Single cell selection and isolation

1 . A method for isolating cells, the method comprising

providing a sample with cells containing a phototagging agent comprising a caged fluorophore moiety, optionally conjugated to a photosensitizer moiety;

imaging the sample to identify at least one target cell to be isolated;

selectively irradiating the identified target cell in the sample with photo-activating light for selectively activating the phototagging agent in the target cell to change its fluorescence response; and

isolating the irradiated target cell from other cells in the sample based on a difference in its fluorescence response compared to non-activated phototagging agent in the other cells.

2 . The method according to claim 1 , wherein the sample is three dimensional sample with multiple layers of cells along a beam direction and the phototagging agent is activated by multi-photon absorption exclusively at a focal spot of the photo-activating light in a subset of one or more of the layers.

3 . The method according to claim 1 , wherein the phototagging agent comprises the photosensitizer moiety, and the photo-activating light is in a wavelength range above 360 nm to activate the photosensitizer moiety.

4 . The method according to claim 1 , wherein isolating the target cell comprises

illuminating cells from the sample with probe light

measuring a fluorescence response of the illuminated cells; and

physically separating the target cell from other cells in the sample based on a difference in the fluorescence response of the target cell compared to the other cells.

5 . The method according to claim 1 , using a microscope system comprising

a sample holder configured to hold the sample with cells containing the phototagging agent;

one or more image light sources configured to illuminate the sample with imaging light;

at least one light detector configured to detect a sample image of the illuminated sample for spatially resolving cells in the sample;

a controller configured to

determine a target selection of an imaged target cell based on the sample image, and

determine target coordinates of a corresponding target cell in the sample based on the target selection;

one or more photo-activation light sources configured to generate photo-activating light for activating the phototagging agent to change its fluorescence response; and

beam patterning and/or steering optics configured to

receive the target coordinates of the target cell, and

selectively direct the photo-activating light to irradiate a target location of the target cell in the sample based on the target coordinates.

6 . The method according to claim 5 , wherein at least one of the photo-activation light sources comprises a pulsed laser which is focused at a specific location inside the sample for photo-activating the phototagging agent by multi-photon absorption, wherein the sample holder comprises a moveable stage configured to vary a coordinate of the laser focus in the sample along an optical axis of the microscope system based on at least one of the target coordinates of the target cell.

7 . The method according to claim 6 , wherein the microscope system comprises a light patterning device arranged in a light path between at least one of the image light sources and the sample holder for applying a pattern of the imaging light, wherein the microscope system is configured to reconstruct a 3D image based on structured illumination imaging or Hadamard imaging method.

8 . The method according to claim 7 , wherein the light patterning device is further arranged in a light path between at least one of the photo-activation light sources and the sample holder for selectively applying the photo-activating light onto the selected target cell of a two-dimensional sample.

9 . The method according to claim 5 , wherein the controller is configured to

analyze one or more sample images to automatically identify and/or track one or more target cells based on a predetermined criterion, and

control the beam patterning and/or steering optics to direct the photo-activating light to irradiate one or more target locations in the sample corresponding to the one or more target cells.

10 . The method according to claim 5 , wherein the microscope system comprises

a sensor configured to measure a fluorescence response from cells in the sample; and

a mechanism configured to physically isolate the irradiated target cell from other cells in the sample based on a difference in its fluorescence response compared to non-activated phototagging agent in the other cells.

11 . The method according to claim 1 , wherein the phototagging agent is cell permeable.

12 . The method according to claim 11 , wherein the photosensitizer moiety is configured to absorbs light of a wavelength 360 nm-420 nm, while the caged fluorophore moiety is essentially insensitive to light of a wavelength >400 nm.

13 . The method according to claim 11 , wherein

the caged fluorophore moiety is based on a fluorophore selected from the group consisting of fluorone-based fluorophores, coumarin-based fluorophores, cyanine-based fluorophores, DCDHF-based fluorophores, and combination thereof; and

the photosensitizer moiety is based on a photosensitizer selected from the group consisting thioxanthones, thiopyrylium, eosin, acridinedione derivatives, and combinations thereof.

14 . The method according to claim 11 , wherein the phototagging agent has a structure according to any one of structures I, II, III, or IV,

wherein a core of said structure represents the caged fluorophore moiety,

R 1 and R 2 individually represent a cage moiety and individually comprise a photo-liable group,

R 3 represents the photosensitizer moiety, and

each R 4 is individually selected from the group consisting of H, halide, C 1 -C 6 -alkyl, C 1 -C 6 -alkoxy, C 1 -C 6 -alkylenes and C 1 -C 6 -alkoxylenes;

wherein R 3 comprises the structure V,

wherein n is an integer >0.

15 . The method according to claim 11 , wherein the phototagging agent is selected from the group consisting of chemical compounds A, B and C

and combinations thereof.

16 . The method of claim 1 , wherein the phototagging agent is formed by a chemical compound inside the cells of the sample, the chemical compound comprising a caged fluorophore moiety conjugated to a photosensitizer moiety.

17 . The method of claim 16 , wherein the photosensitizer moiety absorbs light of a wavelength 360 nm-420 nm, while the caged fluorophore moiety is insensitive to light of a wavelength >400 nm.

18 . The method of claim 16 , wherein

the caged fluorophore moiety is based on a fluorophore selected from the group consisting of fluorone-based fluorophores, coumarin-based fluorophores, cyanine-based fluorophores, DCDHF-based fluorophores, and combination thereof; and

the photosensitizer moiety is based on a photosensitizer selected from the group consisting thioxanthones, thiopyrylium, eosin, acridinedione derivatives, and combinations thereof.

19 . The method of claim 16 , wherein the chemical compound has a structure according to any one of structures I, II, III or IV,

wherein a core of said structure represents the caged fluorophore moiety,

R 1 and R 2 individually represent a cage moiety and individually comprise a photo-liable group;

R 3 represents the photosensitizer moiety, and

each R 4 is individually selected from the group consisting of H, halide, C 1 -C 6 -alkyl, C 1 -C 6 -alkoxy, C 1 -C 6 -alkylenes and C 1 -C 6 -alkoxylenes;

wherein R 3 comprises the structure V,

wherein n is an integer >0.

20 . The method of claim 16 , wherein the chemical compound is selected from the group consisting of chemical compounds A, B and C

and combinations thereof.