In view of a pharmacological test system with living cells we examined Förster Resonance Energy Transfer (FRET) between fluorescent proteins upon Total Internal Reflection (TIR) of a laser beam. We measured in particular HeLa cells expressing membrane associated EGFR-CFP and Grb2-YFP prior to and subsequent to stimulation with the epidermal growth factor EGF. In addition, HeLa cells expressing a membrane associated Green Fluorescent Protein (HeLa hFR-GPI-GFP) were incubated with the energy acceptor Nile Red for further validation. Measurements range from fluorescence microscopy to Fluorescence Lifetime Imaging (FLIM) of a larger number of samples in a TIR reader.
Laser absorption spectroscopy utilizes a tunable infrared source, providing the necessary selectivity, to detect the characteristic fingerprint spectral absorption of an abundant gas. In a simple embodiment such as single-pass absorption, sensitivity is limited as attenuation becomes minuscule for trace level concentrations; a problem exacerbated in the midinfrared region due to significant detector noise. Sensitivity can be improved by increasing interaction between the optical field and molecular ensemble with methods such as a multiple-pass Herriot cell or resonant cavity ring-down spectroscopy but these techniques have a substantial overhead in instrumentation. An alternative approach to this problem is Phase Fluctuation Optical Heterodyne (PFLOH) spectroscopy. Here, interferometric effects are used to detect the minute heating of the sample gas when incident laser light of the appropriate wavelength is absorbed. More specifically, by placing the absorption chamber within one arm of a Mach-Zehnder interferometer, heat-induced changes in the optical path length can be detected with great sensitivity through the resulting fringe modulation. A secondary benefit is that although excitation occurs in the infrared, its effects can be detected using visible lasers and silicon detectors, thereby obviating the need for cooled, infrared detectors. We will present our results used to detect ethane using absorption in the 3.33-3.37 μm region. The Mach-Zehnder interferometer used a Helium Neon laser for the probe laser, and a broadly tunable Optical Parametric Oscillator (OPO) for spectroscopic excitation. We have demonstrated detection levels at parts per billion with further sensitivity possible by implementing several identified improvements.
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