A high-content screening platform utilizing polarization anisotropy and FLIM microscopy

An automated high-content screening microscope has been developed which uses fluorescence anisotropy imaging and fluorescence lifetime microscopy to identify Förster resonant energy transfer between eGFP and mRPF1 in drug screening assays. A wide-field polarization resolved imager is used to simultaneously capture the parallel and perpendicular components of both eGFP and mRFP1 fluorescence emission to provide a high-speed measurement of acceptor depolarization. Donor excited state lifetime measurements performed using laser scanning microscopy is then used to determine the FRET efficiency in a particular assay. A proof-of-principle assay is performed using mutant Jurkat human T-cells to illustrate the process by which FRET is first identified and then quantified by our high-content screening system.

[1]  J. Siegel,et al.  Wide-Field Time-Resolved Fluorescence Anisotropy Imaging (TR-FAIM) , 2003 .

[2]  R. Pepperkok,et al.  The potential of high‐content high‐throughput microscopy in drug discovery , 2007, British journal of pharmacology.

[3]  K. Aoki,et al.  Monitoring spatio-temporal regulation of Ras and Rho GTPase with GFP-based FRET probes. , 2005, Methods.

[4]  Borivoj Vojnovic,et al.  Spatially Distinct Binding of Cdc42 to PAK1 and N-WASP in Breast Carcinoma Cells , 2005, Molecular and Cellular Biology.

[5]  F. Wouters,et al.  Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells , 1999, Current Biology.

[6]  V. Subramaniam,et al.  Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM). , 2002, Biophysical journal.

[7]  B. Vojnovic,et al.  Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions. , 2005, Biophysical journal.

[8]  P. So,et al.  Comparing the quantification of Forster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging. , 2006, Journal of biomedical optics.

[9]  P. J. Reynolds,et al.  Use of a beta1 integrin-deficient human T cell to identify beta1 integrin cytoplasmic domain sequences critical for integrin function. , 1998, Molecular biology of the cell.

[10]  M. Chalfie.  GREEN FLUORESCENT PROTEIN , 1995, Photochemistry and photobiology.

[11]  Marion Peter,et al.  Imaging molecular interactions by multiphoton FLIM , 2004, Biology of the cell.

[12]  R R Duncan,et al.  Multi‐dimensional time‐correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells , 2004, Journal of microscopy.

[13]  Hans C Gerritsen,et al.  Innovating lifetime microscopy: a compact and simple tool for life sciences, screening, and diagnostics. , 2006, Journal of biomedical optics.

[14]  P. Bastiaens,et al.  PKCα regulates β1 integrin‐dependent cell motility through association and control of integrin traffic , 1999 .

[15]  J. Nicolas,et al.  Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins. , 2001, Biophysical journal.

[16]  Peter J. Parker,et al.  Imaging Protein Kinase Cα Activation in Cells , 1999 .

[17]  T. Jovin,et al.  FRET imaging , 2003, Nature Biotechnology.

[18]  L. Stryer.  Fluorescence energy transfer as a spectroscopic ruler. , 1978, Annual review of biochemistry.

[19]  Michiyuki Matsuda,et al.  A FRET-based probe for epidermal growth factor receptor bound non-covalently to a pair of synthetic amphipathic helixes. , 2005, Experimental cell research.

[20]  D. Piston,et al.  High-contrast imaging of fluorescent protein FRET by fluorescence polarization microscopy. , 2005, Biophysical journal.

[21]  F. Wouters,et al.  Imaging biochemistry inside cells. , 2001, Trends in cell biology.

[22]  A. Hoppe,et al.  Polarized fluorescence resonance energy transfer microscopy. , 2004, Biophysical journal.

[23]  R. Tsien,et al.  A monomeric red fluorescent protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Borivoj Vojnovic,et al.  Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy. , 2003, The Biochemical journal.

[25]  J. Siegel,et al.  Time-resolved fluorescence anisotropy imaging applied to live cells. , 2004, Optics letters.

[26]  Yoji Shimizu,et al.  Use of a β1 Integrin-deficient Human T Cell to Identify β1 Integrin Cytoplasmic Domain Sequences Critical for Integrin Function , 1998 .

[27]  S W Hell,et al.  Four-dimensional multiphoton microscopy with time-correlated single-photon counting. , 2000, Applied optics.

[28]  R. Tsien,et al.  green fluorescent protein , 2020, Catalysis from A to Z.