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Two‐Focus Fluorescence Correlation Spectroscopy: A New Tool for Accurate and Absolute Diffusion Measurements
Abstract
We present a new method to measure absolute diffusion coefficients at nanomolar concentrations with high precision. Based on a modified fluorescence correlation spectroscopy (FCS)‐setup, this method is improved by introducing an external ruler for measuring the diffusion time by generating two laterally shifted and overlapping laser foci at a fixed and known distance. Data fitting is facilitated by a new two‐parameter model to describe the molecule detection function (MDF). We present a recorded MDF and show the excellent agreement with the fitting model. We measure the diffusion coefficient of the red fluorescent dye Atto655 under various conditions and compare these values with a value achieved by gradient pulsed field NMR (GPF NMR). From these measurements we conclude, that the new measurement scheme is robust against optical and photophysical artefacts which are inherent to standard FCS. With two‐focus‐FCS, the diffusion coefficient of 4.26×10−6 cm 2 s−1 for Atto655 in water at 25 °C compares well with the GPF NMR value of 4.28×10−6 cm 2 s−1.
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Cited by
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Borgia, A., Borgia, M. B., Bugge, K., Kissling, V. M., Heidarsson, P. O., Fernandes, C. B., Sottini, A., Soranno, A., Buholzer, K. J., Nettels, D., et al. (2018) · Nature · doi:10.1038/nature25762 · Cited by 653
Fluorescence correlation spectroscopy in living cells
Kim, S. A., Heinze, K. G., & Schwille, P. (2007) · Nature Methods · doi:10.1038/nmeth1104 · Cited by 444
Fluorescence Correlation Spectroscopy: Past, Present, Future
Elson, E. L. (2011) · Biophysical Journal · doi:10.1016/j.bpj.2011.11.012 · Cited by 438
Extreme dynamics in a biomolecular condensate
Galvanetto, N., Ivanović, M. T., Chowdhury, A., Sottini, A., Nüesch, M. F., Nettels, D., Best, R. B., & Schuler, B. (2023) · Nature · doi:10.1038/s41586-023-06329-5 · Cited by 365
Accurate Single-Molecule FRET Studies Using Multiparameter Fluorescence Detection
Sisamakis, E., Valeri, A., Kalinin, S., Rothwell, P. J., & Seidel, C. A. M. (2010) · Methods in Enzymology · doi:10.1016/s0076-6879(10)75018-7 · Cited by 268
Metadata
- Authors
- Dertinger, T., Pacheco, V., von der Hocht, I., Hartmann, R., Gregor, I., & Enderlein, J.
- Venue
- ChemPhysChem
- Publisher
- Wiley
- Published
- 2007-02-08
- Volume
- 8
- Issue
- 3
- Pages
- 433-443
- ISSN
- 1439-4235 (ISSN-L)
- 1439-7641
- OpenAlex
- W2167258646
- Cited by
- 345
- References
- 0
- FWCI
- 16.81
- Type
- journal article
- Language
- en
- Open access
- closed
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- Dertinger, T., Pacheco, V., von der Hocht, I., Hartmann, R., Gregor, I., & Enderlein, J. (2007). Two‐Focus Fluorescence Correlation Spectroscopy: A New Tool for Accurate and Absolute Diffusion Measurements. ChemPhysChem. https://doi.org/10.1002/cphc.200600638
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