Optical selection of cells
Provided herein are photochemical crosslinkers and photocleavable crosslinkers and their uses in methods for cell selection from cell cultures. The photochemical crosslinkers comprise a fluorescent dye and a radical generator. The photocleavable crosslinkers comprise a photocleavable linker linking two electrophilic groups to each other. Also provided are systems for imaging cells comprising a plurality of cells crosslinked to extracellular matrix proteins using a crosslinker as described, an imaging apparatus, an illuminating apparatus, and software for image processing.
1. A method of selecting cells with features of interest comprising:
a) providing a culture of cells in a culture dish;
b) imaging the cells to identify a cell of interest;
c) adding a photochemical crosslinker comprising a fluorescent dye linked to a radical generator to the culture of cells; and
d) illuminating the cell of interest with light so that the radical generator produces a radical which covalently cross-links the cell of interest to the surface of the culture dish.
2. The method of claim 1 further comprising washing away cells that are not attached to the surface of the culture dish through the crosslinker.
3. The method of claim 2 , wherein the step of washing away cells comprises incubating the culture of cells with an enzyme.
4. The method of claim 3 , wherein the enzyme is a proteolytic enzyme.
5. The method of claim 3 , wherein the enzyme is an enzyme of marine origin having proteolytic and collagenolytic activity.
6. The method of claim 3 , wherein the enzyme istrypsin.
7. The method of claim 1 further comprising isolating the cell of interest from the culture dish.
8. The method of claim 1 , wherein the culture dish is coated with cell adhesion proteins.
9. The method of claim 8 , wherein the cell adhesion protein is an extracellular matrix protein.
10. The method of claim 9 , wherein the extracellular matrix protein is fibronectin, collagen, laminin, fibrillin, vitronectin, thrombospondins, tenascins, entactins (or nidogens), nephronectin, or fibrinogen, osteopontin, agrin, aggrecan, decorin, F-Spondin, matrix extracellular phosphoglycoprotein (MEPE), nidogen-1, testican, poly-L-lysine, poly-D-lysine, poly-L-orinthine, or a combination thereof.
11. The method of claim 1 , wherein the step of illuminating the cell of interest comprises using patterned illumination.
12. The method of claim 11 , wherein the patterned illumination is performed with a digital micromirror device, galvanometer mirror, acousto-optical beam deflector, or spatial light modulator.
13. The method of claim 1 , wherein the step of illuminating the cell of interest comprises illuminating the cell with light having a wavelength of about 360 nm to about 440 nm.
14. The method of claim 1 , wherein the step of illuminating the cell of interest comprises illuminating the cell with light having a wavelength of about 440 nm to about 500 nm.
15. The method of claim 1 , wherein the step of imaging comprises imaging the cell with a wide-field optical system comprising an objective; a means of illumination; and a camera.
16. The method of claim 15 , wherein the means of illumination is fluorescent illumination.
17. The method of 15 , wherein the means of illumination utilizes transmitted light.
18. The method of claim 1 , wherein the step of imaging is performed with a high-speed camera.
19. The method of claim 1 , wherein the step of imaging is performed using epifluorescensce microscopy, confocal microscopy, differential interference contrast microscopy, phase contrast microscopy, or Raman microscopy.
20. The method of claim 1 further comprising continuing to grow the cell of interest.
21. The method of claim 1 further comprising fixing the cell of interest.
22. The method of claim 1 further comprising removing the cell of interest from the culture.
23. The method of claim 1 , wherein the cell of interest is subjected to DNA sequencing, RNA sequencing, or proteomic analysis.