# US 2018/0024111 A1 · Method for multiplexed sample analysis by photoionizing secondary sputtered neutrals > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2018/0024111 A1 - Document ID: US20180024111A1 - Status: Published application - Kind: A1 - Application number: 15/709,136 - Filed: 2017-09-19 - Published: 2018-01-25 - Earliest claimed: 2015-04-23 - Assignees: The Board of Trustees of the Leland Stanford Junior University - Applicants: The Board of Trustees of the Leland Stanford Junior University - Inventors: Robert M. Angelo - Web page: https://patentgrep.com/patent/US20180024111A1 ## CPC - G01N 33/4833: {of solid biological material, e.g. tissue samples, cell cultures (tissue in vivo A61B5/00; cell suspensions G01N33/48735)} - G01N 23/2258: Measuring secondary ion emission, e.g. secondary ion mass spectrometry [SIMS] (mass-to-charge ratio analysis aspects of SIMS for material analysis G01N27/62) - H01J 49/0004: {Imaging particle spectrometry} - H01J 49/142: {using a solid target which is not previously vapourised} - H01J 49/162: {Direct photo-ionisation, e.g. single photon or multi-photon ionisation} - 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 Disclosed herein is a method of generating a high resolution image of a cellular sample, the method including i) labeling a cellular sample with at least one mass tag, thereby producing a labeled sample in which a biological feature of interest is associated with the at least one mass tag, ii) scanning the sample with a continuous or near-continuous primary ion beam to generate sputtered secondary ions and sputtered neutral species, iii) photoionizing the sputtered neutrals to generate ionized neutral species, wherein the sputtered neutrals are photoionized at a site that is proximal to their source on the sample, iv) detecting the ionized neutral species by mass spectrometry, thereby obtaining spatially addressed measurements of the abundance of the at least one mass tag across an area of the sample, and v) producing an image of the sample using the measurements. A system for performing the method is also provided. ## Claims ### Claim 1 A method of generating a high resolution image of a cellular sample, comprising: i) labeling a cellular sample with at least one mass tag, thereby producing a labeled sample in which a biological feature of interest is associated with said at least one mass tag; ii) scanning the sample with a continuous or near-continuous primary ion beam to generate sputtered secondary ions and sputtered neutral species; iii) photoionizing the sputtered neutrals to generate ionized neutral species, wherein the sputtered neutrals are photoionized at a site that is proximal to their source on the sample; iv) detecting the ionized neutral species by mass spectrometry, thereby obtaining spatially addressed measurements of the abundance of said at least one mass tag across an area of said sample; and v) producing an image of the sample using the measurements. ### Claim 2 (depends on 1) The method of claim 1, wherein the at least one mass tag is a plurality of distinguishable mass tags, and the method comprises obtaining spatially addressed measurements of the abundance of said plurality of distinguishable mass tags across an area of said sample by detecting the ionized neutral species by mass spectrometry. ### Claim 3 (depends on 1) The method of claim 1, wherein the photoionizing step comprises irradiating the neutral species with radiation, thereby photoionizing the neutral species. ### Claim 4 (depends on 3) The method of claim 3, wherein the irradiating comprises irradiating with high power-density optical radiation. ### Claim 5 (depends on 3) The method of claim 3, wherein the radiation is produced by a laser or a light emitting diode (LED). ### Claim 6 (depends on 3) The method of claim 3, wherein the radiation is ultraviolet, visible, or infrared radiation. ### Claim 7 (depends on 3) The method of claim 3, wherein the average power of the radiation is in the range of 1 mW to 100 W. ### Claim 8 (depends on 5) The method of claim 5, wherein the laser or LED operates in continuous wave (CW), quasi-continuous wave (quasi-CW), or pulsed modes of operation. ### Claim 9 (depends on 8) The method of claim 8, wherein the radiation is produced by a single LED or an LED array. ### Claim 10 (depends on 1) The method of claim 1, wherein the photoionizing comprises using resonant ionization to ionize the neutral species. ### Claim 11 (depends on 1) The method of claim 1, wherein the photoionizing comprises using nonresonant ionization to ionize the neutral species. ### Claim 12 (depends on 1) The method of claim 1, wherein the photoionizing comprises applying radiation whose path is parallel to a surface of the sample and over a region of the sample impinged upon by the primary ion beam, to ionize the sputtered neutral species. ### Claim 13 (depends on 3) The method of claim 3, wherein the irradiating comprises intensifying the radiation by an optical resonator located outside a radiation source used for the irradiating. ### Claim 14 (depends on 13) The method of claim 13, wherein the optical resonator is configured to maximize optical resonance of the radiation over a region of the sample impinged upon by the primary ion beam. ### Claim 15 (depends on 3) The method of claim 3, wherein the radiation is intensified by a multipass spectroscopic absorption cell. ### Claim 16 (depends on 1) The method of claim 1, wherein the method comprises applying a voltage to conductive members disposed on the sample, thereby controlling the electric potential of said sample. ### Claim 17 (depends on 1) The method of claim 1, wherein the primary ion beam comprises a beam of oxygen, cesium, gold, argon, bismuth, xenon, C60, SF6, indium, gallium ions, or a combination thereof. ### Claim 18 (depends on 1) The method of claim 1, wherein the primary ion beam has an ion current density of 1 nA/cm2 or more. ### Claim 19 (depends on 1) The method of claim 1, wherein the primary ion beam has an ion density of 1×1013 primary ions/cm2 or more. ### Claim 20 (depends on 1) The method of claim 1, wherein the primary ion beam has an energy of 1 keV or more. ### Claim 21 A system for generating a high resolution image of a cellular sample, comprising: a) a holder for retaining a substrate comprising a sample; b) a continuous or near-continuous primary ion beam source configured to scan the sample and sputter secondary ions and neutral species from the sample; c) a photoionization source configured to ionize the neutral species at a site that is proximal to their source on the sample; d) an orthogonal time-of-flight mass spectrometer configured to detect the ionized neutral species and obtain spatially addressed measurements of the abundance of at least one mass tag associated with said sample; and e) a computer comprising an image analysis module that processes said measurements to produce an image of said sample.