Fluorescence signal level, photobleaching and phototoxicity are major problems encountered ubiquitously in live fluorescence microscopy applications. Because of limited of fluorophore signals, long term monitoring of live cells presents a major challenge to existing fluorescent microscopy technologies. Not only does low signal level affect detection precision over the auto-fluorescence background, the associated photobleaching and phototoxicity also restrict the temporal resolution and observation duration. Inspired by the recent development of the SunTag, we propose to develop a protein labeling scheme using split-fluorescent proteins. By arranged them into tandem arrays and demonstrated proportional signal amplification. This signal amplification can not only solve the signal strength challenge, but also reduce photobleaching / phototoxicity by correspondingly lowering the excitation intensity. Specifically, we plan to develop split-FP tags for multicolor imaging and apply it to live cell imaging of chromosome organization.
To solve the photobleaching and phototoxicity problem that is ubiquitously associated with fluorescence microscopy, we plan to develop protein labeling methods based on multimerized split fluorescent proteins to amplify the signal. We will also apply it to the study of chromosome organization change after mitosis. This project will not only provide a widely applicable tool for biomedical research, but also provide understandings of key principles regulating the establishment of interphase chromosome organization.
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| Irannejad, Roshanak; Pessino, Veronica; Mika, Delphine et al. (2017) Functional selectivity of GPCR-directed drug action through location bias. Nat Chem Biol 13:799-806 |
