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Intensity of the fluorescence signal

Several factors influence the amount of fluorescence emitted from the sample, treated with a certain amount of fluorochrome.
These include:

-) The concentration of dye in the sample sections;
-) The thickness of the sample;
-) The extinction coefficient of the dye;
-) The quantum efficiency of the dye;
-) The amount of colored material actually present in the visual field of the microscope.

The concentration of the dye in the sample is determined by a proper use of the dye in the operations of "marking" of the sample. A greater amount of coloring material, in fact, not always leads to a better outcome with regard to the 'fluorescence emission from the sample, in fact, often this is due to phenomena such as loss of contrast and introduction of "artifacts" in the subject.

The thickness of the sample is critical for success in the analysis of a subject investigated by the technique of fluorescence. Samples are too thick, in fact, retain larger amounts of dye leading to phenomena such as those described above. Samples are too thin, however, does not allow the dye to "mark" in an optimal way to examine the subject, this is a result of a weak or almost no fluorescence emission.

The extinction coefficient tells us the amount of light that is absorbed by a given dye and reflects the absorption characteristics, dependent on the wavelength, indicated by the spectrum of excitation of the fluorophore. The emission increases with a higher absorption of incident light, which means that fluorophores with higher extinction coefficients tend to emit more intensely, and require less energy to excite the sample properly. Although many fluorochromes have high extinction coefficients at wavelengths of excitation peak, sample preparation techniques often limit the maximum permitted concentration in the sample, thus reducing the overall amount of light actually absorbed by the sample marked. Experimentally, the advantage of fluorochromes with a high extinction coefficient is the ability to use reasonable amount of excitation light, while avoiding the negative processes as "fading" and "photobleaching".

The quantum efficiency, which is the ratio of light energy absorbed and emitted fluorescence, determines the amount of light energy that is converted to fluorescence. The fluorochromes commonly used today have a quantum efficiency of about 0.3 to 0.6, but the actual value can be reduced by the phenomenon of "quenching".

The product of these factors, in addition to the fact that many preparations have very small amounts of labeled material observed in the field of view, gives the ratio of intensity of fluorescence emitted by the intensity of excitation light, between 1 / 10000 and 1 / 1000000. Current techniques (eg fluorescence in situ hybridization), using small amounts of fluorescent material, may have lower ratios (one billionth or 1 / 0000000000). Thus, to obtain a fluorescent image of adequate contrast, the fluorescence microscope must be able to attenuate the excitation light up to 1 / 100000000000 (very weak fluorescence) without lowering the fluorescence signal. The use of appropriate optical filters, together with a proper configuration of the intrinsic fluorescence microscope, are an essential component that contributes enormously to the filtering process.