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Time-resolved fluorescence spectroscopy is an extension of fluorescence spectroscopy. Here, the fluorescence of a sample is monitored as a function of time after excitation by a flash of light. The time resolution can be obtained in a number of ways, depending on the required sensitivity and time resolution:
Homogeneous, mix-and-read TR-FRET assays offer advantages over other biomolecular screening assays, such as fluorescence polarization (FP) or TRF assays. [3] In FP assays, background fluorescence due to library compounds is normally depolarized and background signal due to scattered light (e.g. precipitated compounds) is normally polarized.
Steady-state fluorescence anisotropy only give an "average" anisotropy. Much more information can be obtained with time-resolved fluorescence anisotropy [7] where the decay time, residual anisotropy and rotational correlation time can all be determined from fitting the anisotropy decay. Typically a vertically pulsed laser source is used for ...
Many of the detection modes (absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization) are available stand-alone in dedicated plate readers, but are very often found today combined into one instrument (multi-mode plate reader).
In 2013, Hafi et al. [2] developed a novel super-resolution technique through sparse deconvolution of polarization-modulated fluorescent images (SPoD). Because the fluorescent dipole is an inherent feature of fluorescence, and its polarization intensity can be easily modulated with rotating linear polarized excitation, the polarization-based super-resolution technique therefore holds great ...
For allowing anisotropy measurements, the addition of two polarization filters is necessary: One after the excitation monochromator or filter, and one before the emission monochromator or filter. As mentioned before, the fluorescence is most often measured at a 90° angle relative to the excitation light.
At some point in time, the flow is suddenly stopped, and the reaction is monitored using a suitable spectroscopic probe, such as absorbance, fluorescence or fluorescence polarization. The change in spectroscopic signal as a function of time is recorded, and the rate constants that define the reaction kinetics can then be obtained by fitting the ...
Fluorescence polarization immunoassay (FPIA) is a class of in vitro biochemical test used for rapid detection of antibody or antigen in sample. FPIA is a competitive homogenous assay , that consists of a simple prepare and read method, without the requirement of separation or washing steps.