PCH resolves mixtures of different oligomers accurately. In practice, single color FCS cannot detect dimerization of membrane receptors. However, since the diffusion time only scales with the cubic root of the mass, FCS is not sufficiently sensitive to detect changes in molecular mass 21. Typically, the diffusion coefficient of the fluorophores and the average number of molecules (inversely related to the G(0) value) within the illumination volume can be obtained by FCS 20. The amplitudes of the intensity fluctuations can be uniquely described by the molecular brightness of the fluorophore (ε) and the average number of fluorophores (n) within the illumination volume 17 ( Figure 2).
These methods analyze the fluorescence intensity fluctuations that can be observed in time when the fluorophores diffuse in and out of an illumination volume. Alternatively, moment methods, such as fluorescence-correlation spectroscopy (FCS) 16, photon counting histogram (PCH) 17, and Number and Brightness (N&B) 18, 19, are suitable for quantitative oligomer measurements. It is applicable to both chemically fixed and live cells, and allows the analysis of several regions of interest of the cell simultaneously using standard fluorescence microscopy 15. One of the most versatile methods to measure density and oligomerization of fluorescently tagged proteins within single images is the spatial intensity distribution analysis (SpIDA), which relies on spatial sampling. Single molecule step-bleaching resolves the stoichiometry of protein oligomers only if they are immobile 14. Moreover, their temporal resolution (in the order of minutes per image) cannot follow kinetics in the range of seconds. The sub-unit composition of protein oligomers cannot be resolved on a purely spatial basis even by super-resolution methods at x,y resolution of 20-70 nm such as PALM 11, STORM 12, and STED 13. The confocal volume (x,y ≈ 300 nm, z ≈ 900 nm) is insufficiently resolved for proving molecular interaction and stoichiometry, even after optimization by image restoration algorithms 10. Despite the importance and ubiquity of receptor oligomerization, recognized as a key event in cellular signaling 1, 2, 3, 4, 5, 6, 7, few methods can detect clustering events and measure the degree of clustering experimentally 8, 9. Upon extracellular ligand binding, receptors initiate the intracellular signal transduction depending on their conformation, oligomerization, potential co-receptors and membrane composition. We describe a Total Internal Reflection Fluorescence-Number and Brightness (TIRF-N&B) imaging approach for determining the average oligomeric state of receptor molecules at the plasma membrane of live cells, aiming at linking the receptor assembly dynamics to the biological function of the proteins ( Figure 1). Finally, large cell areas as well as sub-cellular compartments can be explored. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics.
INTERFACE ANDOR SOLIS TO MATLAB SERIES
Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. Because only two moments are considered, N&B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. This is, at the same time, the strength and the weakness of the method. Thus, N&B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. In particular, N&B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. N&B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&B) analysis.
Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering.
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