Though much of the interest in fluorescence in the past has been on measuring spectral qualities such as wavelength and intensity, there are two other highly useful intrinsic properties of fluorescence: lifetime (or decay) and anisotropy (or polarization). Each has its own set of unique advantages, limitations, and challenges in detection when it comes to use in biological studies. This review will focus on the property of fluorescence lifetime, providing a brief background on instrumentation and theory, and examine the recent advancements and applications of measuring lifetime in the fields of high-throughput fluorescence lifetime imaging microscopy (HT-FLIM) and time-resolved flow cytometry (TRFC). In addition, the crossover of these two methods and their outlooks will be discussed.
Here we present a simple, high-throughput, time-resolved flow cytometer to detect changes in NAD(P)H autofluorescence lifetime. The lifetime of this metabolite is characterized by its binding state, when bound to its coenzyme the metabolite exhibits a longer lifetime (1-7 ns) indicating a preferential energy generation state of oxidative phosphorylation. A shorter lifetime, 0.1-1 ns, however, indicates an unbound coenzyme which has been correlated to the ATP energy pathway of glycolysis. The work here in is to demonstrate the capabilities of simple, time-resolved cytometry to show subtle changes in autofluorescence lifetime detection. Flow cytometry results are validated by the use of the Agilent Seahorse HS Mini. Results include the method of ATP energy generation measured by the Seahorse as a comparison to flow cytometry lifetime shifts. Measurements were made using an estrogen receptor positive breast cancer cell line and the same cell line under developed Tamoxifen resistance. The outcome of this work is a map of the metabolic profile of tamoxifen resistance in breast cancer.
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