Brightness of Fluorescence Signal
Several factors influence the amount of fluorescence emitted by a stained specimen with a given amount of excitation intensity. These include:
- The dye concentration within stained sections of the specimen and the thickness of the specimen;
- The extinction coefficient of the dye;
- The quantum efficiency of the dye; and of course,
- The amount of stained material actually present within the field of view of the microscope.
The extinction coefficient tells us how much of the incident light will be absorbed by a given dye, and reflects the wavelength-dependent absorption characteristics indicated by the excitation spectrum of the fluorochrome. Emission is increased with higher incident light absorption, meaning that fluorochromes with greater extinction coefficients tend to emit more intensely, and require less energy to adequately excite them. Although many of the fluorochromes have high extinction coefficients at peak excitation wavelengths, practical sample preparation techniques often limit the maximum concentration allowed in the sample, thus reducing the overall amount of light actually absorbed by the stained specimen. Experimentally, the benefit fluorochromes with a high extinction coefficient is the ability to use reasonable amounts of excitation light while avoiding negative quenching processes such as photobleaching.
The quantum efficiency, which is the ratio of light energy absorbed to fluorescence emitted, determines how much of this absorbed light energy will be converted to fluorescence. Today's most commonly used fluorochromes have a quantum efficiencies of approximately 0.3-0.6, but the actual value can be reduced by quenching.
The product of these factors, in addition to the fact that many specimens have very small amounts of stained material in the observed field of view, gives a ratio of emitted fluorescence intensity to excitation light intensity in a typical application of between 1/10000 (10-4) for very highly fluorescent samples and 1/1000000 (10-6). Current techniques (e.g. fluorescence in situ hybridization), which utilize minute amounts of fluorescent material, might have ratios as low as 10-9 or 10-10. Thus, in order to see the fluorescent image with adequate contrast, the fluorescence microscope must be able to attenuate the excitation light by as much as 10-11 (for very weak fluorescence) without diminishing the fluorescence signal. This concept is discussed futher in the Handbook for Fluorescence Microscopy How does the fluorescence microscope correct for this imbalance? Optical filters are indeed essential components, but the inherent configuration of the fluorescence microscope also contributes greatly to the filtering process. This unique configuration is the topic of discussion in the Fluorescence Microscopy section, but first we will discuss fluorescent filters and their characteristics.