I hope you’re doing well, haven’t seen each other in a while. As far as receptor tracking in slices go, we’ve not progressed much. As you’ve done, we routinely use FRAP of phluorin-tagged receptors to evaluate mobility in slices, and this works well, except for the over-expression issue.
As for quantum dot tracking in slices, our own trials have been quite unsuccessful, most QDs being generally too sticky and not diffusing well in tissue. Thus, as for tracking endogenous receptors, I think it’s quite hopeless. I do have seen in a few other labs people using GFP tagged proteins and managing to track them with anti-GFP coated QDs, but I have no direct experience with this approach as if I’m to use a tagged receptor, I prefer then to use FRAP in slice as it’s less prone to artifacts I think.
Sorry I can’t [[help]] more, sure I’d wish we could do that……
Error creating thumbnail: File missingSong and Huganir 2002. Use of yeast two-hybrid method to identify AMPA-receptor-interacting proteins was crucial for the rapid progress in this field and has helped to identify a large complex of such proteins. AMPA-receptor-associated protein complex. AMPA receptors are associated with a large protein network. The GluR2 subunit specifically binds to several proteins, including glutamatereceptor- interacting protein (GRIP) 1, GRIP2, protein interacting with C kinase (PICK1) and N-ethylmaleimide sensitive factor (NSF). GRIP1, GRIP2 and PICK1 in turn bind to other proteins, including GRIP-associated proteins (GRASPs), EphB receptor tyrosine kinases, ephrins, kinesin 5 (KIF5) and protein kinase Cα (PKCα). The GluR1 subunit binds to synapse-associated protein 97 (SAP97, also known as hDLG) and protein 4.1 (4.1 N). All four AMPA-receptor subunits bind to neuronal activity regulated pentraxin (NARP) and stargazin. Stargazin binds, in turn, to the synaptic scaffolding protein postsynaptic density 95 (PSD95). These associated proteins appear to play an important role in the membrane trafficking of the receptors by escorting the receptor from the cell body to the synapse. In addition, this large complex might regulate novel downstream signal transduction pathways that emanate from the AMPA receptor. Abbreviations: CC, coiled-coil domain; GK, guanylate kinase domain; PDZ, PSD95/Dlg/ZO1 domain; SH3, SRC homology 3 domain.
There are two major groups of methods for functional super-resolution microscopy:
1. Deterministic super-resolution: The most commonly used emitters in biological microscopy, fluorophores, show a nonlinear response to excitation, and this nonlinear response can be exploited to enhance resolution. These methods include STED, GSD, RESOLFT and SSIM.
2. Stochastical super-resolution PALM STORM: The chemical complexity of many molecular light sources gives them a complex temporal behaviour, which can be used to make several close-by fluorophores emit light at separate times and thereby become resolvable in time. These methods include SOFI and all single-molecule localization methods (SMLM) such as SPDM, SPDMphymod, PALM, FPALM, STORM and dSTORM.
