Fluorescence microscope

The Fluorescence is the property which have certain bodies to emit of the Lumière after having absorbed photons of stronger energy (shorter wavelength).

Since the discovery of the phenomenon at the beginning of the 20th century, many applications were born in varied fields of sciences and technology. Thus the optical Microscopie will make it possible to detect the presence of various compounds by the observation of fluorescence.

The microscopy in fluorescence is part from now on of the methods of research used in a routine way.

Principle of microscopy in fluorescence

Luminescence is a process which can be divided into 2 main categories: fluorescence and phosphorescence.

An enlightened object emits light in all the directions of space to a wavelength different the exiting wavelength. For a given substance if the emission of the light ceases as of the stop of the excitation then one says it fluorescent, if this emission continues the substance then is phosphorescent.

In fluorescence one distinguishes two types of objects: the first emit fluorescent light by themselves, one speaks about primary fluorescence or autofluorescence (chlorophyl, oil…), the others must be combined with a fluorescent substance to emit fluorescence one thus speaks about secondary fluorescence.
En microscopy of fluorescence one can visualize substances, cells, nonfluorescent molecules by marking them with fluorochromes (the DAPI marks the DNA which fluoresce in blue).
Certains genetic markers like the green fluorescent Protein, (in English Fluorescent Green Protein or GFP) also is very much used in biology. In this case, the fluorochrome is a protein produced directly by the cell itself and does not require the addition of substrate, fluorescence can then be visualized directly in the alive cells.

History of microscopy of fluorescence

See also the article Fluorescence for the history of fluorescence itself.

Various types of microscopes

The microscope is a tool of observation which was born at the 17th century. The traditional optical microscope, the electron microscope with sweeping, the electron microscope with transmission, the optical microscope of fluorescence, the confocal are now used in routine in many laboratories.
Le microscope of fluorescence is built in order to produce a radiation of fluorescence and to observe it. In microscopy of fluorescence, one uses data processing to treat the images (déconvolution).

In the absence of method allowing to limit the observation of the fluorophores to a focal plan (TIRFM, Microscope confocal with laser scanning or microscopy let us multiphotons), one will speak about microscopy with épifluorescence.

Limits of the optical microscope: Limit the use of microscopy in fluorescence for the study of the interactions protein-proteins

Had with the fact that the optical microscope uses the light to obtain an increased image of an object makes that it becomes ineffective starting from a certain scale. Indeed, the light is a whole of color having each one a specific wavelength. The smallest wavelength of the visible colors is that of purple which is of 400 Nm.

The limit of resolution in optical microscopy is fixed by the criterion of Rayleigh which states that two points can be separate only if they are distant of at least the wavelength divided by 2.

This limit of resolution, called to be able of resolution, is thus of 200nm approximately in traditional optical microscopy. Microscopy in field near optical with sweeping (SNOM: Scanning Near-field Microscopy) is the alternative. This type of microscope is founded on the use of a probe of nanometric size (of 50 with 100nm) being used of nanosource (or nanocollector) of light. The resolution is not then any more function wavelength of illumination but only manufacturing technique of the probe.

This lack of resolution restricts the use of microscopy in fluorescence for the study of the interactions protein-proteins (see technical BRET and FREIGHT). Indeed, the proteins have a size characteristic from 0,1 to 1 Nm). By using fluorophores to mark proteins, one can observe a possible colocalisation by the superposition of the two images obtained by excitation of each fluorophore which does not make it possible to prove any interaction: there could be to 800 proteins between two studied proteins. This is why, to study the interaction protein-protein, it is necessary to rely on other techniques exploiting the phenomenon of fluorescence (FREIGHT) or bioluminescence (BRET).

In this field (microscopy of fluorescence) of many techniques developed.

  • simple marking is done by affinity between a fluorochrome and the molecule to be marked.

  • the direct or indirect immunomarquage utilizes a marked antibody.
  • the technique of FISH is used to mark nucleotidic sequences.
  • the use of proteins of fusion (typically, GFP) consists in introducing into the cell to observe a recombining protein gene fluorescent (by transfection or infection), the synthesized protein is then fluorescent.
  • FLIP and FRAP consist in irradiating a zone from which fluorescence will disappear. These techniques make it possible to study the diffusion of the labelled molecules, indeed so of the molecules move fluorescence will be distributed.
  • the FREIGHT uses two fluorochromes, a donor which will transmit its energy to another fluorochrome acceptor. It makes it possible to study interactions between two molecules.
  • BRET (Bioluminescence Resonance Energy Transfer), like FREIGHT if not that the donor is bioluminescent (luciférase).
  • TIRFM Total Internal Reflection Fluorescence Microscopy or microscopy of fluorescence by total Réflexion interns
  • the Spectroscopie of correlation of fluorescence (FCS), is used to study the diffusion of molecules.

Microscopy of fluorescence has many scopes of application.

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