Software Package for Multiparameter Fluorescence Spectroscopy,
Full Correlation and Multiparameter Fluorescence Imaging

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We present a software package for fluorescence data analysis in Multiparameter Fluorescence Detection (MFD) [1]. In this technique time-resolved observation of intrinsic properties of chromophores (i.e. spectral properties of absorption and fluorescence, fluorescence quantum yield, fluorescence lifetime, and anisotropy) is recorded. Selective analysis of molecular subensembles and time dependent parameter traces from single molecule are possible [2].

Subsequent software-correlation yields full correlation curves covering more than 12 orders of magnitude in time [3]. An advanced time-correlated single photon counting (TCSPC) technique simultaneously provides traditional autocorrelation fluorescence correlation (FCS) or cross correlation (FCCS) from selected molecules which makes analysis more powerful.

Software gives opportunity for all potential applications of technique using pulsed or cw laser excitation: antibunching effects, rotational diffusion and fluorescence resonance energy transfer (FRET) as are discussed.

The combination of FCS and fluorescence lifetime (FLCS) [4] is implemented into software correlator. Direct generation of necessary filters for polarisation dependent signals is possible.

Software for probability distribution analysis (PDA) is presented for quantitative and precise description of the photon counting histograms (PCH) from fluorescence resonance energy transfer experiments (FRET) [5]. An accurate description of the histogram profile based on the statistical distributions of fluorescence and background signals is sensitive to small changes in fluorescence signal even when signal counts are low. The PDA formalism allows monitoring the changes in the emission spectra of single molecules, dynamical changes of the system, etc. PDA harbours the potential for Molecular Ångström Optics.

Software for confocal Multiparameter Fluorescence Imaging (MFIS) is developed. Each pixel of MFIS corresponds to “single burst” in solution experiments and all MFD type analysis are applied. Intensity, lifetime, anisotropy, correlation times, correlation amplitudes or any MFD parameter images can be reconstructed [6,7].

 References

 

  [1] Kühnemuth et al., Single Molecules, 2, 251 (2001).   

  [5] Antonik M., et al., J. Phys. Chem. B 110, 6970 (2006).

  [2] Widengren J., et al., Anal. Chem. 78, 2039 (2006).

  [6] Gaiduk et al., Chem.Phys.Chem. 5, 976 (2005).

  [3] Felekyan S., et al., Rev. Sci. Instr. 76, 083104 (2005).

  [7] Kudryavtsev et al., Anal.&Bioanal.Chem., in press.

  [4] Böhmer M., et al., Chem. Phys. Lett. 353, 439 (2002).

 

Multiparameter Fluorescence Detection (MFD) data flow. Click on icons to see program description.
Multiparameter Fluorescence Detection and Imaging (MFDI) data flow. Click on icons to see program description.
Description
Verantwortlich für den Inhalt: E-Mail sendenProf. Dr. Claus Seidel