Cellular activity quantification using labeled probes

Patent metadata

Field	Value
Publication number	US-10083341-B2
Application number	US-201514946535-A
Country	US
Kind code	B2
Filing date	Nov 19, 2015
Priority date	Nov 19, 2014
Publication date	Sep 25, 2018
Grant date	Sep 25, 2018

Abstract

Methods and systems for quantifying cellular activity using labeled probes, e.g., quantum dots, are disclosed. In one example approach, a method for quantifying cellular activity in a sample containing intact cells having labeled complexes comprises receiving images of the sample at a plurality of depths and detecting individual intact cells in the images of the sample at the plurality of depths. For each detected cell, discrete labels may be detected and localized in the cell at each depth, a total number of detected and localized labels may be calculated in the cell, and an activity level of the target molecule for the labeled probe in the cell determined.

First claim

The invention claimed is:

1. A computer-implemented method of quantifying the activity level of a target biomolecule in a sample comprising one or more intact cells, said sample having been treated with a reagent, said reagent comprising a label that can label a cellular structure such that the label can be localized within a cell, and a binding component that binds the target biomolecule, said target biomolecule exhibiting a first activity level within the cell and a second activity level within the sample:
receiving a set of images of the sample, wherein the images within the set comprise individual cells and are taken at a plurality of depths;
detecting a first cell in the images of the sample at the plurality of depths;
detecting and localizing the label at individual sites in the first cell at each depth in the plurality of depths comprising applying a spatial band-pass filter, detecting localized maxima, and calculating a position of each label in the cell at each depth in the plurality of depths, wherein detecting localized maxima is performed using centroid localization or radial symmetry localization;
calculating a total number of detected and localized labels within the first cell; and
calculating a first activity level of the target biomolecule within the cell based on the total number of detected and localized labels at individual sites in the cell;
calculating the first activity level of the target biomolecule within a plurality of cells in the sample; and
calculating the activity level of the target biomolecule within the sample based on the number of detected and localized labels in the plurality of cells.

2. The method of claim 1 , further comprising calculating a continuous probability density function of the first activity level in a subset of cells in the sample based on the total number of detected and localized labels in each cell.

3. The method of claim 2 , wherein the second activity level of the target biomolecule is calculated based on the continuous probability density function of the first activity level of a plurality cells in the sample based on the total number of detected and localized labels in each cell in the plurality of cells.

4. The method of claim 2 , wherein the continuous probability density functions is calculated using a Gaussian kernel density estimation.

5. The method of claim 1 , wherein the target biomolecule is a protein that is modified by phosphorylation and the activity of the target biomolecule comprises phosphorylation.

6. The method of claim 1 , wherein the label comprises a quantum dot and the images comprise fluorescent micrographs.

7. The method of claim 1 , wherein detecting the first cell comprises detecting a nucleus and plasma membrane of the first cell via a threshold-based intensity algorithm and a membrane expansion cell segmentation algorithm.

8. The method of claim 1 , wherein the binding component comprises an antibody or antigen binding fragment thereof or a nucleic acid molecule.

9. The method of claim 1 , wherein the images of the sample at the plurality of depths comprise z-stacks at multiple fields of view of the sample.

10. The method of claim 1 , wherein calculating the total number of detected and localized labels in each cell comprises summing pixel values corresponding to the first cell from all depths in the plurality of depths and subtracting a global approach value for each field of view.

11. A computer-implemented method of quantifying the activity level of a target biomolecule in a sample comprising one or more intact cells, said sample having been treated with a reagent, said reagent comprising a label that can label a cellular structure such that the label can be localized within a cell, and a binding component that binds the target biomolecule, said target biomolecule exhibiting a first activity level within the cell and a second activity level within the sample:
receiving a set of images of the sample, wherein the images within the set comprise individual cells and are taken at a plurality of depths, wherein the images of the sample at the plurality of depths comprise z-stacks at multiple fields of view of the sample;
detecting a first cell in the images of the sample at the plurality of depths;
detecting and localizing the label at individual sites in the first cell at each depth in the plurality of depths;
calculating a total number of detected and localized labels within the first cell, comprising summing pixel values corresponding to the first cell from all depths in the plurality of depths and subtracting a global background value for each field of view, wherein the global background value for each field of view is calculated as a mean of a minimum pixel value corresponding to each y-column of the field of view; and
calculating a first activity level of the target biomolecule within the cell based on the total number of detected and localized labels at individual sites in the cell;
calculating the first activity level of the target biomolecule within a plurality of cells in the sample; and
calculating the activity level of the target biomolecule within the sample based on the number of detected and localized labels in the plurality of cells.

12. The method of claim 11 , further comprising calculating a continuous probability density function of the first activity level in a subset of cells in the sample based on the total number of detected and localized labels in each cell.

13. The method of claim 11 , wherein the target biomolecule is a protein that is modified by phosphorylation and the activity of the target biomolecule comprises phosphorylation.

14. A method of identifying a change in activity of a target biomolecule in response to a test compound, the method comprising:
treating a first set of cells with a first concentration of the test compound;
treating a second set of cells with a negative control;
contacting the first set of cells and second set of cells with a first reagent, said first reagent comprising a first label that can label a cellular structure such that the label can be localized within the cell, and a first binding component that binds a first target biomolecule;
calculating the activity of the target molecule in the first set of cells and the second set of cells using the steps of:
a) receiving a set of images of the first set of cells and the second set of cells, wherein the images within each set comprise individual cells and are taken at a plurality of depths;
b) detecting a first cell in the images of the first set of cells and a first cell in the images of the second set of cells at the plurality of depths;
c) detecting and localizing the label at individual sites at each depth in the plurality of depths in the first cell in the first set of cells and in the first cell in the second set of cells comprising applying a spatial band-pass filter, detecting localized maxima, and calculating a position of each label in the cell at each depth in the plurality of depths, wherein detecting localized maxima is performed using centroid localization or radial symmetry localization;
d) calculating a total number of detected and localized labels within the first cell in the first set of cells and in the first cell in the second set of cells;
e) calculating a first activity level of the target biomolecule within i) a plurality of cells in the first set of cells and ii) a plurality of cells in the second set of cells;
f) calculating the activity level of the target biomolecule within the first set of cells and the second set of cells based on the number of detected and localized labels in the plurality of cells; and
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Assignees

• Vu Tania
• Jacob Thomas
• Univ Oregon Health & Science

Inventors

• Vu Tania
• Jacob Thomas

Classifications

• G06T2207/30072

  Microarray; Biochip, DNA array; Well plate · CPC title

• G06T2207/10064

  Fluorescence image · CPC title

• G06K9/00147 Primary

  Physics · mapped topic

• G01N33/5011

  for testing antineoplastic activity · CPC title

• G06K9/0014

  Physics · mapped topic