Browsing by Author "Wicks, Geoffrey"

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A long Stokes shift red fluorescent Ca2+ indicator protein for two-photon and ratiometric imaging
(Nature Publishing Group, 2014-10) Wu, Jiahui; Abdelfattah, Ahmed S.; Miraucourt, Loïs S.; Kutsarova, Elena; Ruangkittisakul, Araya; Zhou, Hang; Ballanyi, Klaus; Wicks, Geoffrey; Drobizhev, Mikhail; Rebane, Aleksander; Ruthazer, Edward S.; Campbell, Robert E.
The introduction of calcium ion (Ca2+) indicators based on red fluorescent proteins (RFPs) has created new opportunities for multicolour visualization of intracellular Ca2+ dynamics. However, one drawback of these indicators is that they have optimal two-photon excitation outside the near-infrared window (650–1,000 nm) where tissue is most transparent to light. To address this shortcoming, we developed a long Stokes shift RFP-based Ca2+ indicator, REX-GECO1, with optimal two-photon excitation at <1,000 nm. REX-GECO1 fluoresces at 585 nm when excited at 480 nm or 910 nm by a one- or two-photon process, respectively. We demonstrate that REX-GECO1 can be used as either a ratiometric or intensiometric Ca2+ indicator in organotypic hippocampal slice cultures (one- and two-photon) and the visual system of albino tadpoles (two-photon). Furthermore, we demonstrate single excitation wavelength two-colour Ca2+ and glutamate imaging in organotypic cultures.
A New Femtosecond Nonlinear Transmission Experiment for Accurate Determination of Intrinsic Two Photon Absorption Spectra of Non-Fluorescent Chromophores
(2013-03) Wicks, Geoffrey; Rebane, Aleksander
We have developed and tested a new femtosecond experiment for measuring the intensity dependent transmission of two photon absorbing (2PA) chromophores in a broad range of wavelengths. This technique offers a very high accuracy (~0.05% transmission change) and is of particular importance for characterization of the 2PA properties of Platinum complexes and other compounds with low fluorescence emission. As a preliminary demonstration of this new technique we have measured the intrinsic 2PA spectra and the 2PA cross sections of some known chromophores that have been previously characterized by the fluorescence method of 2PA spectrum evaluation.
Two-photon directed evolution of green fluorescent proteins
(2015-07) Stoltzfus, Caleb R.; Barnett, Lauren M.; Drobizhev, Mikhail; Wicks, Geoffrey; Mikhaylov, Alexander E.; Hughes, Thomas E.; Rebane, Aleksander
Directed evolution has been used extensively to improve the properties of a variety of fluorescent proteins (FPs). Evolutionary strategies, however, have not yet been used to improve the two-photon absorption (2PA) properties of a fluorescent protein, properties that are important for two-photon imaging in living tissues, including the brain. Here we demonstrate a technique for quantitatively screening the two-photon excited fluorescence (2PEF) efficiency and 2PA cross section of tens of thousands of mutant FPs expressed in E. coli colonies. We use this procedure to move EGFP through three rounds of two-photon directed evolution leading to new variants showing up to a 50% enhancement in peak 2PA cross section and brightness within the near-IR tissue transparency wavelength range.