As explained under Limitations of fluorescence microscopy, the intensity of fluorescence is much lower than the light produced by the microscope lamphouse. Consequently the efficiency and sensitivity of the system is of great concern.
The light source must be powerful and stable and the light it produces must be efficiently coupled into the microscope. The equation is simple: The more light reaches the sample, the more fluorescence is produced.
On the other hand side, intensive light has its downside, photobleaching and cell damage are of great concern. Light should only be irradiated onto the sample when the cells are really observed and images taken. Consequently, the system needs a reasonably fast shutter.
The camera needs to be appropriately sensitive and low in noise to keep the exposure times to a minimum while still assuring good quality images.
The objective should have a high numerical aperture (NA). The NA determines the size of the cone of light that is captured and also the spatial resolution of the image. Most standard objectives do not transmit UV light, consequently, if UV dyes are employed, the right type of objectives has to be used.
The relative weakness of the fluorescence makes it necessary to quantitatively block the excitation light from reaching the detector. This is one of the reasons why modern fluorescence microscopes are episcopic microscopes ("epi-fluorescence microscopes"). This means that the irradiation of the sample is realized via special condensers through the objective and not through the transmission condenser. In such a setup the objective NA additionally determines the amount of incident light that reaches the specimen.
For each fluorochrome a suitable set of fluorescence filters has to be used. The excitation filter has to transmit only that part of the light spectrum produced by the light source that is needed for the excitation of the dyes but at the same time has to block all the remaining wavelength ranges efficiently. A dichroic filter has to separate the excitation light from the emitted light to be observed. Finally, an emission filter has to eliminate residual excitation light that still might reach the camera for example due to reflection from surfaces or the sample and would cause significant background intensity. In most of the fluorochromes the excitation and emission spectra largely overlap, that means, that the Stokes shift is usually considerably smaller than the width of the two peaks. Thus, often enough a better signal-to-background ratio is obtained if the excitation is done off-maximum, at lower wavelength.