CELL ANALYSIS USING DYNAMIC BIOPHYSICAL METHODS

• US 20160238610A1
• Filed: 02/16/2016
• Published: 08/18/2016
• Est. Priority Date: 02/16/2015
• Status: Active Grant

• Current Assignee: BAR ILAN University
• Original Assignee: BAR ILAN University
• Inventors: GARINI, Yuval, BRONSHTEIN BERGER, Irena, KEPTEN, Eldad
• Granted Patent: US 9,885,705 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: G01N 15/1427 with the synchronisation of components, a time gate for operation of components, or suppression of particle coincidences
• CPC Class Codes: G01N 15/1429 Signal processing
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1488 Methods for deciding
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 2333/4712 Muscle proteins, e.g. myosin, actin, protein
• CPC Class Codes: G01N 2333/66 Thymopoietins
• CPC Class Codes: G01N 2333/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• CPC Class Codes: G01N 33/5029 on cell motility

Abstract

A method for identifying the expression or activity of a nuclear protein in a cell, the method comprising: analyzing multiple images of one or more labeled genetic entities; determining from said analysis, a motile property of said one or more labeled genetic entities; and identifying an expression or activity of a nuclear protein of said cell associated with said motile property.

Claims

1. A method for identifying the expression or activity of a protein in a cell, the method comprising using at least one hardware processor for:

analyzing multiple images of one or more labeled genetic entities;

determining from said analysis, a motile property of said one or more labeled genetic entities; and

identifying an expression or activity of a protein of said cell associated with said motile property.

2. The method of claim 1, wherein said genetic entities are selected from the group consisting of:

telomeres, centromeres, a specific gene locus, nuclear bodies, a nucleolus, and one or more proteins bound thereto.

3. The method of claim 1, wherein said protein is different than said genetic entity.

4. The method of claim 1, wherein said protein is selected from the group consisting of Lamin A, Lamin B, Lamin C, Lap2α

, Lap2-beta, BAF, actin and emerin.

5. The method of claim 1, wherein analyzing said multiple images further comprises eliminating a rotational and/or translational movement attributed to a nucleus of said cell.

6. The method of claim 1, wherein said motile property comprises a diffusion characteristic of said labeled genetic entities, wherein determining said diffusion characteristic comprises calculating a space function with respect to time,

wherein said space function is derived from multiple path trajectories calculated from said multiple images of said one or more labeled genetic entities, and is selected from any of:

a) a mean square distance (MSD) function and b) a mean square volume (MSV) function.

7. The method of claim 6, wherein said identifying comprises applying to said space function a rule that associates any of:

a) a linear property of said space function with a normal diffusion characteristic for said labeled genetic entities and a depletion of a concentration of said protein, b) a logarithmic property of said space function with an anomalous sub diffusion characteristic for said labeled genetic entities and a normal concentration of said protein, c) an exponentially increasing property of said space function with an anomalous subdiffusion or superdiffusion characteristic for said protein, and d) a linear property of said space function for a first time period and a logarithmic property of said space function for a second time period with a normal diffusion characteristic for said labeled genetic entities within a restricted volume of said cell.

8. A system for determining a cell characteristic, the system comprising:

a microscope configured to magnify one or more labeled genetic entities;

a camera configured to

capture multiple images of said one or more labeled genetic entities, wherein said camera is coupled to said microscope; and

a processor configured to

analyze said multiple images;

determine from said analysis, a motile property of said one or more labeled genetic entities; and

identify an expression or activity of a nuclear protein of said cell associated with said motile property.

9. The system of claim 8, wherein said processor is further configured to analyze said captured images by eliminating a rotational and/or translational movement attributed to a nucleus of said cell.

10. The system of claim 8, wherein said processor is configured to determine said diffusion characteristic by calculating a space function with respect to time.

11. The system of claim 8, wherein said genetic entities are selected from the group consisting of:

telomeres, centromeres, a specific gene locus, nuclear bodies, a nucleolus, and one or more proteins bound thereto.

12. The system of claim 8, wherein said protein is different than said genetic entity, and wherein said protein is selected from the group consisting of Lamin A, Lamin B, Lamin C, Lap2α

, Lap2-beta, BAF, actin and emerin.

13. The system of claim 8, wherein said motile property comprises a diffusion characteristic of said labeled genetic entities.

14. The system of claim 8, wherein said space function is derived from multiple path trajectories calculated from said multiple images of said one or more labeled genetic entities.

15. The system of claim 8, wherein said space function is selected from a mean square distance (MSD) function and/or a mean square volume (MSV) function.

16. The system of claim 8, wherein said identifying comprises applying to said space function a rule that associates any of:

a) a linear property of said space function with a normal diffusion characteristic for said labeled genetic entities and a depletion of a concentration of said protein, b) a logarithmic property of said space function with an anomalous sub diffusion characteristic for said labeled genetic entities and a normal concentration of said protein, c) an exponentially increasing property of said space function with an anomalous subdiffusion or superdiffusion characteristic for said protein, and d) a linear property of said space function for a first time period and a logarithmic property of said space function for a second time period with a normal diffusion characteristic for said labeled genetic entities within a restricted volume of said cell.

17. A method for determining the ratio of bound to unbound molecules in a cell, comprising:

obtaining, from a selected area of a cell, data comprising time-resolved emitted light intensity measurements and time-resolved fluorescent lifetime measurements of fluorescently labeled bound molecules and fluorescently labeled unbound molecules;

determining from the obtained data

     1) a continuous photobleaching (CP) curve and

     2) fluorescent lifetime histograms for each of the time-resolved emitted light intensity measurements and fluorescent lifetime measurements; and

calculating a ratio of the fluorescently labeled bound molecules to the fluorescently labeled unbound molecules in the selected regions using the CP curve and the fluorescent lifetime histograms.

18. The method of claim 17, further comprising:

fluorescently labeling the molecules;

selecting the area;

illuminating the selected area with light corresponding to the molecules'"'"' absorption spectrum;

detecting the light intensity emitted from the illuminated area; and

measuring the fluorescent lifetimes of the bound molecules to the unbound molecules.

19. The method of claim 18, further comprising synchronizing the illumination and detection steps, wherein synchronizing comprises applying any of a gating technique, time correlated single photon counting technique, and phase modulation technique.

20. The method of claim 17, wherein calculating further comprises correlating the CP curve with the fluorescent lifetime histograms over time.

21. The method of claim 17, wherein illuminating comprises emitting a laser pulse having a duration ranging between 1 and 1000 picoseconds.

22. The method of claim 17, wherein the molecules are fluorescently labeled using fluorescing molecules selected from the group consisting of molecular probes, fluorescent proteins, quantum dots, metallic particles, and a dye measurable via bright field microscopy.

23. The method of claim 18, wherein the measuring step comprises determining any of the fluorescent light intensity and an emitted photon count per a predetermined time unit.

24. The method of claim 23, further comprising repeating performing said obtaining, determining and calculating steps for a time window ranging from one millisecond to one hundred seconds.

25. The method of claim 18 wherein measuring the light intensity comprises optically filtering the spectral range of the excitation spectral band from the emission spectral band of the emitted light.