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Updated: May 2, 2026
Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy
Published on: April 25, 2021
Characterization of the fluorescence correlation spectroscopy (FCS) standard rhodamine 6G and calibration of its
1Max Planck Institute for Intelligent Systems, Heisenbergstr. 3, 70569 Stuttgart, Germany.
Abstract:
Precise diffusion measurements of rhodamine 6G (Rh6G) dissolved in D2O at concentrations between 50 and 200 μM were carried out in the temperature range from 280 to 320 K using pulsed field gradient nuclear magnetic resonance (PFG-NMR). The obtained diffusion coefficients can be used as a calibration reference in fluorescence correlation spectroscopy (FCS). Besides measuring the diffusivity of Rh6G, the diffusion coefficient of the solvent in the same system could be determined in parallel by PFG-NMR as the resonances of water and Rh6G are well separated in the (1)H NMR spectrum. To analyze the differences due to the isotope effect of the solvent (D2O vs. H2O), the correlation time τD of Rh6G was measured by FCS in both D2O and H2O. The obtained isotopic correction factor, τD(D2O)/τD(H2O) = 1.24, reflects the isotope effect of the solvent´s self-diffusion coefficients as determined previously by PFG-NMR.
