The goal of the proposed research is to expand the capabilities and performance of stochastic single- molecule superresolution (SSMS) microscopy through the development of improved fluorescent protein variants. A primary objective of this project will be to enable SSMS imaging in living cells of three or more proteins simultaneously, allowing visualization of multi-protein complex dynamics in a biologically relevant context with single-molecule localization precision of 20 nm or less. Currently, live-cell SSMS imaging is largely limited to one- and two-color labeling, due to the lack of well-performing fluorescent protein variants at appropriate wavelengths. Much of this shortfall in suitable fluorescent protein labels has come from a reliance on fluorescent proteins from a very limited number of species as starting materials for engineering improved variants. This project will take advantage of wild-type fluorescent proteins with novel optical properties to be cloned from a broad range of species, including several Great Barrier Reef corals made available through an international collaboration. The use of this wide array of novel starting materials will maximize the odds of successful engineering of monomeric fluorescent proteins with markedly improved performance over current technology. Specific emphasis will be placed on functional screening in mammalian cells, rather than directed evolution exclusively in bacteria, as has been the standard approach to most past fluorescent protein engineering efforts. This expanded use of image-based screening in mammalian cells will facilitate the selection of variants that perform optimally in th primary end-user applications of this technology. The end products of this project will be complete sets of monomeric fluorescent protein variants in three classes: (1) standard (non-photoactive) fluorescent proteins in the orange, red, and far-red wavelength classes that display improved brightness, photostability, performance as fusion tags, and performance as SSMS imaging tags; (2) photoactivatable and photochromic fluorescent proteins across the entire visual spectrum that display equivalent performance to standard fluorescent proteins, but also include exceptionally high contrast between dark and activated states; (3) photoconvertible proteins, focused on the development of novel wavelength classes including red-to-yellow, blue-to-red, and red-to-far-red, with performance goals similar to the other two classes. For each of these classes, the goal will be development of high-performing variants that allow imaging of at least three distinct protein species simultaneously in living cells, as measured by several biologically relevant assays using SSMS microscopy. The fluorescent protein variants created in the course of this project will also be highly useful as general fluorescent tags, and are expected to be among the brightest and highest- performing fluorescent proteins yet developed.

Public Health Relevance

The goal of the proposed research is to generate a comprehensive set of genetically encoded optical tools for imaging living biological specimens at extremely high resolution, using the rapidly developing technology known as 'superresolution microscopy.' The tools developed in this study will be made widely available to the biological and biomedical research community where they will be highly useful for studying basic biological and disease processes in living cells across a very broad range of medically relevant areas such as cancer, neurodegenerative disease, and aging.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Sammak, Paul J
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Scintillon Institute for Photobiology
San Diego
United States
Zip Code
Hong, Shi-Rong; Wang, Cuei-Ling; Huang, Yao-Shen et al. (2018) Spatiotemporal manipulation of ciliary glutamylation reveals its roles in intraciliary trafficking and Hedgehog signaling. Nat Commun 9:1732
Tewson, Paul; Martinka, Scott; Shaner, Nathan et al. (2018) Assay for Detecting G?i-Mediated Decreases in cAMP in Living Cells. SLAS Discov 23:898-906
Molina, Rosana S; Tran, Tam M; Campbell, Robert E et al. (2017) Blue-Shifted Green Fluorescent Protein Homologues Are Brighter than Enhanced Green Fluorescent Protein under Two-Photon Excitation. J Phys Chem Lett 8:2548-2554
Mastop, Marieke; Bindels, Daphne S; Shaner, Nathan C et al. (2017) Characterization of a spectrally diverse set of fluorescent proteins as FRET acceptors for mTurquoise2. Sci Rep 7:11999
Clavel, Damien; Gotthard, Guillaume; von Stetten, David et al. (2016) Structural analysis of the bright monomeric yellow-green fluorescent protein mNeonGreen obtained by directed evolution. Acta Crystallogr D Struct Biol 72:1298-1307