Fluorescence correlation spectroscopy of finite-sized particles.

A theory is presented to study fluorescence correlation spectroscopy for particles with size comparable to the beam waist of the observation volume. Analytical correlation curves are derived for some experimentally interesting particle geometries. It is found that the finiteness of the particle generally decreases the value of the correlation amplitude and increases the correlation time compared to a point particle model. Furthermore, not only the size but also the distribution of fluorophores affects the shape of the correlation function. This is experimentally demonstrated with surface and internally labeled fluorescent spheres. In addition, experiments are performed on fluorescent spheres of different radii to validate the model by comparing the results to theoretical predictions.

[1]  Jerker Widengren,et al.  Photodynamic properties of green fluorescent proteins investigated by fluorescence correlation spectroscopy , 1999 .

[2]  Rika,et al.  Direct measurement of a distinct correlation function by fluorescence cross correlation. , 1989, Physical review. A, General physics.

[3]  N. Thompson,et al.  Molecular aggregation characterized by high order autocorrelation in fluorescence correlation spectroscopy. , 1987, Biophysical journal.

[4]  J. Buffle,et al.  Applications of Fluorescence Correlation Spectroscopy— Particle Size Effect , 1998 .

[5]  W. Webb,et al.  Thermodynamic Fluctuations in a Reacting System-Measurement by Fluorescence Correlation Spectroscopy , 1972 .

[6]  Yan Chen,et al.  Determining the stoichiometry of protein heterocomplexes in living cells with fluorescence fluctuation spectroscopy , 2007, Proceedings of the National Academy of Sciences.

[7]  E Gratton,et al.  The photon counting histogram in fluorescence fluctuation spectroscopy. , 1999, Biophysical journal.

[8]  Bin Wu,et al.  Time-integrated fluorescence cumulant analysis in fluorescence fluctuation spectroscopy. , 2005, Biophysical journal.

[9]  Liaohai Chen,et al.  Characterizing specific phage–protein interactions by fluorescence correlation spectroscopy , 2004, Protein science : a publication of the Protein Society.

[10]  W. Webb,et al.  Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy. , 2002, Biophysical journal.

[11]  E. Elson,et al.  The kinetics of conformational fluctuations in an unfolded protein measured by fluorescence methods. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[12]  C. Eggeling,et al.  Fluorescence fluctuation spectroscopy in reduced detection volumes. , 2006, Current pharmaceutical biotechnology.

[13]  S. McLaughlin,et al.  Fluorescence correlation spectroscopy studies of Peptide and protein binding to phospholipid vesicles. , 2004, Biophysical journal.

[14]  Christian Eggeling,et al.  Fluorescence fluctuation spectroscopy in subdiffraction focal volumes. , 2005, Physical review letters.

[15]  J. Rädler,et al.  Dynamics of large semiflexible chains probed by fluorescence correlation spectroscopy. , 2003, Physical review letters.

[16]  R. Winkler,et al.  Intramolecular dynamics of linear macromolecules by fluorescence correlation spectroscopy. , 2006, Physical review. E, Statistical, nonlinear, and soft matter physics.

[17]  M. Eigen,et al.  Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[18]  R. Pecora.  Dynamic Light Scattering , 1985 .

[19]  G. Nienhaus,et al.  Endothelin Receptor in Virus-Like Particles: Ligand Binding Observed by Fluorescence Fluctuation Spectroscopy† , 2004 .

[20]  Yan Chen,et al.  Probing protein oligomerization in living cells with fluorescence fluctuation spectroscopy , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Yan Chen,et al.  Position-sensitive scanning fluorescence correlation spectroscopy. , 2005, Biophysical journal.

[22]  W. Webb,et al.  Quantification of α-Synuclein Binding to Lipid Vesicles Using Fluorescence Correlation Spectroscopy , 2006 .

[23]  E Gratton,et al.  Resolving heterogeneity on the single molecular level with the photon-counting histogram. , 2000, Biophysical journal.