Single cell analysis using secondary ion mass spectrometry

Patent metadata

Field	Value
Publication number	US-9766224-B2
Application number	US-201615068338-A
Country	US
Kind code	B2
Filing date	Mar 11, 2016
Priority date	Mar 25, 2015
Publication date	Sep 19, 2017
Grant date	Sep 19, 2017

Abstract

A method of analyzing a population of cells is disclosed. In certain embodiments, the method includes i) obtaining an array of cells on a substrate, wherein the cells are labeled with one or more mass tags and are separated from one another, ii) measuring, using secondary ion mass spectrometry (SIMS), the abundance of the one or more mass tags at a plurality of locations occupied by the cells, thereby generating, for each individual cell measured, a set of data, and iii) outputting the set of data for each of the cells analyzed. Also provided herein are systems that find use in performing the subject method. In some embodiments, the system is an automated system for analyzing a population of cells using SIMS.

First claim

What is claimed is:

1. A method of analyzing a population of cells, comprising:
i) obtaining an array of cells on a substrate, wherein the cells are labeled with one or more mass tags and are separated from one another;
ii) measuring, using a plurality of secondary ion mass spectrometry (SIMS) pulses, the abundance of the one or more mass tags at each of a plurality of locations occupied by the cells and at each of a plurality of depths within the cells, thereby generating, for each individual cell measured, a set of data; and
iii) outputting the set of data for each of the cells analyzed.

2. The method of claim 1 , wherein the array is addressable.

3. The method of claim 1 , wherein the array is random and the locations occupied by the cells are determined by imaging the substrate prior to the measuring step.

4. The method of claim 3 , wherein the imaging is by optical imaging, electron imaging or low resolution SIMS.

5. The method of claim 1 , wherein the measuring step comprises applying a SIMS ion beam with a diameter equal to or greater than half the average diameter of individual cells to measure the abundance of the one or more mass tags at the plurality of depths on a whole cell basis.

6. The method of claim 5 , wherein the SIMS ion beam has a diameter in the range of 1 μm to 50 μm.

7. The method of claim 1 , wherein the measuring step comprises applying a plurality of pulses of a SIMS ion beam at different sites of an individual cell of the array to obtain measurements of the abundance of the one or more mass tags at the plurality of depths at the different sites.

8. The method of claim 7 , wherein the SIMS ion beam has a diameter in the range of 10 nm to 1500 nm.

9. The method of claim 1 , wherein the array of cells is obtained by:
labeling cells with one or more mass tags; and
attaching cells to a substrate;
wherein the labeling is done either prior to or after the cells are attached to the substrate.

10. The method of claim 9 , wherein said labeling is done by administering the mass tag to an animal subject and obtaining labeled cells from said subject.

11. The method of claim 1 , wherein the method comprises:
labeling cells with a first mass tag and a second mass tag, wherein the first mass tag localizes to a known subcellular structure of the cell;
measuring the abundance of the first and second mass tags at the plurality of depths at different sites of an individual cell of the array; and
determining the subcellular localization of the second mass tag based on the measured abundance of the first and second mass tags at the plurality of depths.

12. The method of claim 1 , further comprising:
iv) identifying one or more cells of interest based on the obtained set of data; and
v) recovering the identified cells for further analysis.

13. The method of claim 5 , further comprising:
identifying one or more cells of interest based on the obtained set of data;
remeasuring, using secondary ion mass spectrometry (SIMS), the abundance of the one or more mass tags at a plurality of locations occupied by the identified one or more cells of interest, thereby generating, for each individual cell remeasured, a second set of data; and
outputting the second set of data for each of the identified cells of interest.

14. A method of analyzing a test population of cells, comprising:
i) obtaining an array of cells on a substrate, wherein the cells are labeled with one or more mass tags and are separated from one another;
ii) measuring, using a plurality of SIMS ion beam pulses, the abundance of the one or more mass tags at each of a plurality of locations occupied by the cells and at each of a plurality of depths within the cells, thereby generating, for each individual cell measured, a set of data;
iii) generating a histogram showing the distribution of the mass tags across the test population of cells; and
iv) comparing the histogram to a reference histogram obtained from a reference population of cells.

15. The method of claim 14 , wherein the method further comprises:
v) identifying one or more cells of interest based on a threshold value for the abundance of the one or more mass tags.

16. The method of claim 15 , wherein the threshold value is determined based on the reference histogram.

17. The method of claim 14 , wherein the test population of cells has been contacted with a test agent and the reference population of cells has not been contacted with the test agent.

18. The method of claim 14 , wherein the test population of cells is obtained from a subject diagnosed with a condition and the reference population of cells is obtained from a healthy subject.

19. An automated system for analyzing an array of cells, the system comprising:
a) a SIMS system comprising a holder for retaining a substrate comprising an array of cells, wherein the cells are labeled with one or more mass tags and are separated from one another, wherein the system is programmed to:
(i) measure the abundance of the one or more mass tags at each of a plurality of depths and at each of a plurality of locations occupied by the cells of the array using a plurality of SIMS ion beam pulses;
(ii) generate a data set that comprises the measurements of the abundance of said one or more mass tags; and
(iii) output the data set; and

b) a computer comprising an analysis module that analyzes said data set.

Assignees

• Univ Leland Stanford Junior

Inventors

• Nolan Garry P
• Bendall Sean C
• Angelo Robert M

Classifications

• G01N2458/15

  Non-radioactive isotope labels, e.g. for detection by mass spectrometry · CPC title

• G01N33/5005

  involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025) · CPC title

• H01J49/14

  using particle bombardment, e.g. ionisation chambers · CPC title

• G01N33/6848

  Methods of protein analysis involving mass spectrometry · CPC title

• H01J49/0036

  Step by step routines describing the handling of the data generated during a measurement · CPC title