# US 2015/0090899 A1 · Preparation of Specimen Arrays on an EM Grid > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2015/0090899 A1 - Document ID: US20150090899A1 - Status: Published application - Kind: A1 - Application number: 14/372,274 - Filed: 2013-01-14 - Published: 2015-04-02 - Earliest claimed: 2012-01-17 - Assignees: The Scripps Research Institute - Applicants: The Scripps Research Institute - Inventors: Bridget Carragher; Clinton S. Potter; Tilak Jain - Web page: https://patentgrep.com/patent/US20150090899A1 ## CPC - G01N 1/28: Preparing specimens for investigation {including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q}(mounting specimens on microscopic slides G02B21/34; means for supporting the objects or the materials to be analysed in electron microscopes H01J37/20 {; laboratory gas handling apparatus B01L5/00}) - G01N 1/2813: {Producing thin layers of samples on a substrate, e.g. smearing, spinning-on (G01N1/30 takes precedence)} - G01N 1/30: Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis} - G01N 1/31: Apparatus therefor - G01N 1/312: {for samples mounted on planar substrates} - H01J 37/20: Means for supporting or positioning the object or the material; Means for adjusting diaphragms or lenses associated with the support - H01J 37/261: {Details} - H01J 2237/20: Positioning, supporting, modifying or maintaining the physical state of objects being observed or treated - H01J 2237/2007: Holding mechanisms - H01J 2237/202: Movement - H01J 2237/204: Means for introducing and/or outputting objects - H01J 2237/208: Elements or methods for movement independent of sample stage for influencing or moving or contacting or transferring the sample or parts thereof, e.g. prober needles or transfer needles in FIB/SEM systems - H01J 2237/2602: Details - 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) - H01J: ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS (spark-gaps H01T; electric arc lamps H05B31/00; particle accelerators H05H) ## Abstract The invention provides methods and compositions for preparation of complex specimen arrays for analysis by electron microscopy. These methods and compositions can permit high throughput screening of samples on single EM grid supports using sample volumes in the nanoliter and picoliter range. ## Claims ### Claim 1 A method for preparing an electron microscopy sample on an EM sample grid, comprising: dispensing a plurality of discrete specimens onto an EM sample grid in an ordered array of specimen locations, each specimen of the plurality of discrete specimens being placed into an individual specimen location in the array of locations, and each individual specimen location in the array of locations having an area of between about 2000 μm2 to about 70,000 μm2; and applying a discrete volume of a stain material suitable for contrast enhancement in an electron microscope to each individual specimen location in the array of locations that received a specimen. ### Claim 2 A method for preparing an electron microscopy sample on an EM sample grid, comprising: dispensing a plurality of discrete specimens onto an EM sample grid in an ordered array of specimen locations, each specimen of the plurality of discrete specimens being placed into an individual specimen location in the array of locations, and each individual specimen location in the array of locations having an area of between about 2000 μm2 to about 70,000 μm2 (for example, each location is a circular spot of between about 50 μm and about 300 μm in diameter); applying a discrete volume of a wash solution to each individual specimen location in the array of locations that received a specimen, and removing excess wash solution by contact with a bibulous or porous material at the periphery of each individual specimen location in the array of locations; and applying a discrete volume of a stain material suitable for contrast enhancement in an electron microscope to each individual specimen location in the array of locations that received a specimen. ### Claim 3 (depends on 1, 2) A method according to claim 1 or 2, wherein said discrete specimens are dispensed by applying more than material to each specimen location, wherein each specimen is formed by mixing the more than one material at each specimen location. ### Claim 4 (depends on 1, 2, 3) A method according to one of claims 1-3, wherein the stain material is selected from the group consisting of ammonium molybdate, uranyl acetate, uranyl formate, phosphotungstic acid, osmium tetroxide, osmium ferricyanide and auroglucothionate, commercially available stains such as NanoW and NanoVan and other stains that have been described in the general literature. ### Claim 5 (depends on 1, 2, 3) A method according to one of claims 1-3, wherein the bibulous or porous material is a microfabricated material applied to the surface of the EM sample grid, wherein the microfabricated material comprises a pattern of openings in the bibulous or porous material corresponding to the ordered array of specimen locations. ### Claim 6 (depends on 1, 2, 3) A method according to one of claims 1-3, wherein the bibulous or porous material comprises a dried gel material. ### Claim 7 (depends on 1, 2, 3) A method according to one of claims 1-3, wherein the bibulous or porous material comprises a fibrous material. ### Claim 8 (depends on 1, 2, 3) A method according to one of claims 1-3, wherein the bibulous or porous material comprises a matrix forming one or more capillary spaces. ### Claim 9 An EM specimen grid, comprising: a plurality of discrete specimen locations delimited from one another by peripheral regions comprising a bibulous or porous material, each individual specimen location in the array of locations having an area of between about 2000 μm2 to about 70,000 μm2. ### Claim 10 (depends on 9) A method according to claim 9, wherein the bibulous or porous material comprises a dried gel material. ### Claim 11 (depends on 9) A method according to claim 9, wherein the bibulous or porous material comprises a fibrous material. ### Claim 12 (depends on 9) A method according to claim 9, wherein the bibulous or porous material comprises a matrix forming one or more capillary spaces. ### Claim 13 A system for dispensing aqueous materials onto an EM sample grid at individual specimen locations in an ordered array of specimen locations, each individual specimen location in the array of locations having an area of between about 2000 μm2 to about 70,000 μm2. The systems comprise: a holder for reversibly receiving an EM sample grid; a volume drop dispenser configured to dispense fluid from one or more dispensing elements onto each individual specimen location in the array of locations, at least one dispensing element in the volume drop dispenser configured to dispense picoliter volumes; a drive mechanism to position the EM sample grid relative to the one or more dispensing elements; and one or more reservoirs operably linked to the drop dispenser for holding one or more aqueous solutions to be dispensed onto each individual specimen location in the array of locations.