Sub-diffraction limit image resolution and other imaging techniques
The present invention generally relates to sub-diffraction limit image resolution. In one aspect, the invention is directed to determining and/or imaging light from two or more entities separated by a distance less than the diffraction limit of the incident light. In one set of embodiments, the entities may be selectively activatable, i.e., one entity can be activated to produce light, without activating other entities. The emitted light may be used to determine the positions of the first and second entities, for example, using Gaussian fitting or other mathematical techniques, and in some cases, with sub-diffraction limit resolution. The methods may thus be used, for example, to determine the locations of two or more entities immobilized relative to a common entity, for example, a surface, or a biological entity such as DNA, a protein, a cell, a tissue, etc.
1. A photoswitchable imaging composition, comprising:
a light-emitting molecular entity, capable of being switched between a first state able to emit light at a first, emission wavelength upon excitation and a second state that does not substantially emit light at the first wavelength upon excitation,
wherein the light-emitting entity comprises a first portion that is capable of emitting light at the first wavelength upon excitation when the light-emitting entity is in the first state, and a second portion that activates the first portion upon exposure to an external stimulus, thereby placing the light-emitting entity in the first state,
wherein the first portion is Cy5, Cy5.5, Cy7, Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, DiD, DiR, YOYO-3, YO-PRO-3, TOTO-3, or TO-PRO-3, and wherein the second portion is Alexa Fluor 405, Cy2, Alexa Fluor 488, Cy3, Cy3B, or Cy3.5, and
wherein the first portion and the second portion are each bonded to a common antibody and are not bonded directly to each other.
2. The imaging composition of claim 1 , wherein the second portion activates the first portion upon exposure to light having an activation wavelength.
3. The imaging composition of claim 1 , wherein the first portion is joined to the common antibody via a succinimide-amine linkage.
4. The imaging composition of claim 1 , wherein the second portion is joined to the common antibody via a succinimide-amine linkage.
5. The imaging composition of claim 1 , wherein the first portion is joined to the common antibody via a maleimide-thiol linkage.
6. The imaging composition of claim 1 , wherein the second portion is joined to the common antibody via a maleimide-thiol linkage.
7. The imaging composition of claim 1 , wherein the first portion and/or the second portion is joined to the common antibody via an iodoacetamide-thiol linkage.
8. The imaging composition of claim 1 , wherein the first portion and/or the second portion is joined to the common antibody via an amine-carboxylic acid linkage.
9. The imaging composition of claim 1 , wherein the first portion and/or the second portion is joined to the common antibody via a hydrazide-aldehyde linkage.
10. The imaging composition of claim 1 , where the first portion and/or the second portion is joined to the common antibody via a hyrdazide-ketone linkage.