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Fluorescence Detection Facts
1) Exitation wavelengths are shorter then emission wavelengths. 2) The ratio of excitation energy to the resulting emission energy is greater then 5,000,000 : 1. 3) The radiated emission fluorescence spectrum is constant; the wavelength range ans shape of the spectrum remains the same. 4) The intensity of the emission spectrum is directly proportional to the excitation energy and the concentration of the fluorophore under test. 5) The solvent's (background) signal is the summation of: 1) The solvent's fluorescence spectrum and Roman responce, 2) Noise (dark current plus) of the photomultiplier tube, 3) Excitation wavelengths that pass through the emission filter, 4) Emission wavelengths that pass through the excitation filter. 6) Three of the four sources of solvent signal result in a DC component which can be subtracted. The noise of the PMT is composed of DC and fluctuating components. It is the fluctuating component of the noise that effects the repeatability of the detection system. 7) The fluctuating component of the noise from the PMT is proportional to the square root of the input of the light intensity. A four fold increase in light increases the fluctuating noise by two, but also increases the signal to noise ratio by two. It is very importent to have the maximum light possible to the PMT to obtain the maximum signal to noise ratio. 8) The fluctuating component of the noise from the PMT is also proportional to the square root of the bandwidth of the electronics. A lamp that is turned on for a short duration of time, requires a bandwidth greater then 10,000x that used for a continuously operating lamp. The increased noise due to the increased bandwidth offsets the benefit of the higher light intensity. 9) The higher the ratio of the fluorophore's fluorescence signal to the solvent's DC signal, the lower the limit of fluorescence signal detection. Thus, it is very important to select correct optical filters, low noise PMT tubes, and a solvent with low fluorescence response of it's own. 10) The optimum illumination sources for filter fluorometers are lamps or lasers that provide single line wavelengths. The radiated energy on either side of the line source is low thus duplicating the thoughput of an optical excitation filter. Lasers do not require excitation filters since emission wavelengths are not present in their output spectrum. 11) Mercury lamps radiate many different line sources of energy; major lines are the following wavelengths: 245, 265, 313, 366, 405, 436, 546, and 577nm. 12) The selection of excitation and emission optical filters is based upon
the following factors: 13) Dichroic filters are applicable when there is a relatively large separation between the peak excitation and emission wavelengths. 14) Interference filters are recommended to obtain maximum fluorescence to solvent signal ratio. 15) When using a Spectrofluorometer, at fixed wavelengths, the fluorescence to solvent's DC signal ratio will be improved by using optical filters. 16) Changing the electronic gain (changing range positions) will not change the "Fluorescence Signal / Noise" or the "Fluorescence Signal / Solvent" ratios. (The electronic gain effects each of these parameters the same- thus the ratio remains the same.)
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