Optical Filters and Signal Purity

A major source of enors in all fluorescence measurements is interference due to scattered light, stray light, or sample impurides. This oUmii can be minimized by careful selection of the emisskm filter by the use of optical filters in addition U tbt exdtafion and emission monochromators, and by control periments desired to reveal the presence of unwanted con mnents. The use of optical filtm and control eiqjeriii ts to avoid such artifacts is best illustrated by spedfic examples. 

The most appropriate blank solution is one which is identical to the sampk but does not contain the fluoio-phore. This can be difficult to accomplish with protein or membrane solutions, where the macromokcules themselves are the source of the signal. Such solutions will typically be more strongly scattering than the buffer blanks. In these cases it is useful to add glycogen or colloidal silica (Ludox) to the buffer blank, to mimic the amount of scattering from the sample. This allows one to test whether the chosen filters are adequate Co reject scattered light from the sample. 

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