form an image. The disclosed apparatus and methods can also be used in combination with acoustic waves to manipulate and control the distribution of biological macromolecules within a thin liquid film on the Cryo-EM grid.

[00034] The disclosed apparatus and methods can be implemented using a computer programmable robot to manipulate and control the Cryo-EM grid during sample dispensing, plunging the Cryo-EM grid into liquid ethane, and storage of the grid. The disclosed system and method for transducing electrical signals into acoustic frequencies to coordinate sample mixing and dispensing onto Cryo-EM grid provides better dispersion of the homogenously mixed multiple sample as small droplets of uniform size (nanoliter volumes) over the Cryo-EM grid which facilitates to achieve thin liquid film in the holes for improved image formation. In an illustrative embodiment, a computer programmable robot 106 is configured to control and manipulate a tweezer 136 that holds the Cryo-EM grid after sample dispensation onto the grid 104. [00035] FIG. 1 shows a schematic of an exemplary apparatus 100 for preparing samples for cryogenic electron microscopy. In the illustrative embodiment, apparatus 100 includes an acoustic transducer 118. Acoustic transducer 118 may have a sample receiving area 116 for receiving one or more liquid droplets 122a and/or 122b, and an acoustic signal generator 120 coupled to the acoustic transducer 118. In some embodiments, control circuitry 102 coupled to the acoustic signal generator 120 may be configured to cause the acoustic signal generator 120 to generate a first acoustic signal 124 at a first frequency for a first time period, and subsequently to generate a second acoustic signal 126 at a second frequency for a second time period. According to the present disclosure, the receiving area 116 of acoustic transducer 118 may vibrate to mix the one or more liquid droplets 122a and/or 122b in response to the first acoustic signal 124, thereby generating a mixed liquid sample 128, and vibrates to dispense the mixed liquid sample 128 in response to the second acoustic signal 126.

[00036] As shown in FIG. 1, according to another aspect of the present disclosure, the apparatus 100 also includes a computer apparatus 102 having a user interface 400 and at least one processor. A tweezer/gripper 136 may be configured for gripping a cryogenic electron microscopy sample grid 104, and a robotic arm 106 may be configured to removably retain the gripper 136. In some embodiments, grid 104 may be a copper mesh grid. According to an aspect of the present disclosure, the robotic arm 106 may be configured to move on forceps 136 (at one or more steps 108, as detailed further in process 250) the grid 104 from a home position 222, and subsequently to a sample collection position 224 facing the receiving area 116 of the acoustic transducer 118, subsequently to an ethane bath 130 position, and subsequently to a storage position (not shown) in response to computer executable instructions executable on the processor via computer 102. In an illustrative embodiment, the disclosed apparatus 100 may also include mechanical actuator 110, such as a solenoid 110, which may be configured to move a piece of blotting paper 112 from a retracted position to an extended position proximate the sample collection position of the grid 104 in response to the computer executable instructions.

Embodiments of the present disclosure may optionally include a camera 114, which may be configured to capture a video recording of the grid 104 while the grid 104 is moving from the sample collection to the ethane bath 130 position.

[00037] FIG. 2A is a schematic view of an exemplary method 200 for preparing samples for cryogenic electron microscopy via apparatus 100. FIG. 2B is an exemplary process flow 250 of the exemplary method 200 of FIG. 2A for preparing samples for cryogenic electron microscopy according to apparatus 100. In the illustrative embodiment, the method 200 includes steps 252 of glow-discharging and/or plasma cleaning 202 a cryogenic electron microscopy grid 104 to render the grid 104 hydrophilic. The grid 104 can then be gripped at step 254 with a tweezer 136 and mounted to a robotic arm 106 at step 256. The robotic arm 106 can then robotically move the tweezer 136 from a home position 222 to a sample collection position 224 at step 258. A mechanical actuator 110 may robotically move blotting paper 112 to a position 116 proximate to the sample collection position 224 such that the blotting paper 112 touches the grid 104 at step 259. In some embodiments, two or more microliter sized liquid samples 122a and 122b are deposited at step 260 side by side on a sample receiving area 116 of an acoustic transducer 118 which is connected to function generator 120. The function generator 120 may then be activated at step 262 to first generate one or more low frequencies 124 that premix the samples 122a and 122b for a first time period, and subsequently generate one or more high frequencies 126 that spray the premixed samples 122a and 122b onto the grid 104.

