Simultaneous two color image capture for sub-diffraction localization fluorescence microscopy

A sub-diffraction limit fluorescence localization microscope was constructed using a standard cooled 1.4 mega-pixel fluorescence charge-coupled device (CCD) camera to simultaneously resolve closely adjacent paired quantum dots on a flat surface with emissions of 540 and 630 nm. The images of the overlapping Airy discs were analyzed to determine the center of the point spread function after noise reduction using Fourier transformation analysis. The Cartesian coordinates of the centers of the point spread functions were compared in serial images. Histograms constructed from serial images fit well to Gaussian functions for resolving two quantum dots separated by as little as 10 nm in the x-y coordinates. Statistical analysis of multiple pairs validated discrimination of inter-fluorophore distances that vary by 10 nm. The method is simple and developed for x-y resolution of dilute fluorophores on a flat surface, not serial z sectioning.