Comparison and accuracy of methods to determine the confocal volume for quantitative fluorescence correlation spectroscopy

Abstract

SummarySingle molecule detection based on fluorescent labels offers the possibility to gain not only qualitative but also quantitative insight into specific functions of complex biological systems. Fluorescence correlation spectroscopy is one of the favourite techniques to determine concentrations and diffusion constants as well as molecular brightness of molecules in the pico-to nano-molar concentration range, with broad applications in biology and chemistry. Although fluorescence correlation spectroscopy in principle has the potential to measure absolute concentrations and diffusion coefficients, the necessity to know the exact size and shape of the confocal volume very often hampers the possibility to obtain quantitative results and restricts fluorescence correlation spectroscopy to relative measurements mainly. The determination of the confocal volume in situ is difficult because it is sensitive to optical alignment and aberrations, optical saturation and variations of the index of refraction as observed in biological specimen. In the present contribution, we compare different techniques to characterize the confocal volume and to obtain the confocal parameters by fluorescence correlation spectroscopy curve fitting, a fluorescence correlation spectroscopy dilution series and confocal scanning of fluorescent beads. The results are compared in the view of quantitative fluorescence correlation spectroscopy measurement and analysis. We investigate how unavoidable artefacts caused by a non-ideal confocal volume can be experimentally determined and validated.

Dynamic Organization of Transcription Compartments Is Dependent on Functional Nuclear Architecture

“…The obtained diffusion times (t d ) were used to calculate its diffusion coefficient ðD ¼ u 2 0 =4t d Þ and then to determine the sizes of the UTP aggregates to generate the distribution histogram. The size of the effective volume was predetermined by measuring dyes with known diffusion coefficient (17), and the u 0 was determined to be 0.25 mm for both 488-nm and 543-nm excitation with laser power of 10 mW.…”

Direct Quantification of Solute Diffusivity in Agarose and Articular Cartilage Using Correlation Spectroscopy

“…smaller than the diffraction-limited resolution of the system) and measuring their reconstructed sizes. 34 However, this technique requires specialized reconstruction and analysis software, and is oftentimes difficult to match to the experimental imaging conditions. 34 Conversely, calibration of the PSF geometry via the use of a known diffusion standard is a simple and well-accepted method for PSF determination.…”

Single Molecule 3D Orientation in Time and Space: A 6D Dynamic Study on Fluorescently Labeled Lipid Membranes

“…The structural factor was determined with the asymmetric 2D diffusion model to S = w 0 /z 0 = 0.22 (or the eccentricity of the focal volume k = z 0 /w 0 == 4.4), again in accordance to literature values for (Gaussian) confocal volumes [51]. Although the emitted photons are separated in center and rim with respect to their emission angle, the focal volume is still considered to be close to a 3D Gaussian, and thus the amplitude of the correlation curve remains a reliable measure of the number of fluorescent molecules within the focal volume.…”