# US 2014/0360286 A1 · Apparatus and Method for Producing Specimens for Electron Microscopy > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0360286 A1 - Document ID: US20140360286A1 - Status: Published application - Kind: A1 - Application number: 14/372,277 - Filed: 2013-01-14 - Published: 2014-12-11 - Earliest claimed: 2012-01-17 - Assignees: The Scripps Research Institute; Engineering Arts LLC - Applicants: The Scripps Research Institute; Engineering Arts LLC - Inventors: Bridget Carragher; Clinton S. Potter; Tilak Jain; Peter Kahn; Peter Wiktor - Web page: https://patentgrep.com/patent/US20140360286A1 ## 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 1/42: Low-temperature sample treatment, e.g. cryofixation - H01J 37/261: {Details} - H01J 37/26: Electron or ion microscopes; Electron or ion diffraction tubes - H01J 2237/2001: Maintaining constant desired temperature - H01J 2237/2802: Transmission microscopes - 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 devices for preparing frozen vitrified samples for transmission electron microscopy. By reducing the volume of sample from microliter scale to picoliter scale, the requirement for blotting of excess fluid is minimized or eliminated. ## Claims ### Claim 1 A system for preparing an electron microscopy sample on an EM sample grid, comprising: a. a plunger assembly comprising a shaft operably connected to a shaft drive assembly, a triggering mechanism, and a holder for reversibly receiving an EM sample grid at a first end of the shaft, the shaft drive assembly and triggering mechanism configured to (i) position the EM sample grid at a first position prior to a trigger event, and (ii) displace the shaft in a first direction at a predetermined rate and position the EM sample grid at a second position following a trigger event; b. a fluid dispenser configured to dispense fluid from one or more dispensing elements onto one or more independently addressable subregions of the EM sample grid in the first position or between the first and second positions; c. a sample reservoir operably linked to the drop dispenser; and d. a cryogen reservoir configured to position a cryogenic liquid at the second position. ### Claim 2 (depends on 1) A system according to claim 1, wherein the one or more dispensing elements dispense a picoliter sample volume by thermal inkjet printing, acoustic inkjet printing, or piezoelectric inkjet printing onto the one or more independently addressable subregions of the EM sample grid. ### Claim 3 (depends on 1) A system according to claim 1, wherein the one or more dispensing elements comprise one or more print pins that dispense a picoliter sample volume by physical contact with one or more subregions of the EM sample grid. ### Claim 4 (depends on 1) A system according to claim 1, wherein the drop dispenser is operably connected to a positioning drive configured to provide adjustment of the drop dispenser relative to the EM sample grid in the first position or between the first and second positions. ### Claim 5 (depends on 1) A system according to claim 1, wherein the drop dispenser comprises a plurality of dispensing elements configured to dispense a picoliter sample volume onto different independently addressable subregions of the EM sample grid. ### Claim 6 (depends on 1) A system according to claim 1, further comprising an environmental chamber configured to provide controlled relative humidity and temperature. ### Claim 7 (depends on 1) A system according to claim 1, further comprising one or more of: a first camera system configured to provide monitoring of fluid dispensed from the one or more dispensing elements, and a second camera system configured to provide monitoring alignment of the position of the dispensing elements relative to the EM sample grid in the first position and/or monitoring of droplet characteristics on the EM sample grid. ### Claim 8 (depends on 1) A system according to claim 1, wherein the shaft drive assembly comprises a pneumatic drive. ### Claim 9 (depends on 1) A system according to claim 1, wherein the shaft drive assembly comprises a stepper motor drive. ### Claim 10 (depends on 1) A system according to claim 1, comprising a computer interface configured to control operation of one or more system elements. ### Claim 11 A method for preparing an electron microscopy sample on an EM sample grid, comprising: a. positioning the EM sample grid on a plunger assembly, the plunger assembly comprising a shaft operably connected to a shaft drive assembly, a triggering mechanism, and a holder for reversibly receiving the EM sample grid at a first end of the shaft, the shaft drive assembly and triggering mechanism configured to (i) position the EM sample grid at a first position prior to a trigger event, and (ii) displace the shaft in a first direction at a predetermined rate and position the EM sample grid at a second position following a trigger event; b. with the EM sample grid in the first position or between the first and second positions, dispensing one or more sample volumes onto one or more independently addressable subregions of the EM sample grid using a fluid dispenser configured to dispense fluid from one or more dispensing elements; c. activating the triggering mechanism to displace the shaft to the second position, wherein the EM sample grid is immersed into a cryogenic liquid contained within a cryogen reservoir. ### Claim 12 (depends on 11) A method according to claim 11, wherein the one or more dispensing elements dispense a picoliter sample volume by thermal inkjet printing, acoustic inkjet printing, or piezoelectric inkjet printing onto the one or more independently addressable subregions of the EM sample grid. ### Claim 13 (depends on 11) A method according to claim 11, wherein the one or more dispensing elements comprise one or more print pins that dispense a picoliter sample volume by physical contact with one or more subregions of the EM sample grid. ### Claim 14 (depends on 11) A method according to claim 11, wherein the drop dispenser is operably connected to a positioning drive configured to provide adjustment of the drop dispenser relative to the EM sample grid. ### Claim 15 (depends on 11) A method according to claim 11, wherein the drop dispenser comprises a plurality of dispensing elements that dispense a picoliter sample volume onto different independently addressable subregions of the EM sample grid. ### Claim 16 (depends on 11) A method according to claim 11, further comprising maintaining the EM sample grid in a controlled relative humidity and temperature environment during at least step (b). ### Claim 17 (depends on 11) A method according to claim 11, further comprising one or more of: monitoring of fluid dispensed from the one or more dispensing elements using a first camera system, and monitoring alignment of the position of the dispensing elements relative to the EM sample grid and/or monitoring of droplet characteristics on the EM sample grid in the first position using a second camera system. ### Claim 18 (depends on 11) A method according to claim 11, wherein the shaft drive assembly comprises a pneumatic drive. ### Claim 19 (depends on 11) A method according to claim 11, wherein the shalt drive assembly comprises a stepper motor drive.