Multiparameter Fluorescence And Selection Of Optimal Filter Sets: Mathematics And Computer Program.

In Multi-Parameter Fluorescence microscopy (MPF), as many as five fluorophores have been used simultaneously, to study structural and physiological properties of cells in culturel. Each fluorophore is examined and evaluated by using a filter set (excitation filter, emission filter and dichroic reflector) specific for that fluorophore. The filters for each fluorophore cannot be chosen in isolation, but depend on what other fluorophores are to be used. Thus filter selection depends on a large number of spectra, and the task of optimization becomes complex as the number of fluorophores is increased, so that the excitation and emission wavelengths begin to overlap. This paper presents a mathematical statement of the problem, a method for computer solution, and recommendations for standard presentation of fluorescence spectra. Related efforts by other workers has approached two problems - analysing envelope spectra in terms of their known components, and finding the best quasi-monochromatic wavelengths to quantify components by ratioing. In contrast, the present work considers specific excitation of each fluorophore, considers the filter bandpasses in relationship to the detector sensitivity, and also considers simultaneously the excitation and the emission aspects of fluorescence. Though primarily directed to fluorescence microscopy, this work has application in other fields, such as flow cytometry, where multiple fluorophores are used. The mathematical statement also gives insight into the process of quantifying fluorescence.