Fluorescence correlation spectroscopy: incorporation of probe volume effects into the three-dimensional Gaussian approximation

AbstractThe cell nucleus is a three-dimensional, dynamic organelle that is organized into many subnuclear bodies, such as chromatin and nucleoli. The structure and function of these bodies is maintained by diffusion and interactions between related factors as well as dynamic and structural changes. Recent studies using fluorescent microscopic techniques suggest that protein factors can access and are freely mobile in mitotic chromosomes, despite their densely packed structure. However, the physicochemical properties of the chromosome itself during cell division are not yet fully understood. Physical parameters, such as the refractive index (RI), volume of the mitotic chromosome, and diffusion coefficients of fluorescent probes inside the chromosome were quantified using an approach combining label-free optical diffraction tomography with complementary confocal laser scanning microscopy and fluorescence correlation spectroscopy. Variance in these parameters correlated among various osmotic conditions, suggesting that changes in RI are consistent with those in the diffusion coefficient for mitotic chromosomes and cytosol. Serial RI tomography images of chromosomes in live cells during mitosis were compared with three-dimensional confocal micrographs to demonstrate that compaction and decompaction of chromosomes induced by osmotic change were characterized by linked changes in chromosome RI, volume, and the mobility of fluorescent proteins.

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Single Plane Illumination Fluorescence Correlation Spectroscopy (SPIM-FCS) probes inhomogeneous three-dimensional environments

Optics Express ◽

10.1364/oe.18.010627 ◽

2010 ◽

Vol 18 (10) ◽

pp. 10627 ◽

Cited By ~ 104

Author(s):

Thorsten Wohland ◽

Xianke Shi ◽

Jagadish Sankaran ◽

Ernst H.K. Stelzer

Keyword(s):

Fluorescence Correlation Spectroscopy ◽

Three Dimensional ◽

Correlation Spectroscopy ◽

Single Plane ◽

Fluorescence Correlation

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Calibration of Probe Volume in Fluorescence Correlation Spectroscopy

Applied Spectroscopy ◽

10.1366/000370207781745883 ◽

2007 ◽

Vol 61 (9) ◽

pp. 956-962 ◽

Cited By ~ 1

Author(s):

Yi Gao ◽

Zhenming Zhong ◽

M. Lei Geng

Keyword(s):

Correlation Functions ◽

Fluorescence Correlation Spectroscopy ◽

Time Correlation ◽

Correlation Spectroscopy ◽

Beam Radius ◽

Data Sets ◽

Probe Volume ◽

Calibration Process ◽

Fluorescence Correlation ◽

Dimensional Parameters

In fluorescence correlation spectroscopy (FCS), an accurate evaluation of the probe volume is the basis of correct interpretation of experimental data and solution of an appropriate diffusion model. Poor fitting convergence has been a problem in the determination of the dimensional parameters, the beam radius, ω, and the distance along the optical axis of the probe volume, l. In this work, the instability of fitting during the calibration process is investigated by examining the χ2 surfaces. We demonstrate that the minimum of χ2 in the ω dimension is well defined for both converging and diverging data. The difficulty of fitting comes from the l dimension. The uncertainty in l could be significantly larger than that in ω, as determined by F-statistics. A modified calibration process is recommended based on examining two data treatment methods, combining several short data sets into a single long run and averaging the correlation functions of several short data sets. It is found that by using the mean of several converging correlation functions from short data sets instead of a long time correlation, more stable and consistent dimensional parameters are extracted to define the probe volume.

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