Abstract
In living cells the transport and diffusion of molecules is constrained by compartments of various sizes. This paper is an attempt to show that the size of these compartments can in principle be estimated experimentally from Fluorescence Correlation Spectroscopy (FCS) combined with the measurement of the photobleaching rate. In this work, confocal fluorescence microscopy experiments have been carried out on giant unilamellar vesicles, a system that mimics cellular compartmentalisation. We have developed numerical and analytical models to describe the fluorescence decay due to photobleaching in this geometry, which has enabled us to point out two regimes depending on the value of the parameter p B = σ P/D (where σ B is the photobleaching cross section of the dye, D its diffusion constant, and P the laser power (in photon/s)). In particular, when p B ≪ 1 (i.e. in the fast diffusion regime), the photobleaching rate is independent of the diffusion constant and scales like σ B P/R 2, in agreement with the experimental results. On the other hand, the standard diffusion models used to analyse the FCS data do not take into account the effects of the fluorescence decay on the autocorrelation curve. We show here how to correct the raw data for these drawbacks.
Similar content being viewed by others
REFERENCES
W. W. Webb (2001). Fluorescence correlation spectroscopy: Inception, biophysical experimentation and prospectus. Appl. Opt. 40, 3969-3983.
D. Madge, E. Elson, and W. W. Webb (1972). Thermodynamic fluctuations in a reacting system-measurement by fluorescence correlation spectroscopy. Phys. Rev. Lett. 29, 705-708.
E. L. Elson (2001). Fluorescence correlation spectroscopy measures molecular transport in cells. Traffic 2, 789-796.
E. Haustein and P. Schwille (2003). Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopy. Methods 29, 153-166.
S. T. Hess, S. Huang, A. A. Heikal, and W. W. Webb (2002). Biological and chemical applications of fluorescence correlation spectroscopy: A review Biochemistry 41, 697-705.
P. Schwille, U. Haupts, S. Maiti, and W. W. Webb (1999). Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one-and two-photon excitation. Biophys. J. 77, 2251-2265.
P. S. Dittrich and P. Schwille (2001). Photobleaching and stabilization of fluorophores used for single molecule analysis with one-and two-photon excitation. Appl. Phys. B 73, 829-837.
J. Widengren and R. Rigler (1996). Mechanisms of photobleaching investigated by fluorescence correlation spectroscopy. Bioimaging 4, 149-157.
G. H. Patterson and D. W. Piston (2000). Photobleaching in two-photon excitation microscopy Biophys J. 78, 2159-2162.
T. S. Chen, S. Q. Zeng, Q. M. Luo, Z. H. Zhang, and W. Zhou (2002). High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy. Biochem. Biophys. Res. Commun. 291, 1272-1275.
K. Bacia and P. Schwille (2003). A dynamic view of cellular processes by in vivo fluorescence auto-and cross-correlation spectroscopy. Methods 29, 74-85.
K. Bacia, I. V. Majoul, and P. Schwille (2002). Title probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis. Biophys. J. 83, 1184-1193.
L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke (1995). Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy. Biophys. J. 68, 2588-2600.
L. Song, C. A. G. O. Varma, J. W. Verhoeven, and H. J. Tanke (1996). Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy. Biophys. J. 70, 2959-2968.
L. Song, R. P. van Gijlswijk, I. T. Young, and H. J. Tanke (1997). Influence of fluorochrome labeling density on the photobleaching kinetics of fluorescein in microscopy. Cytometry 27, 213-223.
C. Eggeling, L. Brand, and C. A. M. Seidel (1997). Laser-induced fluorescence of coumarin derivatives in aqueous solutions: Photochemical aspects for single molecule detection. Bioimaging 5, 105-115.
C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel (1998). Photobleaching of fluorescence dyes under conditions used for single-molecule detection: Evidence of two-step photolysis. Anal. Chem. 70, 2651-2659.
C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel (1999). In W. Rettig (Ed.), Applied Fluorescence in Chemistry, Biology and Medicine, Springer-Verlag, Berlin, pp. 193-240.
R. Peters, A. Brüngers, and K. Schulten (1981). Continuous fluorescence microphotolysis: A sensitive method for study of diffusion processes in single cells Proc. Nat. Acad. Sci. U.S.A. 78, 962-966.
U. Kubitscheck, P. Wedekind, and R. Peters (1998). Three-dimensional diffusion measurements by scanning microphotolysis. J. Microsc. (USA) 192, 126-138.
M. Wachsmuth, T. Weidemann, G. Müller, U. W. Hoffmann-Rohrer, T. A. Knoch, W. Waldekc, and J. Langowski (2003). Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching. Biophys. J. 84, 3353-3363.
G. Carrero, D. McDonald, E. Crawford, G. de Vries, and M. J. Hendzel (2003). Using FRAP and mathematical modeling to determine the in vivo kinetics of nuclear proteins. Methods 29, 14-28.
J. Lippincott-Schwartz, N. Altan-Bonnet, and G. H. Patterson (2003). Photobleaching and photoactivation: Following protein dynamics in living cells. Suppl. Nat. Cell Biol. 5, S7-S14.
M. I. Angelova, S. Soléau, P. Méléard, J. Faucon, and P. Bothorel (1992). Preparation of giant vesicles by external A. C. electric field Progr. Coll. Pol. Sci. 89, 127.
J. Enderlein (1996). Path integral approach to fluorescence correlation experiments. Phys. Lett. A 221, 427-433.
R. Courant and D. Hilbert (1989). Methods in Mathematical Physics, Wiley, New York, Vol. I, ch. V.
J. Mertz (1998). Molecular photodynamics involved in multi-photon excitation fluorescence microscopy. Eur. Phys. J. D 3, 53-66.
N. L. Thompson (1991). In J. R. Lakowicz (Ed.), Topics in Fluorescence Spectroscopy, Plenum Press, New York, pp. 337-378.
J. Widengren, U. Mets, and R. Rigler (1995). Fluorescence correlation spectroscopy of triplet states in solution: A theoretical and experimental study. J. Phys. Chem. 99, 13368-13379.
Y. Chen, J. D. Müller, Q. Ruan, and E. Gratton (2002). Molecular brightness characterisation of EGFP in vivo by fluorescence fluctuation spectroscopy Biophys. J. 82, 133-144.
D. M. Benson, J. Bryan, A. L. Plant, A. M. Gotto, Jr., and L. C. Smith (1985). Digital imaging fluorescence microscopy: Spatial heterogeneity of photobleaching rate constants in individual cells. J. Cell Biol. 100, 1309-1323
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Delon, A., Usson, Y., Derouard, J. et al. Photobleaching, Mobility, and Compartmentalisation: Inferences in Fluorescence Correlation Spectroscopy. Journal of Fluorescence 14, 255–267 (2004). https://doi.org/10.1023/B:JOFL.0000024557.73246.f9
Issue Date:
DOI: https://doi.org/10.1023/B:JOFL.0000024557.73246.f9