Ultrahigh-resolution multicolor colocalization of single fluorescent probes

An optical ruler based on ultrahigh-resolution colocalization of single flu orescent probes is described in this paper. It relies on the use of two uni que families of fluorophores, namely energy-transfer fluorescent beads (Tra nsFluoSpheres) and semiconductor nanocrystal quantum dots, that can be exci ted by a single laser wavelength but emit at different wavelengths. A multi color sample-scanning confocal microscope was constructed that allows one t o image each fluorescent light emitter, free of chromatic aberrations, by s canning the sample with nanometer scale steps with a piezo-scanner. The res ulting spots are accurately localized by fitting them to the known shape of the excitation point-spread function of the microscope. We present results of two-dimensional colocalization of TransFluoSpheres (40 nm in diameter) and of nanocrystals (3-10 nm in diameter) and demonstrate distance-measurem ent accuracy of better than 10 nm using conventional far-field optics. This ruler bridges the gap between fluorescence resonance energy transfer, near - and far-field imaging, spanning a range of a few nanometers to tens of mi crometers.