Phiv, plateau d'histologie végétale

Microscopy with epifluorescence

Principe

Fluorescence: wavelength-activated molecule emits light in all the directions of a higher wavelength (with less energy).
In fluorescence microscopy two types of objects can be distinguished : the first ones, when excited, emit fluorescent light by themselves (primary fluorescence or autofluorescence : chlorophyl, oil…), the others must be bound to a fluorescent marker to emit fluorescence (secondary fluorescence).
In the absence of method allowing to focus the observation of the fluorophores to a focal plan (confocal microscope with laser scanning or multiphoton microscopy), the technique is just called epifluorescence microscopy.
The deconvolution is used to treat the images in order to limit the image to a focal plan (= an optical cut).

Use

In fluorescence microscopy, cells, organelles, non fluorescent molecules can be visualized by labelling them with fluorochromes (with the DAPI the DNA fluoresces in blue). Genetic markers like the GFP (Green Fluorescent Protein) are also very much used in biology. In this case, the protein is translated directly from a transgene by the cell itself and does not require any substrate addition. Fluorescence can then be visualized directly in the living cells.

Examples