Scanning‐less wide‐field single‐photon counting device for fluorescence intensity, lifetime and time‐resolved anisotropy imaging microscopy

A scanning‐less single‐photon counting system for FLIM and fluorescence anisotropy wide‐field imaging is described and characterized in this paper. The two polarizations of the fluorescence are divided by a Glan prism and acquired at the same time by the QA detector. Fluorescence decay profiles can be reconstructed for any desired area up to each pixel and used to calculate time‐resolved fluorescence anisotropy decays.

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

[2]  T Ragan,et al.  New time-resolved techniques in two-photon microscopy. , 1998, Cellular and molecular biology.

[3]  T. Főrster,et al.  10th Spiers Memorial Lecture. Transfer mechanisms of electronic excitation , 1959 .

[4]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[5]  R. Pansu,et al.  Characterization of a confocal microscope for time-resolved photon counting fluorescence , 1997, Journal of Fluorescence.

[6]  Alessandro Esposito,et al.  Fluorescence Lifetime Imaging Microscopy , 2004, Current protocols in cell biology.

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

[8]  J Enderlein,et al.  Theoretical study of detection of a dipole emitter through an objective with high numerical aperture. , 2000, Optics letters.

[9]  T. Laurence,et al.  Polarization Spectroscopy of Single Fluorescent Molecules , 1999 .

[10]  M. Tramier,et al.  Restrained torsional dynamics of nuclear DNA in living proliferative mammalian cells. , 2000, Biophysical journal.

[11]  Douglas Magde,et al.  Fluorescence Quantum Yields and Their Relation to Lifetimes of Rhodamine 6G and Fluorescein in Nine Solvents: Improved Absolute Standards for Quantum Yields¶ , 2002, Photochemistry and photobiology.

[12]  N. Tripathi.  Densities, Viscosities, and Refractive Indices of Mixtures of Hexane with Cyclohexane, Decane, Hexadecane, and Squalane at 298.15 K , 2005 .

[13]  R. Pansu,et al.  Confocal microscopic study on fluorescence quenching dynamics of single latex beads in poly(vinyl alcohol) film , 2000 .

[14]  A. Hoek.  Time-resolved fluorescence spectroscopy , 2000 .

[15]  Robert M. Clegg,et al.  Fluorescence lifetime imaging microscopy (FLIM): Spatial resolution of microstructures on the nanosecond time scale , 1993 .

[16]  Valentina Emiliani,et al.  Low-intensity two-dimensional imaging of fluorescence lifetimes in living cells , 2003 .

[17]  Hans C. Gerritsen,et al.  Fluorescence lifetime imaging using a confocal laser scanning microscope , 1992 .

[18]  R. Tsien,et al.  Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin , 1997, Nature.

[19]  Andreas Volkmer,et al.  Identification of Single Molecules in Aqueous Solution by Time-Resolved Fluorescence Anisotropy , 1999 .

[20]  D. Axelrod.  Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization. , 1979, Biophysical journal.

[21]  Enrico Gratton,et al.  Time-resolved fluorescence microscopy using two-photon excitation , 1995 .

[22]  R. Pansu,et al.  Development of Intracellular Calcium Measurement by Time‐resolved Photon‐counting Fluorescence , 1999, Photochemistry and photobiology.

[23]  R. J. Robbins,et al.  Picosecond dynamics of electronic energy transfer in condensed phases , 1981 .

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

[25]  V. Agranovich,et al.  Electronic excitation energy transfer in condensed matter , 1982 .

[26]  B. Valeur,et al.  Fluorescence depolarization by electronic energy transfer in donor–acceptor pairs of like and unlike chromophores , 1991 .

[27]  A. Periasamy,et al.  Fluorescence Lifetime Imaging Microscopy (FLIM): Instrumentation and Applications , 1992 .

[28]  M. Gee,et al.  Time-resolved evanescent wave-induced fluorescence anisotropy for the determination of molecular conformational changes of proteins at an interface , 2004, European Biophysics Journal.