[00038] As shown in FIGs. 2A and 2B, in illustrative method embodiment 200, the low frequencies and high frequencies 124 and 126 are in the 90 kHz to 110 kHz range, and the first time period and second time period are in the millisecond range. The robotic arm 106 may then robotically plunges the grid 104 into an ethane bath 130 at step 264, and subsequently, robotically transfers the grid 104 into a grid box submerged in nitrogen for long term storage (not shown). In the illustrative method embodiment 200, camera 114 is triggered to capture a video recording of the grid 104 while the grid 104 is moving from the sample collection to the ethane bath 130. In some embodiments, the grid 104 may be subject to a stream of humid air such that the relative humidity proximate to the grid 104 is approximately 80%. This can be enabled by humidifier 304, as shown in FIG. 3A.

[00039] FIG. 3A is a photographic view of an exemplary robotic system setup 300 for cryogenic electron microscopy. In some embodiments, setup 300 may include apparatus 100, method 200, and respective components therein, as previously introduced. In setup 300, SCARA Robot 302 may function similarly to robotic arm 106, and may be directed by function generator 120. Setup 300 may further include humidifier 304, which in some embodiments, may subject grid 104 may to a stream of humid air such that the relative humidity proximate to the grid 104 is approximately 80%. An emergency stop 306 may include a button and/or other activation device configured to halt operation of the systems and methods described herein.

[00040J FIG. 3B includes a photographic view 325 of the exemplary robotic system setup 300 for cryogenic electron microscopy of FIG. 3A, illustrating a closeup of the shroud setup 300. This setup 300, as shown in view 325, details a camera, such as camera 114, for real time evaluation of the grid 104, blotting solenoid/mechanism 110, and transducer 118, according to an aspect of the present disclosure. FIG. 3C includes a photographic view 350 of the exemplary robotic system setup 300 for cryogenic electron microscopy as introduced in FIGs. 3A and 3B, illustrating a sample 122a and/or 122b being sprayed by an acoustic transducer 118 to a grid 104. This sample 122a may then be mixed with sample 122b via vibration, as previously discussed, within the receiving area 116 of acoustic transducer 118 in response to the first acoustic signal 124, thereby generating a mixed liquid sample 128, followed by the vibration to dispense the mixed liquid sample 128 in response to the second acoustic signal 126.

[00041] FIG. 4A illustrates a photographic view of an exemplary graphical user interface 400 relating to the exemplary robotic system setup 300 for cryogenic electron microscopy of the present disclosure. In embodiments, the disclosed graphical user interface 400 may also be seen in FIG. 1, and can enable easier control over computer systems, such as 102, can include a log for note keeping, allow a user to set variables, and calibrate the robot, such as 302, within one program. [00042] FIG. 4B illustrates a schematic of an exemplary acousto-fluidics setup 425 for cryogenic electron microscopy, according to an aspect of the present disclosure. Setup 425 may include a Raspberry Pi 4, which may be used for activating various parts of system 100 and/or 300, including the acoustic transducer 118, within computer 102. For example, GPIO PIN 402 may operate the blotting solenoid 110 within system 100 and/or 300. A function generator, such as 120, may be controlled by Raspberry Pi 425 via USB, whereby function generator 120 control the transducer 118 for mixing and/or spraying samples 122a and/or 122b. In some embodiments, the Raspberry Pi 425 can be easily programmable for different applications and/or processes within system 100 and/or 300.

