[Illinois] ECE 416 Fluorescence III and Homogeneous Assays I

By Brian Cunningham

University of Illinois at Urbana-Champaign

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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;In this lecture, we continue the discussion of Fluorescence. Examples of results are shown from the fluorescent dyes and how they may be used from a fluorescent microscope. Getting results and images from Fluorescence Scanners are then discussed next. The laser is focused down on a point in the sensor and that point will give off light that can be detected. Then, we move on to the applications of homogeneous assays which are the same surface type the whole time (not half liquid and then half solid), but can only be performed for a small subset of assays. The fluorescence dyes can be placed on this or a heterogeneous assay which has one or both of the assay components immobilized on a sort of solid surface. The interaction occurs on the surface of the solid phase. The dyes are like antennas and when an electric field for a photon of light propagates, electrons are excited and the dye will light up. However, it depends on the direction of the propagation and polarization. With fluorescence polarization, the dye molecule will most strongly absorb light whose vector is parallel to the long axis of the molecule. The molecules have a preferred direction of physical orientation that may result in the polarized filter not becoming absorbed. If the dye molecule is attached to the protein, only the dye molecule oriented parallel to the Electric field of the incident light will be excited. It must also be stated that there is a time delay between the excitation and emission. When measuring polarization it is between 0 and 1, if we have high viscosity or volume, the molecule will rotate more slowly. Then, an example of a polarized fluorescence sensor is shown.





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  • Brian Cunningham (2013), "[Illinois] ECE 416 Fluorescence III and Homogeneous Assays I," http://nanohub.org/resources/17666.

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