Analog mean-delay (AMD) method is a new powerful alternative method in determining the lifetime of a fluorescence molecule for high-speed confocal fluorescence lifetime imaging (FLIM). The major advantage of this method is that the mean delay effect caused by a slow measurement system can be completely removed. The measurement speed can be very fast compared to the conventional TCSPC method because the AMD method can detect multiple photons simultaneously for a single excitation pulse. More accurate fluorescence lifetimes can be determined with more photons such that an accurate fluorescence lifetime image can be acquired quickly by the AMD method. In this study, we demonstrated cancer discrimination based on real-time AMD(Analog Mean-Delay)-FLIM(Fluorescence Lifetime Imaging Microscopy). We subcutaneously injected MDA-MB-231 breast cancer cell lines into nude mice. After subcutaneous (SC) injection of sodium fluorescein, the fluorescence lifetime of sodium fluorescein was measured by real-time AMD-FLIM. The fluorescence lifetime of sodium fluorescein depends on the local pH and pH differs between abnormal and normal tissues, cancer tissue can be discriminated from normal tissue by measuring the fluorescence lifetime of pH-sensitive sodium fluorescein. The measured fluorescence lifetime of sodium fluorescein inside the normal and abnormal tissues were 4.15~4.28 ns and 2.36~3.18 ns. Since the measured fluorescence lifetime for abnormal tissues were well differentiated from those for normal tissues, the fluorescence lifetime of sodium fluorescein could be used as an indicator to increase the accuracy of cancer detection with confocal microscopy or endoscopy.
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