Method for multiplexed sample analysis by photoionizing secondary sputtered neutrals

• US 10,408,814 B2
• Filed: 09/19/2017
• Issued: 09/10/2019
• Est. Priority Date: 04/23/2015
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Angelo, Robert M.
• Primary Examiner(s): Ippolito, Nicole M
• Application Number:
  US15/709,136
• Publication Number:
  US 20180024111A1
• Time in Patent Office: 721 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 comprising:

scanning a biological sample labelled with at least one mass tag with a primary ion beam to generate sputtered neutral species;

photoionizing the sputtered neutrals to generate photoionized species; and

detecting the photoionized species by mass spectrometry.

2. The method of claim 1, wherein the sputtered neutrals are photoionized at a site that is proximal to their source on the mass tag-labeled biological sample.

3. The method of claim 1 further comprising obtaining spatially-addressable measurements of an abundance of the mass tag at a plurality of specific positions across an area of the biological sample.

4. The method of claim 1, wherein the photoionizing step comprises irradiating the neutral species using an optical radiation source.

5. The method of claim 4, wherein the optical radiation source comprises a laser or a light emitting diode (LED).

6. The method of claim 5, wherein the optical radiation source produces ultraviolet, visible, or infrared radiation.

7. 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.

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

9. The method of claim 1, further comprising labeling a feature of interest in the biological sample with the mass tag.

10. The method of claim 1, wherein a plurality of distinguishable mass tags are bound to a plurality of biological features in the biological sample, the method further comprising obtaining spatially-addressable measurements of the plurality of biological features.

11. The method of claim 10, further comprising producing an image of the biological sample using the measurements.

12. The method of claim 1, wherein photoionizing comprises applying radiation in a path that is substantially parallel to a surface of the biological sample and over a region of the sample impinged upon by the primary ion beam, to ionize the sputtered neutral species.

13. The method of claim 4, wherein the irradiating comprises intensifying the radiation by an optical resonator.

14. The method of claim 1, further comprising applying a voltage to conductive members disposed on the biological sample, thereby controlling the electric potential of the biological sample.

15. 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.

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

17. The method of claim 1, wherein the primary ion beam is continuous.

18. A system, comprising:

an ion source configured to generate a primary ion beam and to scan the primary ion beam on a sample to generate neutral species from the sample;

an ionization source configured to generate radiation to ionize the neutral species to generate a plurality of ions;

a detector configured to receive the plurality of ions; and

a computational device connected to the detector and configured so that during operation of the system, the computational device determines information about the sample based on measurements of the plurality of ions by the detector.

19. The system of claim 18, wherein the primary ions comprise at least one member selected from the group consisting of oxygen ions, cesium ions, gold ions, argon ions, xenon ions, bismuth ions, C₆₀ ions, SF₆ ions, indium ions, and gallium ions.

20. The system of claim 18, wherein the plurality of ions comprise ions of at least one lanthanide element.

21. The system of claim 18, wherein the neutral species comprise at least one metallic element conjugated to a polydentate ligand.

22. The system of claim 18, wherein the neutral species comprise at least one mass tag.

23. The system of claim 18, wherein the detector comprises an orthogonal time-of-flight detector.

24. The system of claim 18, wherein a duty cycle of the primary ion beam is 80% or more.

25. The system of claim 18, wherein:

the information about the sample comprises abundance information for at least one mass tag at multiple sample locations; and

the computational device is configured to generate an image of the sample based on the abundance information.

26. The system of claim 25, wherein the information about the sample comprises abundance information for multiple distinguishable mass tags at multiple sample locations, and wherein the mass tags correspond to metallic elements.