High-content data evaluation by means of confocal fluorescence spectroscopy

Abstract

With the development of fluorescence correlation spectroscopy (FCS) in the 1990s, a fundamental milestone was set in the field of highly resolved and quantitative fluorescence detection. Moreover, the increasing knowledge about the meaning of confocal fluorescence detection and its experienced handling enabled unrivalled degrees of detection sensitivity. In the end of the decade, hence the possibility of detecting single fluorescent molecules initiated a productive scientific rush for a comprehensive exploitation of fluorescence properties on the single molecule level. Meanwhile, confocal fluorescence spectroscopy has overcome its predominantly scientific meaning in basic research, and rather found wide applications even in the life science industry. However, biological assay systems relevant for industrial dedication mainly require reagent concentrations above those of classical single molecule detection. They rather lie within the fluctuation range, which means that in the temporal average a plurality of fluorescent particles rather than only one is present within the confocal detection area at a time. Thus, although individual molecules may not longer be resolved, their diffusive fluctuations are furthermore visible and contain a valuable amount of information. On the other hand FCS, a typical fluctuation evaluation technique, is restricted to the quantification of translational molecular diffusion, which in a number of cases is not sufficient to characterize the biological system of interest. Hence, a series of additional complementary fluctuation and non-fluctuation based confocal spectroscopic techniques was developed during recent years and named FCS + plus. They provide simultaneous access to a multitude of molecular properties like concentration, translational and rotational diffusion, molecular brightness, coincidence and fluorescence lifetime and thus meet the needs of both scientific research and industrial application. In the following particular aspects of molecular polarization will be shortly described and illustrated by a comparison of stationary and time-resolved anisotropy. Another valuable subject in especially industrial application of fluorescence is that of artificially interfering effects. The most prominent of these disturbances is given with the potential auto-fluorescence of non-labeled biological molecules. In these - frequently appearing - cases the wanted signal of fluorescently labeled material will be superimposed by artifacts, making a proper data interpretation rather difficult. However, the FCS + plus' abilities of decomposing a fluorescence signal into the molecular species' fractional contributions enables a sophisticated consideration of unwanted interferences.