Tsuyoshi Sonehara

Dual-view imaging system using a wide-range dichroic mirror for simultaneous four-color single-molecule detection.

A dual-view imaging system for simultaneous four-color single-molecule (SM) detection was developed. As for the detection procedure, four species of SM fluorophores, namely, Alexa 488, 555, 647, and 680, are immobilized on different slides and excited by evanescent-wave illumination. Fluorescence emitted from an SM fluorophore is split by a wide-range dichroic mirror (WR DM) in a dual-view optics and imaged as two SM fluorescence spots (SM spots) on an electron-multiplying charge-coupled device (EM-CCD) at 100 Hz. The transmittance of the WR DM changes gradually over the wavelength range of 500 to 700 nm so that the signal ratios of the two SM spots for the four fluorophore species differ. A method for classifying SM fluorophores into four species in accordance with their signal ratios was developed. It was used to classify 597 SM fluorophores at an accuracy of above 98% for all the species. This accuracy is comparable to that of a conventional four-color SM detection system. To classify four species, the conventional system disperses SM fluorescence with a prism and provides an elongated SM spot that uses more pixels of an EM-CCD chip than that of the developed system. The developed system can thus detect 1.5-fold more SM spots with the same-size EM-CCD chip, so it can achieve 1.5-fold higher throughput. Moreover, the developed system is based on a simple and practical approach, namely, replacing an ordinary dichroic mirror in a commercially available dual-view optics with a WR DM. This replacement transforms a dual-view imaging system for two-color detection into a system for four-color detection. The developed system is suitable for detection systems of next-generation DNA sequencers and DNA microarray-chip analyzers.

Simultaneous four-color imaging of single molecule fluorophores using dichroic mirrors and four charge-coupled devices

We developed a total-internal-reflection (TIR) fluorescence microscopy using three dichroic mirrors and four charge-coupled devices (CCDs) to detect simultaneously four colors of single-molecule (SM) fluorophores. Four spectrally distinct species of fluorophores (Alexa 488, Cy3, Cy5, or Cy5.5) were each immobilized on a different fused silica slide. A species of fluorophores on the slide was irradiated simultaneously, by two excitation beams from an Ar ion laser (488 and 514.5 nm) and a diode laser (642 nm) through TIR on the slide surface. Fluorescence emitted from the fluorophores was spectrally resolved into four components by the dichroic mirrors, and four images were generated from them simultaneously and continuously, with the four CCDs at a rate of 10 Hz. A series of images was thus obtained with each CCD. Fluorescence spots for a species were observed mainly in the series of images recorded by its respective-color CCD. In the first image in the series, we picked out the spots as continuous pixel regions that had the values greater than a threshold. Then we selected only those spots that exhibited single-step photobleaching and regarded them as SM fluorescence spots. Pixel values of SM fluorescence spots widely differed. Some SM fluorophores had pixel values smaller than the threshold, and were left unpicked. Assuming the pixel values of SM fluorescence spots differed with a Gaussian profile, we estimated the ratios of unpicked fluorophores to be less than 20% for all the species. Because of the spectral overlaps between species, we also observed cross-talk spots into CCDs other than the respective-color CCDs. These cross-talk SM fluorescence spots can be mistaken for correct species. We thus introduced the classification method and classified SM fluorescence spots into correct species in accordance with two kinds of four-dimensional signal vectors. The error rates of fluorophore classification were estimated to be less than 3.2% for all the species. Our system is suitable for the biological studies that desire to simultaneously monitor the four colors of SM fluorophores.

Multicomponent Analysis by Submillimeter-Effective-Length Capillary Electrophoresis using Patterned Fluorescence Correlation Spectroscopy

A multicomponent system is analyzed by using a combination of capillary electrophoresis and patterned fluorescence correlation spectroscopy. In this combination, fluorescent analyte molecules dispersed continuously in a capillary migrate through a stationary interference pattern created by two intersecting excitation laser beams, fluorescence emission from which is monitored with a single detector. The power spectrum of fluctuations in the detector photocurrent gives a virtual electropherogram. Using an effective length of 740 μm, we obtained a resolution of 1.0 for a two-component injection of TOTO-1-stained pUC18 DNA and λ DNA.