Design of ultra-sensitive fluorescence detection system for applications in molecular biology

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Authors
Tovkach, Ivan
Issue Date
1-Aug-12
Type
Thesis
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en_US
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Abstract
I present design of a new ultra-sensitive data acquisition system for fluorescence detection applications. Based on single photon detection, the developed system comprises a unique single photon spectrometer with high detection speed and wide linearity dynamic range, and a fiberized capillary sample analyzer. The system is capable of detecting multicolor fluorescence produced by mixtures of fluorescent dyes as well as by micro and nano-objects. In the system, the analyzed sample is forced into the capillary by applying either air pressure or electric field. When the sample passes through a detection area, a laser beam induces multi-color fluorescence. Polychromatic fluorescence is collected by a fiber and directed to the single photon spectrometer. The spectrometer performs color separation and the measurement of fluorescence in the range of wavelengths from 480nm to 720nm. The fluorescence which comes to the spectrometer through the input fiber is coupled to a collimator which produces a parallel polychromatic beam of 10mm diameter. The parallel beam passes through laser rejection filters and undergoes separation on the diffraction grating into constituent wavelength components. The separated monochromatic beams are focused onto channels of the 32-channel PMT by the system comprising spherical mirror. The received photons produce very short current pulses in channels of the 32-channel PMT. The pulses undergo amplification and counting. The obtained photon count is transferred to computer for recording and data processing. The unique combination of fluorescent dyes with known spectra is recognized in each individual measurement by using color deconvolution of the dye's spectra. The developed data acquisition system and its component modules can be used for many applications in Life Sciences, Physics, and other fields. I applied the developed data acquisition system for detection of multicolor fluorescence of mixtures of quantum dots, high speed detection of micro-beads encoded with quantum dots and fluorescent dyes, and DNA sequencing by capillary electrophoresis.
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67 pg.
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The Graduate School, Stony Brook University: Stony Brook, NY.
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