Compact Time-Correlated Single Photon Counting Fluorescence Lifetime Analysis System for Point-of-Care Applications

應用於即時檢驗的緊湊型時間相關單光子計數熒光壽命分析系統研究

Student thesis: Doctoral Thesis

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Award date5 Sept 2019

Abstract

Fluorescence analysis plays a key role in many areas such as biology, chemistry, and medicine research. Commercially available fluorescence analysis instrumentations based on conventional design typically use complex optical and electrical subsystems to provide high-accuracy lifetime measurement, rendering the entire system bulky and expensive. However, well-designed optics and electronics are necessary to maintain a good level of performance for practical applications. To mitigate the above shortcomings, this thesis presents design techniques for realizing portable, low-cost time-correlated single photon counting (TCSPC) system for fluorescence lifetime analysis applications. The design techniques specifically focus on effective excitation rejection, component size reduction, detection limit improvement, and fluorescence lifetime extraction accuracy, suitable for a variety of chemical and biomedical applications. A prototype has been made to demonstrate the design techniques.

First, we present the hardware and firmware design for the proposed TCSPC system. Each optical and electrical component has been carefully selected to meet the requirements of compactness, excitation rejection, and lifetime extraction accuracy. The time-to-digital module of the proposed TCSPC system achieves a root mean square (RMS) differential non-linearity (DNL) of 4% of the least significant bit (LSB) and a full width at half maximum (FWHM) temporal resolution from 121 ps to 145 ps within the 500 ns full-scale range (FSR).

Second, we present the design of optics, with a liquid-core waveguide (LCW) at its core, which is capable of efficiently propagating the emission to the detector and simultaneously attenuating the excitation. The LCW is integrated into the proposed TCSPC analysis system, which enables significant size reduction while delivering good performance. The optimal length of the LCW is determined based on Monte Carlo simulations to satisfy both temporal dispersion and emission-to-excitation ratio requirements.

Third, we present experimental results from the proposed TCSPC prototype in the extraction of fluorescence lifetime of fluorescein, coumarin 6, and rhodamine 6G, down to the concentration of 1 nM, 1 nM, and 2.5 nM, respectively, significantly outperforming similar fluorescence lifetime analysis systems in terms of detection limit. The proposed system has also been verified by fluorescence lifetime measurements of a V-carbazole-DNA conjugated bioassay, showing a detection limit of 6.25 nM. The average lifetime of V-carbazole-DNA conjugated bioassay exhibits a linear response in the range of 1.0 ns to 6.5 ns, providing a wide dynamic range for lifetime measurement. The proposed TCSPC system demonstrates the enabling technology for rapid, point-of-care (POC) diagnostics.