Prism-based spectral imaging of four species of single-molecule fluorophores by using one excitation laser.

A prism-based imaging system for simultaneously detecting four species of single-molecule (SM) fluorophores was developed. As for the detection method, four spectrally distinct species of BigDye fluorophores were bound to 50-nm-diameter gold nanoparticles (AuNPs) to form AuNP/BigDye complexes. Four species of complexes were randomly immobilized on different fused-silica slides. BigDyes were excited by an argon-ion-laser (excitation wavelengths: 488 and 514.5 nm) beam through total internal reflection on the slide surface. SM fluorescence emitted from a complex was spectrally dispersed through a prism to form an SM spot elongated in the spectral direction on a charge-coupled device. A scattered light spot generated by the AuNP of the same complex under 594-nm laser illumination was used as a wavelength reference, and the SM fluorescence spectrum was obtained from the pixel-intensity pattern of the elongated SM spot. Peak locations of fluorescence spectra of all the observed SM spots were obtained, and their histograms were distinctly separated according to species. SM spots can thus be classified as one of four species according to their peak locations. By statistically analyzing the histograms, the classification accuracy was estimated to be above 93.8 %. The number of pixels in the spectral direction required for classifying four species of SM fluorophores was estimated to be 10. As for the conventional system (which uses two excitation lasers), 15 pixels are required. Using BigDyes as the four fluorophores (which consist of donors linked to acceptors and can be excited by just an argon-ion laser) is the reason that such a small number of pixels was achieved. The developed system can thus detect 1.5 times more SM fluorophores per field of view; that is, its throughput is 1.5 times higher. The approach taken in this study, namely, using BigDye with a prism-type system, is effective for increasing the throughput of DNA microarray-chip analysis and SM real-time DNA sequencing.