Method for multiplexed sample analysis by photoionizing secondary sputtered neutrals

• US 9,797,879 B2
• Filed: 04/20/2016
• Issued: 10/24/2017
• Est. Priority Date: 04/23/2015
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Angelo, Robert M.
• Primary Examiner(s): Ippolito, Nicole
• Application Number:
  US15/134,074
• Publication Number:
  US 20160313295A1
• Time in Patent Office: 552 Days
• Field of Search: 250281, 250282, 250283
• CPC Class Codes: 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
• CPC Class Codes: G01N 33/4833 of solid biological material, e.g. tissue samples, cell cultures tissue in vivo A61B5/00; cell suspensions G01N33/48735
• CPC Class Codes: H01J 49/0004 Imaging particle spectrometry
• CPC Class Codes: H01J 49/142 using a solid target which is not previously vapourised
• CPC Class Codes: H01J 49/162 Direct photo-ionisation, e.g. single photon or multi-photon ionisation

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

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.

2. 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.

3. The method of claim 1, wherein the photoionizing step comprises irradiating the neutral species with radiation, thereby photoionizing the neutral species.

4. The method of claim 3, wherein the irradiating comprises irradiating with high power-density optical radiation.

5. The method of claim 3, wherein the radiation is produced by a laser or a light emitting diode (LED).

6. 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.

7. The method of claim 6, wherein the radiation is produced by a single LED or an LED array.

8. The method of claim 3, wherein the radiation is ultraviolet, visible, or infrared radiation.

9. The method of claim 3, wherein the average power of the radiation is in the range of 1 mW to 100 W.

10. 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.

11. The method of claim 10, 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.

12. The method of claim 3, wherein the radiation is intensified by a multipass spectroscopic absorption cell.

13. The method of claim 1, wherein the photoionizing comprises using resonant ionization to ionize the neutral species.

14. The method of claim 1, wherein the photoionizing comprises using nonresonant ionization to ionize the neutral species.

15. 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.

16. 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.

17. 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.

18. The method of claim 1, wherein the primary ion beam has an ion current density of 1 nA/cm² or more.

19. The method of claim 1, wherein the primary ion beam has an ion density of 1×

10¹³ primary ions/cm² or more.

20. The method of claim 1, wherein the primary ion beam has an energy of 1 keV or more.

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.