METHOD FOR DETECTING CELLS

• US 20160069903A1
• Filed: 09/10/2014
• Published: 03/10/2016
• Est. Priority Date: 09/10/2014
• Status: Abandoned Application

• Current Assignee: Fundacio Centre De Regulacio Genomica, Institucio Catalana De Recerca I Estudis Avancats
• Original Assignee: Fundacio Centre De Regulacio Genomica, Fundacio Institut de Ciencies Fotoniques, Institucio Catalana De Recerca I Estudis Avancats
• Inventors: COSMA, Maria Pia, LAKADAMYALI, Melike, MANZO, Carlo, RICCI, Maria Aurelia
• Application Number:
  US14/482,586
• Publication Number:
  US 20160069903A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 2201/12 Circuits of general importance; Signal processing
• CPC Class Codes: G01N 2333/47 Assays involving proteins of known structure or function as defined in the subgroups
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G01N 33/6875 Nucleoproteins
• CPC Class Codes: G06T 7/0012 Biomedical image inspection

Abstract

The present invention relates to methods for detecting the chromatin state of a cell based on recording a super resolution image of nucleosome organization and correlating said imaged with size of nucleosomal clutches, nucleosomal density and/or number of nucleosomes per nucleosomal clutches. Additionally, the invention relates to a kit comprising a first antibody capable of specifically binding to a histone protein and a photo switchable fluorophore linked-secondary antibody and the use of the kit of the invention for detecting the chromatin state of a cell and isolating a cell in an open chromatin state or in a close chromatin state. The invention also relates to a device adapted to detect the chromatin state of a cell.

Claims

1. A method for detecting the chromatin state of a cell comprising

a) contacting a sample containing cells with a first antibody capable of specifically binding to a histone protein,

b) contacting the antibody:

histone complex formed in step a) with a secondary antibody having at least one photoswitchable fluorophore adapted to be optically excited at a certain wavelength λ

₂ and to emit light at a wavelength λ

₂ different from λ

₂,

c) recording a super resolution image of nucleosome organization by means of a sensor being sensitive at least to the wavelength of emission of the photoswitchable fluorophore by exciting the sample with an optical radiation having a wavelength λ

₂,

d) correlating the image obtained in step c) with size of nucleosomal clutches, nucleosomal density and/or number of nucleosomes per nucleosomal clutches, and

e) comparing data obtained in step d) with a corresponding reference value to obtain a score based on size of nucleosomal clutches, nucleosomal density and/or number of nucleosomes per nucleosomal clutch,

wherein if the cell comprises smaller clutches, less densely compacted nucleosomes or less nucleosomes per clutches compared to the corresponding reference value then it is indicative that said cell is in an open chromatin state and wherein if the cell comprises bigger clutches, more densely compacted nucleosomes or more nucleosomes per clutches compared to the corresponding reference value then it is indicative that said cell is in a closed chromatin state.

3. The method according to claim 1, wherein the secondary antibody further comprises a second fluorophore adapted to be optically excited at a wavelength λ

₃ and reactivate the first fluorophore by bringing it from its dark state back to its ground state, upon which the first fluorophore can be excited again at its excitation wavelength λ

₂ and emit light at its emission wavelength λ

₂.

4. The method according to claim 3, wherein a plurality of super resolution images are taken by means of a sensor being sensitive at least to the wavelength of emission of the second fluorophore λ

₂ rendering a further super resolution image by collecting the sensed light emissions recorded in the plurality of images.

5. The method according to claim 4, wherein the power of the optical radiation having a wavelength λ

₂ is monotonically increased.

6. The method according to claim 4, wherein, before recording each super resolution image of the plurality of super resolution images, the sample is excited one or more times with an optical radiation having a wavelength λ

₂ and subsequently excited one or more times with an optical radiation having a wavelength λ

₂.

7. The method according to claim 4, wherein the super resolution image is rendered from a list of locations (x,y) determined as the coordinates in the sample where an optical emission of a photoswitchable fluorophore adapted to emit light at a wavelength λ

₂ is present.

8. The method according to claim 7, wherein

a density image of resolution lower than or equal to the rendered high resolution image and representing the same area as said rendered high resolution image is provided wherein each pixel of the density image has a value proportional to the number of locations of the location list falling within the area represented by said pixel,

a binary image representing the same area as the density image comprising zero value pixels if the corresponding value represented by the density image in the same location is lower than a predefined threshold;

and, nonzero if said value is higher, is provided,

identifying connected regions of pixels representing values higher than the predefined threshold,

for each connected region, providing a list of clutch positions by grouping the localization coordinates within said connected region according to a distance-based criterion being the position of the clutch the centroid position of the localization coordinates associated with said clutch.

9. The method according to claim 8, wherein the size of each clutch is calculated as a measure of the spreading of the positions of all the localization coordinates associated with said clutch and/or the number of nucleosomes within said clutch.

10. The method according to claim 8, wherein the density of nucleosomes within a clutch calculated as the number of nucleosomes within that clutch divided by the area occupied by said clutch.

11. The method according to claim 1, wherein the histone protein is H2B.

12. A method for isolating a cell in an open chromatin state comprising

a) detecting the chromatin state of a cell by a method according to claim 1, and

b) isolating a cell having smaller clutches, less densely compacted nucleosomes or less nucleosomes per clutches.

13. The method for isolating a cell in an open chromatin state according to claim 12 wherein the cell in an open chromatin state is selected from the group consisting of transcriptionally active cell, pluripotent cell, cancer cell and drug-perturbed cell.

14. The method for isolating a cell in a close chromatin state

a) detecting the chromatin state of the cell by a method according to claim 1, and

b) isolating a cell having bigger clutches, more densely compacted nucleosomes or more nucleosomes per clutches.

18. A device adapted to detect the chromatin state of a cell comprising

a source of optical radiation adapted to emit light at a wavelength, over an interrogation area adapted to receive a biological sample,

an optical sensor sensible to a second wavelength adapted to measure the optical radiation at λ

₂,

a control unit connected to the optical sensor and to the source of optical radiation wherein said control unit is adapted to carry out the method according to claim 1.

19. A device adapted to detect the chromatin state of a cell comprising

a first source of optical radiation adapted to emit light at a wavelength λ

₂ over an interrogation area adapted to receive a biological sample,

a further second source of optical radiation adapted to emit light at a wavelength λ

₂ over an interrogation area adapted to receive a biological sample,

an optical sensor sensible to a second wavelength λ

₂ adapted to measure the optical radiation at λ

₂,

a control unit connected to the optical sensor and to the first and to the second source of optical radiation wherein said control unit is adapted to carry out the method according to claim 3.

2. The method according to claim wherein the cell in an open chromatin state is selected from the group consisting of a transcriptionally active cell, a pluripotent stem cell, a cancer cell and a drug perturbed cell.

15. A kit comprising a first antibody capable of specifically binding to a histone protein and a photoswitchable fluorophore linked-secondary antibody.

16. The kit according to claim 15, wherein the histone protein is histone H2B.

17. Use of the kit according to claim 15 for detecting the chromatins state of a cell and isolating a cell in an open chromatin state or in a close chromatin state.