Methods and compositions relating to optical super-resolution patterning
This disclosure provides methods for generating super-resolution patterns of molecules on substrates. In one aspect, disclosed herein is a method comprising contacting a plurality of transiently binding nucleic acid probes to their respective targets wherein the targets are immobilized on a substrate, detecting a binding event in a select region or set of select regions within a diffraction limited region of the substrate, and irradiating the diffraction limited region of the substrate, wherein the probes comprise a photocrosslinker. In one embodiment, the photocrosslinker is 3-cyanovinylcarbazole.
1. A method comprising
contacting a plurality of transiently binding nucleic acid probes to their respective targets, wherein the targets are located within 200 nm of each other and immobilized in a select region of a substrate, and
detecting binding of a probe to a target in the select region of the substrate, and
then irradiating the select region of the substrate to immobilize the probe to the select region of the substrate,
wherein the probes comprise a photocrosslinker and a fluorophore.
2. The method of claim 1 , wherein the fluorophore emits at least 10 4 -10 6 photons, optionally wherein the fluorophore is ATTO655.
3. The method of claim 1 , wherein the probes further comprise a functional group or a moiety.
4. The method of claim 3 , wherein the functional group or the moiety is a chemical handle.
5. The method of claim 3 , wherein the functional group or the moiety is biotin, avidin, or a nanoparticle.
6. The method of claim 3 , wherein the functional group or the moiety is an alkyne or azide.
7. The method of claim 1 , wherein the select region of the substrate is irradiated with 366 nm to 405 nm light for less than 1 second or less than 0.5 seconds.
8. The method of claim 1 , wherein the select region of the substrate is a select area or a select volume of the substrate.
9. The method of claim 1 , wherein the photocrosslinker is 3-cyanovinylcarbazole.
10. The method of claim 1 , wherein the method is automated.
11. The method of claim 1 , wherein the binding of the probe to the target is detected using a CCD or EMCCD camera.
12. The method of claim 1 , wherein the select region of the substrate is irradiated using a laser spot illuminator or a DMD array.
13. The method of claim 1 , wherein the transiently binding nucleic acid probes have a length of 8 or 9 nucleotides.
14. The method of claim 1 , wherein the target is or is conjugated to a nucleic acid having a length of about 8 or 9 nucleotides.
15. A method comprising
contacting a plurality of transiently binding nucleic acid probes to their respective targets located within 200 nm of each other and immobilized in a select region of a substrate,
detecting binding of a probe to a target in the select region of the substrate, and
then irradiating the select region of the substrate,
wherein the probes are hairpin probes that comprise a photocleavable linker and a fluorophore.
16. The method of claim 15 , wherein the fluorophore emits at least 10 4 -10 6 photons, optionally wherein the fluorophore is ATTO655.
17. The method of claim 15 , wherein the probes further comprise a functional group or a moiety.
18. The method of claim 17 , wherein the functional group or the moiety is a chemical handle.
19. The method of claim 17 , wherein the functional group or the moiety is biotin, avidin, or a nanoparticle.
20. The method of claim 17 , wherein the functional group or the moiety is an alkyne.
21. The method of claim 15 , wherein the select region of the substrate is irradiated with light having a wavelength of equal or less than 405 nm for less than 1 second or less than 0.5 seconds.
22. The method of claim 15 , wherein the select region of the substrate is a select area or a select volume of the substrate.
23. The method of claim 15 , wherein the photocleavable linker comprises 1-(2-nitrophenyl)ethyl.
24. The method of claim 15 , wherein the method is automated.
25. The method of claim 15 , wherein the binding of the probe to the target is detected using a CCD camera.
26. The method of claim 15 , wherein the select region of the substrate is irradiated using a laser spot illuminator or a DMD array.