[00043] FIG. 5 includes photographic views 510, 520, 530, and 540 of an exemplary acoustic transducer 118 in an exemplary process 500 of mixing and spraying a sample, such as 122a and/or 122b, according to an aspect of the present disclosure. In view 510, a 3pL droplet of distilled water and a 3pL droplet of dye are placed separately on the acoustic transducer. In view 520, the droplets are acoustically merged. In view 530, the acoustic mixing is continued for less than 30 msec using low frequencies. Subsequently, in view 540, high frequencies are applied to spray the sample out of the plane of transducer surface. In some embodiments, 30 msec of spray time is applied for each grid and a spray to plunge time is less than 150 msec. In the illustrative process 500 embodiment, the timing parameters may be optimized to complete an entire process from mixing to storing in less than 50 msec. This method 500 can achieve rapid homogenous mixing, such as 128, compared to previous and/or other methods used for time-resolved experiments. [00044] FIGs. 6A and 6B show preliminary testing results using the disclosed systems and methods 100-300. FIG. 6A is a Cryo-EM photographic view 600 of exemplary preliminary testing results for an Apoferritin sample on a Cryo-EM grid as obtained by simple blot and plunge method. The Apoferrtin sample may include sample 122a, and the Cryo-EM grid may include grid 104. Each pane of views in view 600 includes various levels of magnification detailing these results; for example, view 602 is the sample 122a at 200 pm, view 604 is the sample 122a at 10 pm, view 606 is the sample 122a at 2 pm, and view 608 is the sample 122a at 100 nm. FIG. 6B is a Cryo-EM photographic view 625 of exemplary preliminary testing results for the mixed Apoferritin and dGTPase samples 122a and 122b (as shown previously as mixture 128) on the same grid 104, mixed and dispensed using an acoustic transducer 118, according to the present disclosure. Each pane of views in view 600 includes various levels of magnification detailing these results; for example, view 612 is the sample 122a at 200 pm, view 614 is the sample 122a at 10 pm, view 616 is the sample 122a at 2 pm, and view 618 is the sample 122a at 100 nm. For each of FIGs. 6A and 6B, arrow marks in each of the views point to the Apoferritin samples 122a.

[00045] FIG. 7 is an exemplary 3D rendering 700 of the Apoferritin sample 122a, illustrating the efficacy of the disclosed systems and methods 100-300 via successful determination of Apoferritin by manual application of the sample, blot, and plunge method, as previously discussed. Successful mixing of Apoferritin and dGTPase samples 122a and 122b has been demonstrated using the disclosed acoustic transducer 118, highlighting its versatility and potential for various applications.

[00046J FIG. 8A is a schematic 800 of an exemplary implementation of an alternative embodiment of the present disclosure. In this exemplary embodiment, a robotic arm, which may include robotic arm 106, may be used to sequentially present a grid, such as grid 104, to multiple transducers 118 that acoustically dispense, and optionally mix, additional liquid sample components onto a grid 104. FIG. 8B is a photographic view 825 of the exemplary schematic 800 of FIG. 8A. This embodiment may be used for mixing and liquid thinning of samples on a grid 104, for example. In some embodiments, an acoustic transducer 118 can be used to dispense precise nL droplets on the grid. By placing multiple dispensers, multiple samples can be mixed on the grid 104 and transient protein-protein interactions can be observed with high time resolution by mixing the samples using acoustics on the way to ethane (e.g., 1 ms).

[00047] FIG. 9A includes a concentrated image 925 of exemplary implementation 900, according to the present disclosure. In this exemplary embodiment 900, which is an alternative embodiment of method 200, on-grid mixing on grid 104 takes place, in contrast to method 200. In method 200, samples 122a and 122b are applied onto an acoustic transducer 118 that can be operated at different frequencies to either mix samples rapidly and homogenously, or to spray them onto a grid 104 immediately prior to plunging it into cryogen 130, increasing the precision and reducing the lag in time-resolved experiments. Similarly, method 900 may include components of system 100. In FIG. 9A, the first sample 122a may be ~1 pL, and may be placed on grid 104. In image 950 of FIG. 9B, method 900 further illustrates that while blotting, second sample 122b may sprayed onto the grid 104 for on-grid mixing. The spraying of sample 122b may for a second time period of 500 msec on grid 104 while blotting. In this example 900, a second acoustic transducer 902 may be attached to the blotting paper 112, and may also be coupled to and operated at a particular frequency by the function generator 120 and/or control circuitry in addition to first acoustic transducer 118 for rapid homogeneous on-grid mixing. This step of method 900 may be followed by robotic arm 106 then robotically plunging the grid 104 into an ethane bath 130 at after a period of 3 seconds, for example. Subsequently, robotic arm 106 may robotically transfer the grid 104 into a grid box submerged in nitrogen for long term storage (not shown). This method 900 may thereby optimize consumption of samples 122a and/122b, and can allow initiating the reactions of the present disclosure on grid 104 by rapid mixing at a precise time point before vitrification.

