Retractable ion guide, grid holder, and technology for removal of cryogenic sample from vacuum

Patent metadata

Field	Value
Publication number	US-11728146-B2
Application number	US-202217575135-A
Country	US
Kind code	B2
Filing date	Jan 13, 2022
Priority date	Jan 13, 2021
Publication date	Aug 15, 2023
Grant date	Aug 15, 2023

Abstract

Imaging by cryo-electron microscopy (cryo-EM) requires that a sample be encased in an amorphous solid, such as amorphous ice. In current cryo-EM preparation systems, once the sample has been deposited on an EM grid and coated in the amorphous solid, the EM grid must be removed from vacuum and then transferred into the vacuum of the cryo-EM system. As a result, samples deposited on the grid are exposed to damage and contamination. The present invention provides improved EM grid handling systems and devices compatible with advanced cryo-EM sample preparation techniques and which reduce or eliminate exposure of the sample on the grid to atmosphere and elevated temperatures. These methods and devices will also significantly reduce handling time and complexities associated with cryo-EM sample preparation.

First claim

The invention claimed is:

1. A cryo-electron microscopy (cryo-EM) sample preparation system comprising:
a) a sample vacuum chamber;
b) a sample holder positioned within the sample vacuum chamber, wherein said sample holder comprises a receiving surface;
c) a temperature control means able to provide a temperature of −100° C. or less to the receiving surface of the sample holder;
d) an ion or particle generator having an internal pressure, wherein the ion or particle generator is able to produce a controllable analyte beam containing charged or uncharged analyte particles and direct said analyte beam to contact the receiving surface of the sample holder, thereby depositing analyte particles onto the receiving surface; and
e) a beam doser able to produce a controllable vapor stream and direct said vapor stream to contact the receiving surface of the sample holder, wherein the vapor stream generates an amorphous solid,
wherein the sample vacuum chamber is able to be isolated from the ion or particle generator and produce an internal pressure different from the internal pressure of the ion or particle generator.

2. The system of claim 1 wherein the ion or particle generator comprises an ion or particle guide able to transmit said analyte beam to the sample vacuum chamber.

3. The system of claim 2 further comprising a vacuum gate valve able to isolate the ion or particle guide from the sample vacuum chamber and allow the ion or particle guide to have an internal pressure independent from the sample vacuum chamber when isolated.

4. The system of claim 2 wherein the ion or particle guide is a retractable ion guide having a first end connected to a cell or chamber of the ion or particle generator and a second end comprising a linear slide, wherein the second end is able to be moved within to the sample vacuum chamber.

5. The system of claim 1 wherein the ion or particle generator is a mass spectrometer device and the ion or particle guide is an ion guide.

6. The system of claim 5 wherein the mass spectrometry device comprises:
a) a first ion guide having first and second ends, wherein the first end of the first ion guide is connected to a cell or chamber of the mass spectrometry device, and the second end of the first ion guide is connected to a first vacuum gate valve;
b) a second ion guide having first and second ends, wherein the first end of the second ion guide is connected to the first vacuum gate valve, and the second end of the second ion guide is connected to the sample vacuum chamber,
wherein the first vacuum gate valve is able to isolate the first ion guide from the second ion guide and allow the first ion guide to have a different pressure than from the second ion guide when isolated.

7. The system of claim 6 wherein the first ion guide comprises a linear slide able to move the first ion guide and connect and disconnect the first ion guide to the first vacuum gate valve.

8. The system of claim 6 wherein the second ion guide comprises a linear slide able to move the second ion guide toward and away from the first vacuum gate valve, wherein the linear slide is able to connect and disconnect the first ion guide to the first vacuum gate valve.

9. The system of claim 1 wherein the temperature control means comprises a container in fluid communication with the sample vacuum chamber, wherein the container is able to provide a cooling fluid to the sample vacuum chamber and reduce the temperature receiving surface of the sample holder.

10. The system of claim 1 further comprising a storage container connected to the sample vacuum chamber and means for transporting the sample holder from the sample vacuum chamber into the storage container, wherein the storage container is able to be closed and isolated from the sample vacuum chamber after the sample holder is received into the storage container.

11. The systems of claim 1 wherein the sample holder comprises:
a) a transmission electron microscopy (TEM) grid;
b) an annular clip ring having a center cavity or depression able to hold the TEM grid,
wherein the TEM grid is adhered to the clip ring;
c) a button front plate having a central opening extending through the button front plate,
wherein the button front plate contacts a front and/or outer surface of the clip ring; and
d) a button back plate contacting a back and/or outer surface of the clip ring,
wherein the button front plate and back plate enclose the clip ring and TEM grid, thereby forming a solid button assembly, wherein a receiving surface of the TEM grid is aligned with the central opening of the button front plate.

12. The system of claim 11 further comprising a clip ring restraint, which at least partially surrounds the outer surface of the clip ring and extends radially outward from the button assembly.

13. The system of claim 11 further comprising a cold box positioned within the sample vacuum chamber, said cold box comprising: a thermal mass front plate having an opening extending through the thermal mass front plate, a thermal mass rear plate in contact with the thermal mass front plate, and a cavity between the thermal mass front plate and rear plateable to hold the button assembly, wherein the opening of the thermal mass front plate is aligned with the central opening of the button front plate and the receiving surface of the TEM grid.

14. The system of claim 13 further comprising a mechanical actuator able to select and position a button assembly into the cold box so that the opening of the thermal mass front plate is aligned with the central opening of the button front plate and the receiving surface of the TEM grid.

15. The system of claim 13 wherein the thermal mass rear plate is in thermal contact with the TEM grid through the button assembly, and contains one or more regions in thermal contact with a circulating cooling fluid.

16. A method for preparing a sample for cryo-electron microscopy (cryo-EM) comprising the steps of:
a) generating charged or uncharged analyte particles and collecting said analyte particles in an ion guide, wherein the ion guide comprises a vacuum valve separating the ion guide into a first region and a second region, wherein the first region has a first internal pressure;
b) positioning a sample holder having a receiving surface in a sample vacuum chamber having a second pressure when the vacuum valve is closed, wherein the sample vacuum chamber is connected to the second region of the ion guide;
c) cooling the receiving surface in the sample vacuum chamber to a temperature of −100° C. or less;
d) producing a controllable vapor stream, wherein the vapor stream is able to generate an amorphous solid;
e) opening the vacuum valve and transmitting the analyte particles from the first region of the ion guide, through the second region, and into the sample vacuum chamber;
f) transmitting the vapor stream into the sample vacuum chamber; and
g) contacting the cooled receiving surface with the analyte particles and vapor stream, thereby forming a layer of analyte particles on the receiving surface, wherein the analyte particles are coated with the amorphous solid.

17. The method of claim 16 wherein the ion guide is part of a mass spectrometer.

18. The method of claim 17 further comprising purifying the analyte particles before the analyte particles enter the sample vacuum chamber.

19. The method of claim 16 wherein the first pressure in the first region of the ion guide and the second pressure in the sample vacuum chamber, independently from one another, are equal t

Assignees

• Wisconsin Alumni Res Found

Inventors

• Westphall Michael Scott
• Coon Joshua Jacques

Classifications

• H01J49/0004 Primary

  Imaging particle spectrometry · CPC title

• G01N1/42 Primary

  Low-temperature sample treatment, e.g. cryofixation · CPC title

• H01J49/04

  Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components · CPC title

• H01J49/0468

  with means for heating or cooling the sample · CPC title

• H01J49/0486

  with means for monitoring the sample temperature · CPC title