THIN-ICE GRID ASSEMBLY FOR CRYO-ELECTRON MICROSCOPY
원문보기
IPC분류정보
국가/구분
United States(US) Patent
공개
국제특허분류(IPC7판)
H01J-037/16
C23F-004/00
C23F-001/40
H01J-037/26
출원번호
US-0233176
(2016-08-10)
공개번호
US-0351374
(2016-12-01)
발명자
/ 주소
Wang, Liguo
출원인 / 주소
Wang, Liguo
인용정보
피인용 횟수 :
0인용 특허 :
0
초록▼
A grid assembly for cryo-electron microscopy may be fabricated using standard nanofabrication processes. The grid assembly may comprise two support members, each support member comprising a silicon substrate coated with an electron-transparent silicon nitride layer. These two support members are pos
A grid assembly for cryo-electron microscopy may be fabricated using standard nanofabrication processes. The grid assembly may comprise two support members, each support member comprising a silicon substrate coated with an electron-transparent silicon nitride layer. These two support members are positioned together with the silicon nitride layers facing each other with a rigid spacer layer disposed therebetween. The rigid spacer layer defines one or more chambers in which a biological sample may be provided and fast frozen with a high degree of control of the ice thickness.
대표청구항▼
1. A system for cryo-electron microscopy, comprising: a first support member comprising a first substrate coupled to a first electron-transparent layer, wherein the first substrate comprises one or more lower openings;a second support member comprising a second substrate coupled to a second electron
1. A system for cryo-electron microscopy, comprising: a first support member comprising a first substrate coupled to a first electron-transparent layer, wherein the first substrate comprises one or more lower openings;a second support member comprising a second substrate coupled to a second electron-transparent layer, wherein the second substrate comprises one or more upper openings; anda rigid spacer layer disposed between the first support member and the second support member. 2. The system of claim 1, wherein: the one or more lower openings comprise one or more substantially rectangular channels extending in a first direction; andthe one or more upper openings comprise one or more substantially rectangular channels extending in a second direction substantially orthogonal to the first direction. 3. The system of claim 1, wherein: the one or more lower openings comprise a first plurality of channels extending in a first direction; andthe one or more upper openings comprise a second plurality of channels extending in a second direction substantially orthogonal to the first direction such that each channel in the first plurality of channels overlaps with each of the second plurality of channels, wherein the portions of overlapping channels form a plurality of apertures through which electrons may pass. 4. The system of claim 1, wherein: the rigid spacer layer comprises silicon dioxide or metal deposited on the first support member. 5. The system of claim 1, wherein: the rigid spacer layer forms one or more chambers, each chamber being aligned with one of the lower openings and one of the upper openings to form a aperture such that electron beams may pass through the one of the lower openings, the chamber, and the one of the upper openings. 6. The system of claim 5, wherein: the rigid spacer layer comprises silicon dioxide or metal deposited on the first support member; andthe one or more chambers are etched from the silicon dioxide or metal. 7. The system of claim 5, further comprising: a vitrified biological sample contained in the one or more chambers. 8. The system of claim 5, wherein: each of the chambers includes an exit portion aligned with an edge of the first support member and an edge of the second support member. 9. The system of claim 1, wherein: the first substrate and the second substrate comprise silicon; andthe one or more lower openings are etched in the silicon of the first substrate; andthe one or more upper openings are etched in the silicon of the second substrate. 10. The system of claim 9, wherein: the first electron-transparent layer comprises a first silicon nitride layer deposited on the first substrate; andthe second electron-transparent layer comprises a second silicon nitride layer deposited on the second substrate. 11. The system of claim 1, wherein: the first substrate is less than approximately 300 μm thick; andthe second substrate is less than approximately 300 μm thick. 12. The system of claim 1, wherein: the rigid spacer layer is between about 10 nm to about 300 nm thick. 13. The system of claim 1, wherein: the first electron-transparent layer and the second electron-transparent layer are between about 10 nm to about 30 nm thick. 14. A method of performing cryo-electron microscopy, comprising: providing a first support member comprising a first substrate coupled to a first electron-transparent layer, wherein the first substrate comprises one or more lower openings;depositing a biological sample on the first electron-transparent layer of the first support member;providing a second support member comprising a second substrate coupled to a second electron-transparent layer, wherein the second substrate comprises one or more upper openings;positioning the second electron-transparent layer of the second support member on the biological sample, whereby the second electron-transparent layer is separated from the first electron-transparent layer by a rigid spacer layer; andfreezing the biological sample to produce a vitrified biological sample between the first electron-transparent layer and the second electron-transparent layer. 15. The method of claim 14, further comprising: imaging the biological sample by directing an electron beam through the one or more lower openings in the first substrate, the first electron-transparent layer, the vitrified biological sample, the second electron-transparent layer, and the one or more upper openings in the second substrate. 16. The method of claim 14, further comprising: prior to freezing the biological sample, withdrawing a portion of the biological sample until the second support member is supported by the rigid spacer layer to retain the first support member at a predetermined distance from the second support member. 17. The method of claim 16, wherein: the withdrawing the portion of the biological sample comprises positioning an absorptive material adjacent to an exit portion of a chamber formed by the rigid spacer layer. 18. The method of claim 14, wherein: the one or more lower openings comprise one or more substantially rectangular channels extending in a first direction;the one or more upper openings comprise one or more substantially rectangular channels extending in a second direction; andthe positioning the second electron-transparent layer of the second support member on the biological sample comprises positioning the second support member on the first support member such that the first direction is substantially orthogonal to the second direction. 19. A method of manufacturing a cryo-electron microscopy grid assembly, comprising: providing a first support member comprising a first substrate coupled to a first electron-transparent layer, wherein the first substrate comprises one or more lower openings; anddepositing a rigid spacer layer on the first electron-transparent layer, the rigid spacer layer forming one or more chambers, each chamber being aligned with one or more of the lower openings. 20. The method of claim 19, wherein the providing the first support member comprises: depositing the first electron-transparent layer on a silicon wafer;etching the one or more lower openings in the silicon wafer; anddicing the silicon wafer into a plurality of first support members. 21. The method of claim 20, wherein: the depositing the first electron-transparent layer on the silicon wafer comprises depositing silicon nitride on the silicon wafer. 22. The method of claim 20, wherein the depositing the rigid spacer layer comprises: depositing the rigid spacer layer comprising silicon dioxide or metal on the first electron-transparent layer at a predetermined thickness. 23. The method of claim 22, wherein the depositing the rigid spacer layer further comprises: etching the rigid spacer layer to form the one or more chambers.
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