[00048] FIG. lOA and 10B show preliminary testing results using the disclosed systems and methods 100 and 900. FIG. lOAis a Cryo-EM photographic view 1000 of exemplary preliminary testing results of method 900, when ACE2 and spike protein samples 122a and 122b, respectively, are mixed by applying ACE2 122a on the grid 104 followed by spraying the spike sample 122b during blotting. Each pane of views in view 1000 includes various levels of magnification detailing these results; for example, view 1002 is the samples 122a and 122b at 200 pm, view 1004 is the samples 122a and 122b at 10 pm, view 1006 is the samples 122a and 122b at 2 pm, and view 1008 is the samples 122a and 122b at 100 nm. FIG. 10B shows in image 1050 in view 1008, detailing 2D class averages showing the SARSCOV2 spike interacting with

ACE2 to provide improved results.

[00049J Although this description contains many specifics, these should not be construed as limiting the scope of the invention but as merely providing illustrations of some of the presently preferred embodiments thereof, as well as the best mode contemplated by the inventor of carrying out the invention. The invention, as described and claimed herein, is susceptible to various modifications and adaptations as would be appreciated by those having ordinary skill in the art to which the invention relates.

Claims

CLAIMS What is claimed is:

1. An apparatus for preparing samples for cryogenic electron microscopy, the apparatus comprising: an acoustic transducer having a sample receiving area for receiving one or more liquid droplets; an acoustic signal generator coupled to the acoustic transducer; and control circuitry coupled to the acoustic signal generator, the control circuitry configured to cause the acoustic signal generator to generate a first acoustic signal at a first frequency for a first time period and subsequently to generate a second acoustic signal at a second frequency for a second time period.

2. The apparatus of claim 1, further comprising: a computer apparatus comprising a user interface and at least one processor; and a gripper configured for gripping a cryogenic electron microscopy sample grid.

3. The apparatus of claim 1 and/or 2, further comprising: a robotic arm configured to removably retain the gripper and to move the grid from a home position, and subsequently to a sample collection position facing the receiving area of the acoustic transducer, and subsequently to an ethane bath position; and subsequently to a storage position in response to computer executable instructions executable on the processor.

4. The apparatus of any of claims 1-3, further comprising: a mechanical actuator configured to move a piece of blotting paper from a retracted position to an extended position proximate the sample collection position of the grid in response to the computer executable instructions.

5. The apparatus of claim 4, wherein the receiving area of the acoustic transducer vibrates to mix the one or more liquid droplets in response to the first acoustic signal thereby generating a mixed liquid sample.

6. The apparatus of claim 5, wherein the receiving area of the acoustic transducer vibrates to dispense the mixed liquid sample in response to the second acoustic signal.

7. The apparatus of any of claims 1-4, further comprising: a camera configured to capture a video recording of the grid while the grid is moving from the sample collection to the ethane bath position.

8. The apparatus of any of claims 1-7, further comprising: a humidifier, wherein the humidifier provides a stream of humid air to the grid such that the relative humidity proximate to the grid is approximately 80%.

9. The apparatus of any of claims 1-8, wherein the low frequencies and high frequencies are in the 90 kHz to 110 kHz range, and the first time period and second time period are in the millisecond range.

