Fluorescence microscope

The fluorescence microscope is one of the large family of light microscopes.

Fluorescence microscope technique

Fluorescence is the property that some bodies or molecules have of emitting a light after being excited by light of a higher energy. An object excited by a wavelength will therefore emit fluorescence at a higher wavelength.

The fluorescence microscope is therefore the same as a light microscope except that the light used is not white but has a defined wavelength range. However, in general the light reaches the specimen from above (epi-fluorescence) and not from below.

After emission, lasers with a single wavelength or excitation filters only allow light of the desired wavelength to pass onto the sample. After the sample has been excited, it then emits a light at a different wavelength. Filters can be used to study only the desired wavelength, i.e. the emission photons by blocking the excitation photons.

Use of the fluorescence microscope

The fluorescence microscope can be used to visualise objects which are naturally fluorescent (chlorophyl etc.) or molecules which have been made fluorescent in order to examine them (proteins bound to GFP, DAPI for DNA, fluorophores, etc.).

Several techniques have been developed using this microscope :

• immunofluorescence (labelling with an antibody bound to a fluorophore);
• FISH or fluorescent in situ hybridisation to label nucleotide sequences due to oligonucleotides binding to fluorophores ;
• FRET or fluorescent energy transfer is used to visualise whether or not two molecules bind;
• BIFC or bimolecular fluorescence complementation is used to visualise binding by reconstituting the integrity of the fluorophore with two molecules.