Principle of Luminescence Microscopy

The photometric throughput of a luminescence microscope is estimated as follows assuming linear and isotropic response and illumination low enough to avoid saturation [25]. Excitation fight with wavelength X is absorbed by the chromophores in the object field with a probability governed by their absorption cross section taW in cm molecule defined as  

For calculating the intensity of the signal at the detector one has to take into account the solid angle of collection for the chromophore embedded into a medium with refractive index n and acting as a non-Lambertian diffuse point source (i.e. the intensity of which in a given direction is not proportional to the cosine of the angle with respect to the direction of maximum irradiance). This solid angle is equal to  

Further assuming an overall transmittance toptiX) of the entire optical setup, a measuring spectral bandwidth and a spectral sensitivity qdeti. ) of the detector, one finally gets the photoelectron flux (in photoelectrons s molecule ) in the image plane taking into 

There arc various techniques for recording luminescence microscopy images and for improving the contrast and/or the resolution. Their description is out of the scope of this chapter. It is howevCT worth mentioning confocal microscopy. In this technique, conventional illumination in which the observed object is completely and evaily irradiated by a light source (wide field illumination) is replaced by point laser illumination. A scanning unit is inserted 

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