10. A method for preparing samples for cryogenic electron microscopy, the method comprising: glow discharging a cryogenic electron microscopy grid to render the grid hydrophilic; gripping the grid with a tweezer; mounting the tweezer to a robotic arm; robotically moving the tweezer from a home position to a sample collection position; robotically move blotting paper to a position proximate the sample collection position such that the blotting paper touches the grid; depositing two or more microliter sized liquid samples side by side on a sample receiving area of an acoustic transducer, wherein the transducer is connected to a function generator; activating the function generator to first generate low frequencies that premix the samples for a first time period and to subsequently generate high frequencies that spray the premixed samples onto the grid; and robotically plunging the grid into an ethane bath and subsequently robotically transferring the grid into a grid box submerged in nitrogen for long term storage.

11. The method of claim 10, further comprising: triggering a camera to record a video of the grid while the grid is moving from the sample collection position to the ethane bath.

12. The method of claims 10 and/or 11, further comprising: robotically moving the blotting paper to a position proximate to the sample collection position via a mechanical actuator.

13. The method of claim 12, wherein the robotic arm is configured to operate in response to computer executable instructions executable on a computer apparatus.

14. The method of claim 13, wherein the computer apparatus includes a user interface and at least one processor.

15. The method of claim 14, wherein a receiving area of the acoustic transducer vibrates to mix the one or more liquid droplets in response to the first acoustic signal thereby generating a mixed liquid sample.

16. The method of claim 15, wherein the receiving area of the acoustic transducer vibrates to dispense the mixed liquid sample in response to the second acoustic signal.

17. The apparatus of claim 16, wherein the low frequencies and high frequencies are in the

90 kHz to 110 kHz range, and the first time period and second time period are in the millisecond range.

18. The method of claim 10, further comprising: providing a stream of humid air to the grid such that the relative humidity proximate to the grid is approximately 80%.

19. An apparatus for preparing samples for cryogenic electron microscopy, the apparatus comprising: a first acoustic transducer having a sample receiving area for receiving one or more liquid droplets; a second acoustic transducer coupled to a piece of blotting paper; an acoustic signal generator coupled to the first and the second acoustic transducer; control circuitry coupled to the first acoustic signal generator and the second acoustic signal generator, the control circuitry configured to cause the second acoustic signal generator to generate an acoustic signal at a first frequency for a first time period; a computer apparatus comprising a user interface and at least one processor; a gripper configured for gripping a cryogenic electron microscopy sample grid; a robotic arm configured to removably retain the gripper and to move the grid from a home position, and subsequently to a sample collection position facing the receiving area of the acoustic transducer, and subsequently to an ethane bath position; and subsequently to a storage position in response to computer executable instructions executable on the processor; a mechanical actuator configured to move the piece of blotting paper from a retracted position to an extended position proximate the sample collection position of the grid in response to the computer executable instructions; a camera configured to capture a video recording of the grid while the grid is moving from the sample collection to the ethane bath position; and a humidifier, wherein the humidifier provides a stream of humid air to the grid such that the relative humidity proximate to the grid is approximately 80%.

20. The apparatus of claim 19, wherein the receiving area of the first acoustic transducer vibrates to mix the one or more liquid droplets in response to the acoustic signal thereby generating a mixed liquid sample, wherein the receiving area of the first acoustic transducer dispenses the mixed liquid sample after the first time period.

21. A method for preparing samples for cryogenic electron microscopy, the method comprising: glow discharging a cryogenic electron microscopy grid to render the grid hydrophilic; gripping the grid with a tweezer; mounting the tweezer to a robotic arm; robotically moving the tweezer from a home position to a sample collection position; robotically move blotting paper to a position proximate the sample collection position such that the blotting paper touches the grid; depositing two or more microliter sized liquid samples side by side on a sample receiving area of an acoustic transducer, wherein the transducer is connected to a function generator; activating the function generator to generate a frequency that mixes the samples on the grid; and robotically plunging the grid into an ethane bath and subsequently robotically transferring the grid into a grid box submerged in nitrogen for long term storage.