AI Article Synopsis
- The IRIS technique enables advanced super-resolution imaging by using fast-exchanging fluorescent probes that can label many targets at once.
- Previous methods for creating specific probes were time-consuming, but a new mutagenesis strategy has been developed to streamline this process across various antibody sequences.
- The research successfully created IRIS probes from several antibody types, allowing for detailed visualization of proteins in neurons, enhancing the ability to study small synaptic connections.
Article Abstract
Image reconstruction by integrating exchangeable single-molecule localization (IRIS) achieves multiplexed super-resolution imaging by high-density labeling with fast exchangeable fluorescent probes. However, previous methods to develop probes for individual targets required a great amount of time and effort. Here, we introduce a method for generating recombinant IRIS probes with a new mutagenesis strategy that can be widely applied to existing antibody sequences. Several conserved tyrosine residues at the base of complementarity-determining regions were identified as candidate sites for site-directed mutagenesis. With a high probability, mutations at candidate sites accelerated the off rate of recombinant antibody-based probes without compromising specific binding. We were able to develop IRIS probes from five monoclonal antibodies and three single-domain antibodies. We demonstrate multiplexed localization of endogenous proteins in primary neurons that visualizes small synaptic connections with high binding density. It is now practically feasible to generate fast-dissociating fluorescent probes for multitarget super-resolution imaging.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9606137 | PMC |
| http://dx.doi.org/10.1016/j.crmeth.2022.100301 | DOI Listing |
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