Fluorescence optical microscopy imaging is a rapidly growing field and has become an essential method for biological research. Indeed, recent technological advances have led to the development of faster and higher resolution microscopes for collecting three-dimensional images of a living biological sample. At the same time, significant progress has been made in the development of stable and luminous fluorescent probes, which has revolutionised the ability to track individual cells in vivo.
The confocal microscope is a special photonic microscope device that allows the localization of specific molecules, in the cellular context, and makes important contributions to the functional study of biological systems at different levels of organization: sub-cellular, cellular, tissue or embryonic. Thanks to its very shallow depth of field (about 400 nm), this technique makes it possible to section a sample into an optical slice of very good quality without further processing. The use of very specific fluorescent tracers allows in situ localisation with a spatial resolution of a few hundred nanometres of macromolecular components such as nucleic acids, proteins, but also small molecules such as peptides, lipids and even ions.
Spectral basis of organic fluorochromes
(P. Leclerc comp. 03-02-15)
Before choosing a fluorochrome, it is essential to know on which instrument(s) the fluorescence signal will be studied in order to determine its spectral possibilities in terms of excitation and emission.
With the SP5-AOBS confocal microscope, the laser excitation lines are available: 405, 458, 476, 488, 496, 514, 561 and 633nm. The AOBS system will send the light of the chosen excitation wavelength to the object and recover all the fluorescence emitted by the sample on the diffraction prism. The positioning of the detection slits in front of the different detectors allows to select at will the emission spectral band in which an image is to be produced. Thus the choice of fluorochromes is mainly determined by the excitation wavelength and, in the case where several markers are combined, by emission wavelengths sufficiently spaced to avoid the risk of crosstalk.
Other criteria to be taken into account are :
- The molar extinction coefficient ε (also known as molar absorbance) which is an absorption parameter derived directly from Beer-Lambert’s law and which expresses the variation in light intensity (D.O.) as a function of the molar concentration of the absorbent molecule and the length of the optical path.
- the quantum yield Φf (QE: quantum efficiency or quantum yield) which is equal to the ratio between the number of photons emitted and the number of photons received in the excitation spectral region of the fluorochrome. Its value therefore varies between 0 and 1, so a very efficient fluorochrome will emit many fluorescence photons in relation to the number of excitation photons it will have received.
- the product of the 2 previous values ε.Φf which will determine the brightness of the fluorochrome.
- the temporal stability of the brilliance of the fluorochrome when subjected to prolonged excitation. All organic fluorochromes lose brilliance when excited. This is the phenomenon of photobleaching (photo-bleaching or bleaching). Very dependent on the conditions of the environment: pH, presence of free radicals, etc., it is only by experiment that this parameter can be understood more or less well. This phenomenon can be partially attenuated by using anti-bleaching mounting media but, here again, it is only experience that will allow us to determine the right conditions.
The following list is not exhaustive, but it does offer a fairly complete choice.