T Luminescence Imaging and Biosensing in Cells and Tissues
thesisposted on 2018-07-25, 00:00 authored by Ting Chen
Time-gated luminescence imaging and detection using brightly emitting complexes of the lanthanide elements Tb(III) and Eu(III) offer several distinct advantages for immunofluorescence microscopy and for designing biosensors of protein function. Lanthanide complexes emit in multiple, well separated bands, and lanthanide emission lifetimes are on the order of milleseconds. These features can be leveraged to develop highly sensitive, multiplexed protein imaging and screening assays based on Förster resonance energy transfer (FRET) between a Tb(III) or Eu(III) complex donor and a fluorescent protein acceptor. The donor emission can be easily filtered from sensitized acceptor emission, and is able to sensitize multiple differentially colored acceptors. While short-lived non-specific background, directly excited acceptor fluorescence, and autofluorescence are eliminated by time-gating, where pulsed light excites specimens, and a short delay is implemented before the detector starts collecting light. The main objectives of the studies in this dissertation were to expand the application of time-gated luminescence imaging in live cells and tissues, and to develop lanthanide-based biosensors for quantitative FRET imaging and HTS of PPIs in living cells with high sensitivity and signal-to-noise ratio. The feasibility of extracellular Tb-to-QD FRET biosensing was demonstrated by immunostaining different epitopes of epidermal growth factor receptor (EGFR) with QD- and Tb-antibody conjugates in A431 cells. By eliminating the non-specific background, time-gated luminescence microscopy (TGLM) enables the visualization of various markers and intermolecular, Tb(III)-to-dye FRET on FFPE tissue. Results also indicate the compatibility of TGLM with H&E staining in certain protocols. On the other hand, quantitative TGLM results from single chain lanthanide-based FRET biosensors were provided. Time-gated detection of PPI and/or its inhibition were performed in multi-well plate with lysised cells or live mammalian cells. High quality data were obtained by the indication of Z’ factor and strictly standardized mean difference (SSMD). The results strongly implicate the potential of lanthanide-based single-chain biosensors for hit selection in HTS assays. For future work, the interaction between protein phosphatase 1 regulatory subunit 12C (PPP1R12C) and its catalytic subunit alpha isozyme (PPP1CA), and their inhibition with a short peptide or other regulatory domain will be investigated to validate the ability of the biosensors to be utilized in HTS to discover effective small molecule inhibitors of PPIs. Finally, a new method, which combines split-DHFR, FRET, and time-gated detection, is proposed for mapping protein network or identifying potential hits of designated PPIs in living mammalian cells.