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This article describes the use of confocal fluorescence correlation microscopy as a way to measure the translocation and dynamics of molecules on a sub-cellular scale. Using this technique it is possible with a single measurement to determine not only the size of labelled molecules in terms of their translational diffusion coefficient (DT) but also their number density within a sample1,2. The sample volume is defined precisely by confocal optics, and in our system is a quasi cylindrical space of approximately 1 fL, with a length to width ratio of between 6 to 8. The probe volume is therefore of subcellular dimensions. FCM has a great potential in cell biology, since most cellular events involve changes in the physical size and/or number of individual sub-cellular components e. g., the binding and release of sub-units or effectors, changes in the level of gene expression, the processes of polymerisation and depolymerisation, and changes in microviscosity or compartmentation. In such complex systems time-dependent molecular interactions can be resolved from the correlated intensity fluctuations of individual specifically-labelled, or intrinsically fluorescent, molecules in the probe volume. |
