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Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy
Published on: January 18, 2017
High-precision and wide-range feature wavelength quantitative analysis (FWQA) method for fluorescent substances based
Tianyu Guo1, Tianshuo Lan1, Xinna Yu1
1State Key Laboratory of Submarine Geoscience, School of Automation and Intelligent Sensing, Shanghai Jiao Tong University, Shanghai 200240, China.
None:
Conventional fluorescence spectrometry has a limited linear range and insufficient detection accuracy at high concentrations in wide-range analysis. To address this problem, a novel feature wavelength quantitative analysis method (FWQA) for fluorescent substances is proposed. This method integrates feature wavelength extraction which utilizes inner filter effect (IFE) induced concentration dependent red shift (CDRS) with a full-spectrum analysis. The wavelengths λmax, (corresponding to the peak intensity Imax), λl and λr (corresponding to the intensity nImax on the left and right sides of the peak in the fluorescence spectrum) serve as feature wavelengths. A linear model was established to link the concentration of substances with these wavelength combinations. By dynamically optimizing of n and wavelength combination strategies, adaptively matching the spectral morphology of different fluorescent substances, the optimal fitting model is obtained. The experimental results show that for sodium fluorescein, rhodamine B, and chlorophyll-a, this method extends the linear detection range to more than five times that of traditional methods, with a relative average error of less than 1 %, improving the range and accuracy. The testing of light source fluctuations and receiver position changes found that the impact on characteristic wavelengths was minimal. This confirms the stability of the method, providing valuable solutions for high-precision and wide range fluorescent substance analysis.

