Fluorescence Microscopy
This is a great tool with which to visualize many biological phenomena in both live and fixed specimens. Fluorescence either endogenous or induced, emitted from the specimen, can be detected as an image as single photons and thus can be used as a quantitative and qualitative method to assess a biological process.
What is fluorescence?
Fluorescence is the visible or invisible radiation emitted by certain substances as a result of incident radiation of a shorter wavelength the delay between excitation and emission is typically less than a nanosecond. When there is a short (~microsecond) delay between absorption and emission this is known as delayed fluorescence. When this delay is greater than a microsecond this is termed phosphorescence.
The process of fluorescence is described in figure 1. When the fluorescent molecule absorbs a photon (green) electrons are excited from the ground state to a higher energy level or excited state. Subsequently, as the excited electron relaxes and returns to a lower energy level the energy is emitted in the form of a photon (red). The shift in emission photon wavelength from that of the exciting photon is known as the Stokes shift.
The use of fluorescence in microscopy
The principles of fluorescence microscopy are similar to that of conventional brightfield microscopy, with an obvious difference in the source and the manipulation of the illuminating light. Ideally, the fluorescence microscope must maximize the collection of the emission light whilst at the same time excluding that used for excitation. The emission light must then be efficiently passed to detector typically a CCD camera (charge coupled device) or EMCCD (electron multiplication CCD) camera for low light applications, for visualisation.
The properties of many fluorophores are such that they have characteristic excitation and emission profiles (see link to probes website). So that the fluorophore in question is only excited and it’s emitted light is only collected we must restrict the excitation and emission light paths to allow the transmission of the desired areas of the visible spectrum. This is achieved through the use of a number excitation and emission filters in combination with diachronic mirrors (these reflect light below a certain wavelength while allowing transmission above it).
