High content data evaluation by means of confocal fluorescence spectroscopy

摘要

With the development of fluorescence correlation spectroscopy (FCS) in the 1990s, a fundamental milestone was set inthe field of highly resolved and quantitative fluorescence detection. Moreover, the increasing knowledge about themeaning of confocal fluorescence detection and its experienced handling enabled unrivalled degrees of detectionsensitivity. In the end of the decade, hence the possibility of detecting single fluorescent molecules initiated aproductive 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 thoseof classical single molecule detection. They rather lie within the 'molecular fluctuation range', which means that in thetemporal average a plurality of fluorescent particles rather than only one is present within the confocal detection area ata time. Thus, although individual molecules may not longer be resolved, their diffusive fluctuations are furthermorevisible and contain a valuable amount of information. On the other hand FCS, a typical fluctuation evaluationtechnique, is restricted to the quantification of translational molecular diffusion, which in a number of cases is notsufficient to characterize the biological system of interest.Hence, a series of additional complementary fluctuation and non-fluctuation based confocal spectroscopic techniqueswas developed during recent years and named FCS+plus. They provide simultaneous access to a multitude of molecularproperties like concentration, translational and rotational diffusion, molecular brightness, coincidence and fluorescencelifetime 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 ofstationary and time-resolved anisotropy.Another valuable subject in especially industrial application of fluorescence is that of artificially interfering effects. Themost prominent of these disturbances is given with the potential 'auto-fluorescence' of non-labeled biologicalmolecules. In these - frequently appearing - cases the wanted signal of fluorescently labeled material will besuperimposed by artifacts, making a proper data interpretation rather difficult. However, the FCS+plus´ abilities of decomposinga fluorescence signal into the molecular species´ fractional contributions enables a sophisticatedconsideration of unwanted interferences.