NDLI: Prism-based Spectral Imaging of Four Species of Single-molecule Fluorophores by Using One Excitation Laser

Content Provider	Springer Nature Link
Author	Haga, Takabu Sonehara, Tsuyoshi Fujita, Takeshi Takahashi, Satoshi
Copyright Year	2013
Abstract	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.
Starting Page	591
Ending Page	597
Page Count	7
File Format	PDF
ISSN	10530509
Journal	Journal of Fluorescence
Volume Number	23
Issue Number	3
e-ISSN	15734994
Language	English
Publisher	Springer US
Publisher Date	2013-03-08
Publisher Place	Boston
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Prism Spectral imaging Four colors Single molecule BigDye Fluorescence-resonance energy transfer Biomedicine general Biophysics and Biological Physics Biotechnology Biochemistry Analytical Chemistry
Content Type	Text
Resource Type	Article
Subject	Sociology and Political Science Spectroscopy Law Biochemistry Clinical Biochemistry Clinical Psychology

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
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3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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