# US 10,309,881 B2 · Methods and apparatus for preparing aqueous specimens for electron microscopy using volatile surfactants > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,309,881 B2 - Document ID: US10309881B2 - Status: Granted - Kind: B2 - Application number: 15/194,777 - Filed: 2016-06-28 - Published: 2017-01-05 - Granted: 2019-06-04 - Earliest claimed: 2015-06-30 - Assignees: The Regents of the University of California - Applicants: The Regents of the University of California - Inventors: Robert M. Glaeser; Bong-Gyoon Han - Web page: https://patentgrep.com/patent/US10309881B2 ## CPC - G01N 1/42: Low-temperature sample treatment, e.g. cryofixation - G01N 1/40: Concentrating samples - G01N 1/405: {by adsorption or absorption} - G01N 2001/4027: {evaporation leaving a concentrated sample} - G01N 2001/4061: {Solvent extraction} - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract This disclosure provides systems, methods, and apparatus related to the preparation of electron microscopy specimens. In one aspect, a method includes providing an electron microscopy grid. An aqueous suspension including an electron microscopy sample is deposited on the electron microscopy grid. A portion of the aqueous suspension is removed from the electron microscopy grid. A volatile surfactant is contacted to the aqueous suspension. A thickness of aqueous suspension is allowed to decrease over a period of time. The aqueous suspension is then frozen. ## Claims ### Claim 1 A method comprising: (a) providing an electron microscopy grid; (b) depositing an aqueous suspension including an electron microscopy sample on the electron microscopy grid; (c) removing a portion of the aqueous suspension from the electron microscopy grid; (d) after operation (c), contacting a volatile surfactant to the aqueous suspension remaining on the electron microscopy grid; (e) after operation (d), allowing a thickness of the aqueous suspension to decrease over a period of time; and (f) freezing the aqueous suspension to generate a film of the aqueous suspension having a uniform thickness disposed on the electron microscopy grid. ### Claim 2 (depends on 1) The method of claim 1, wherein a volume of aqueous suspension deposited on the electron microscopy grid is about 3 microliters or less. ### Claim 3 (depends on 1) The method of claim 1, wherein operation (c) comprises contacting an edge of the electron microscopy grid with an absorbent material. ### Claim 4 (depends on 3) The method of claim 3, wherein the absorbent material comprises filter paper. ### Claim 5 (depends on 1) The method of claim 1, wherein the electron microscopy grid includes a first surface and a second surface, and wherein operation (c) comprises contacting the first surface with an absorbent material. ### Claim 6 (depends on 1) The method of claim 1, wherein a thickness of the aqueous suspension on the electron microscopy grid is about 10 microns to 100 microns after operation (c) and before operation (d). ### Claim 7 (depends on 1) The method of claim 1, wherein operation (d) comprises contacting the volatile surfactant to the aqueous suspension using a capillary. ### Claim 8 (depends on 1) The method of claim 1, wherein operation (d) comprises exposing the aqueous suspension to vapor of the volatile surfactant. ### Claim 9 (depends on 1) The method of claim 1, wherein the volatile surfactant comprises a surfactant selected from a group consisting of chloroform and nonafluorobutyl methyl ether. ### Claim 10 (depends on 1) The method of claim 1, wherein operation (d) further comprises contacting a non-violatile surfactant to the aqueous suspension. ### Claim 11 (depends on 10) The method of claim 10, wherein the non-volatile surfactant is dissolved in the volatile surfactant. ### Claim 12 (depends on 10) The method of claim 10, wherein the non-volatile surfactant comprises a surfactant selected from a group consisting of ligands, hydrophilic polymers, phospholipid vesicles, and amphipols. ### Claim 13 (depends on 1) The method of claim 1, wherein the period of time in operation (e) is about 1 second to 300 seconds. ### Claim 14 (depends on 1) The method of claim 1, wherein operation (f) includes contacting liquid ethane to the electron microscopy grid. ### Claim 15 (depends on 1) The method of claim 1, wherein operation (f) is performed when the thickness of the aqueous suspension on the electron microscopy grid is about 100 nanometers or less. ### Claim 16 (depends on 1) The method of claim 1, wherein the electron microscopy grid includes a carbon film disposed on a first surface of the electron microscopy grid, and wherein the aqueous suspension is deposited on the carbon film. ### Claim 17 (depends on 1) The method of claim 1, wherein the electron microscopy grid has a diameter of about 3 millimeters. ### Claim 18 (depends on 1) The method of claim 1, wherein operations (b), (c), (d), and (e) are performed at a specific temperature and a specific relative humidity. ### Claim 19 (depends on 18) The method of claim 18, wherein the specific temperature is about 0° C. to 40° C., and wherein the specific relative humidity is about 50% to 100%. ### Claim 20 An apparatus for preparing an electron microscopy grid, comprising: a chamber, the chamber defining a plurality of ports; a rod disposed in the chamber; and a sample holder attached to the rod; the apparatus operable to perform a method comprising: (a) receiving an electron microscopy grid in the sample holder; (b) receiving a deposition of an aqueous suspension including an electron microscopy sample on the electron microscopy grid; (c) receiving an absorbent material to remove removing a portion of the aqueous suspension from the electron microscopy grid; (d) after operation (c), receiving a volatile surfactant to contact contacting a volatile surfactant to the aqueous suspension remaining on the electron microscopy grid; (e) after operation (d), allowing a thickness of the aqueous suspension to decrease over a period of time; and (f) freezing the aqueous suspension to generate a film of the aqueous suspension having a uniform thickness disposed on the electron microscopy grid.