Super-Resolution Immunofluorescence with Diffraction-Limited Preview

Abstract

An imaging agent comprises (a) at least one target recognition moiety; (b) at least one observable moiety non-transiently bound to the target recognition moiety, and (c) at least one docking moiety bound to the target recognition moiety, wherein the docking moiety is capable of transiently binding at least one observable moiety. In some embodiments, the at least one target recognition moiety is an antibody or antigen binding fragment thereof.

1 . An imaging agent comprising:
 a. at least one target recognition moiety;   b. at least one first observable moiety non-transiently bound to the target recognition moiety, and   c. at least one docking moiety bound to the target recognition moiety, wherein the docking moiety is capable of transiently binding at least one second observable moiety.   
     
     
         2 . The imaging agent of  claim 1 , wherein the at least one target recognition moiety is an antibody or antigen binding fragment thereof. 
     
     
         3 . The imaging agent of  claim 1 , wherein the first observable moiety non-transiently bound to the target recognition moiety is a signal-emitting moiety. 
     
     
         4 . The imaging agent of  claim 3 , wherein the signal-emitting moiety is a fluorophore. 
     
     
         5 . The imaging agent of  claim 1 , wherein the first observable moiety non-transiently bound to the target recognition moiety is directly attached to the target recognition moiety. 
     
     
         6 . The imaging agent of  claim 1 , wherein the first observable moiety non-transiently bound to the target recognition moiety is indirectly attached to the target recognition moiety. 
     
     
         7 . The imaging agent of  claim 6 , wherein the first observable moiety non-transiently bound to the target recognition moiety is directly attached to the docking moiety. 
     
     
         8 . The imaging agent of  claim 1 , wherein the first observable moiety non-transiently bound to the target recognition moiety is bound covalently. 
     
     
         9 . The imaging agent of  claim 1 , wherein the docking moiety is attached to the imaging agent covalently. 
     
     
         10 . The imaging agent of  claim 1 , wherein the first observable moiety non-transiently bound to the target recognition moiety is bound noncovalently. 
     
     
         11 . The imaging agent of  claim 1 , wherein the docking moiety is attached to the imaging agent noncovalently. 
     
     
         12 . The imaging agent of  claim 11 , wherein
 a. the nucleic acid bound to the first observable moiety is longer than the nucleic acid bound to the target recognition moiety, or   b. the nucleic acid bound to the target recognition moiety is longer than the nucleic acid bound to the first observable moiety, and   
       the longer nucleic acid serves as a toehold for displacement of the shorter strand. 
     
     
         13 . The imaging agent of  claim 1 , wherein the docking moiety comprises nucleic acids. 
     
     
         14 . The imaging agent of  claim 1 , wherein the target recognition moiety is an antibody or antigen binding fragment thereof, an aptamer, or an oligonucleotide. 
     
     
         15 . The imaging agent of  claim 1 , wherein both the first observable moiety non-transiently bound to the target recognition moiety and the second observable moiety transiently bound to the target recognition moiety are attached to a single target recognition moiety. 
     
     
         16 . The imaging agent of  claim 1 , wherein the first observable moiety non-transiently bound to the target recognition moiety and the second observable moiety transiently bound to the docking moiety attached to the target recognition moiety are attached to different target recognition moieties, wherein:
 a. the different target recognition moieties bind to the same epitope on a target;   b. the different target recognition moieties bind to different epitopes on the same target; or   c. the different target recognition moieties bind to different proteins, optionally wherein the proteins are capable of interacting with each other.   
     
     
         17 - 21 . (canceled) 
     
     
         22 . The imaging agent of  claim 10 , wherein the first observable moiety non-transiently bound to the target recognition moiety is bound through a nucleic acid-nucleic acid interaction. 
     
     
         23 . The imaging agent of  claim 22 , wherein
 d. the nucleic acid bound to the first observable moiety is longer than the nucleic acid bound to the target recognition moiety, or   e. the nucleic acid bound to the target recognition moiety is longer than the nucleic acid bound to the first observable moiety, and   
       wherein the longer nucleic acid serves as a toehold for displacement of the shorter strand.