Microscopie Raman stimulée (CARS) : Principes et applications

Abstract

Les avancées récentes en physique des lasers ont permis de mettre en œuvre une technique de microscopie permettant d’observer les liaisons chimiques des molécules présentes dans un échantillon biologique. Cette technique utilise l’effet Raman stimulé comme origine de contraste et permet de générer des images en trois dimensions à partir d’échantillons non marqués et ne nécessitant aucune préparation. Connue sous le nom de CARS (coherent anti-stokes raman scattering), cette nouvelle microscopie optique nécessite, pour l’instant, un appareillage complexe mais des avancées récentes laissent envisager sa commercialisation dans les années à venir. Quels types d’images peut-on obtenir en microscopie CARS ? Quelles sont les limites et les espoirs d’une telle approche en imagerie cellulaire et tissulaire ? A quoi ressemble et ressemblera un microscope CARS ? Même si les réponses à ces questions sont encore en pleine évolution, elles permettent de mieux cerner les enjeux d’une technique qui s’approche du but ultime de la microscopie : voir une molécule ou un assemblage moléculaire évoluer et interagir dans l’échantillon sans avoir recours à aucun marquage.

A new technique in microscopy is now available which permits to image specific molecular bonds of chemical species present in cells and tissues. The so called Coherent Anti-Stokes Raman Scattering (CARS) approach aims at maximazing the light matter interaction between two laser pulses and an intrinsic molecular vibrationnal level. This is possible through a non linear process which gives rise to a coherent radiation that is greatly enhanced when the frequency difference between the two laser pulses equals the Raman frequency of the aimed molecular bond. Similar to confocal microscopy, the technique permits to build an image of a molecular density within the sample but doesn’t require any labelling or staining since the contrast uses the intrinsic vibrationnal levels present in the sample. Images of lipides in membranes and tissues have been reported together with their spectral analysis. In the case of very congested media, it is also possible to use a non invasive labelling such as deuterium which shifts the molecular vibration of the C-H bond down to the C-D bond range which falls in a silent region of the cell and tissue vibrational spectra. Such an approach has been used to study lipid phase in artificial membranes. Although the technique is still under development, CARS has now reach a maturity which will permit to bring the technology at a commercial stage in the near futur. The last remaining bottleneck is the laser system which needs to be simplified but solutions are now under evaluation. When combined with others more conventionnel techniques, CARS should give its full potential in imaging unstained samples and like two photons techniques has the potential of performing deep tissues imaging.