In many cell-signaling pathways information is transmitted via the diffusion of messenger molecules. In most cases, messengers react with other substances and diffuse at the same time. Effective diffusion coefficients may be introduced to characterize the net transport rate that results from the combined effect of these two processes. It was shown in [B. Pando, Proc. Natl. Acad. Sci. U.S.A. 103, 5338 (2006)] that even in the simplest scenario in which one bimolecular reaction is involved, two different effective coefficients are relevant. One gives the rate at which small perturbations spread out with time while the other relates the mean square displacement of a single particle to the time elapsed. They coincide in the absence of reactions but may be very different in other cases. Optical techniques provide a relatively noninvasive means by which transport rates can be estimated. In the above mentioned paper it was discussed why, under certain conditions, fluorescence recovery after photobleaching (FRAP), a technique commonly used to estimate diffusion rates in cells, provides information on one of the two effective coefficients. In the present paper we show that, under the same conditions, another commonly used optical technique, fluorescence correlation spectroscopy (FCS), gives information on the other one. This opens up the possibility of combining experiments to obtain information that goes beyond effective transport rates. In the present paper we discuss different ways to do so.

Knowledge of the basic mechanisms controlling osteogenesis and adipogenesis might provide new insights into the prevention of osteoporosis and age-related osteopenia. With the help of magnetic cell sorting and fluorescence activated cell sorting (FACS), osteoblastic subpopulations of mesenchymal progenitor cells were characterized. Alkaline phosphatase (AP) negative cells expressed low levels of osteoblastic and adipocytic markers. AP positive cells expressed adipocytic markers more strongly than the AP negative cell populations, thus suggesting that committed osteoblasts exhibit a greater adipogenic potential. AP negative cells differentiated to the mature osteoblastic phenotype, as demonstrated by increased AP-activity and osteocalcin secretion under standard osteogenic culture conditions. Surprisingly, this was accompanied by increased expression of adipocytic gene markers such as peroxisome proliferator-activated receptor-g2, lipoprotein lipase and fatty acid binding protein. The induction of adipogenic markers was suppressed by transforming growth factor-b1 (TGF-b1) and promoted by bone morphogenetic protein 2 (BMP-2). Osteogenic culture conditions including BMP-2 induced both the formation of mineralized nodules and cytoplasmic lipid vacuoles. Upon immunogold electron microscopic analysis, osteoblastic and adipogenic marker proteins were detectable in the same cell. Our results suggest that osteogenic and adipogenic differentiation in human mesenchymal progenitor cells might not be exclusively reciprocal, but rather, a parallel event until late during osteoblast development.

The flash photolysis of "caged" compounds is a powerful experimental technique for producing rapid changes in concentrations of bioactive signaling molecules. These caged compounds are inactive and become active when illuminated with ultraviolet light. This paper describes an inexpensive adaptation of an Olympus confocal microscope that uses as source of ultraviolet light the mercury lamp that comes with the microscope for conventional fluorescence microscopy. The ultraviolet illumination from the lamp (350 - 400 nm) enters through an optical fiber that is coupled to a nonconventional port of the microscope. The modification allows to perform the photolysis of caged compounds over wide areas (∼ 200 μm) and obtain confocal fluorescence images simultaneously. By controlling the ultraviolet illumination exposure time and intensity it is possible to regulate the amount of photolyzed compounds. In the paper we characterize the properties of the system and show its capabilities with experiments done in aqueous solution and in Xenopus Laevis oocytes. The latter demonstrate its applicability for the study of Inositol 1,4,5-trisphosphate-mediated intracellular calcium signals.

Many cell signaling pathways involve the diffusion of messengers that bind and unbind to and from intracellular components. Quantifying their net transport rate under different conditions then requires having separate estimates of their free diffusion coefficient and binding or unbinding rates. In this paper, we show how performing sets of fluorescence correlation spectroscopy (FCS) experiments under different conditions, it is possible to quantify free diffusion coefficients and on and off rates of reaction-diffusion systems. We develop the theory and present a practical implementation for the case of the universal second messenger, calcium (Ca^{2+}) and single-wavelength dyes that increase their fluorescence upon Ca^{2+} binding. We validate the approach with experiments performed in aqueous solutions containing Ca^{2+} and Fluo4 dextran (both in its high and low affinity versions). Performing FCS experiments with tetramethylrhodamine-dextran in Xenopus laevis oocytes, we infer the corresponding free diffusion coefficients in the cytosol of these cells. Our approach can be extended to other physiologically relevant reaction-diffusion systems to quantify biophysical parameters that determine the dynamics of various variables of interest.

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