Fluorescence Microscopy

Fluorescence microscopes are designed to detect the fluorophores in a sample; either naturally occurring fluorophores or those used as dyes and labels. These fluorophores absorb energy in specific wavelengths (excitation range), and emit it in a different wavelength (emission range). This allows us to identify fluorophores by absorption and emission. More precisely, as each fluorophore has a unique excitation spectrum and a unique emission spectrum, each peaking at a specific wavelength, they can be distinguished by either their excitation spectrum, by their emission signal, or by their Stokes shift, which is the distance between peak excitation and peak emission. This is the basic principle behind all fluorescence microscopy systems, whether it be a simple system such as a epifluorescent microscope or a more complex setup such as those used in multiphoton systems.

Fluorescence Filter Sets

Each fluorescence filter cube features three distinct optical filters: an excitation filter a dichroic mirror and an emission filter. The excitation filter, otherwise called an exiter, is a bandpass filter designed to pass only the wavelengths of light which are absorbed by the fluorophore. By doing so, it minimizes excitation that might be caused by the light source and other sources of fluorescence and also blocks excitation wavelength light in the flurorecence emission band. The minimum transmission of this filter is what will determine the brightness or brilliance of the final image. In general, an excitation filter should have a minimum of 40% transmission; transmission of greater than 85 percent will give a bright, well-lit image. Optical density determines the background darkness, and should be at minimum 3.0 but preferably closer to 6.0. The excitation filter should be chosen so that the center wavelength (CWL) is close to the peak excitation of the fluorophores, and the bandwith is completely within the fluorophores excitation range. The dichroic mirror is a specialized edge filter sometimes called a dichromatic beamsplitter. When used at a 45 angle of incidence, it is able to reflect light in the wavelengths of the excitation band and transmit light from the emission band to the emitter and on to the detector. The third component of the fluorescence filter is the emitter. This is also an bandpass filter, and passes exclusively light emitted by the fluorophore. Excitation light and all other light outside, both UV an IR, are blocked out effectively by the emitter filter.