Résumés
Résumé
Les avancées récentes en physique des lasers ont permis de mettre en oeuvre 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.
Summary
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.
Parties annexes
Références
- 1. Raman CV, Krishnan KS. A new radiation. Indian J Phys 1928 ; 2 : 387.
- 2. Choo-Smith LP, Edwards HG, Endtz HP, et al. Medical applications of Raman spectroscopy : from proof of principle to clinical implementation. Biopolymers 2002 ; 67 : 1-9.
- 3. Zumbusch A, Holtom G, Xie XS. Three-dimensional vibrational imaging by coherent anti-stokes raman scattering. Phys Rev Lett 1999 ; 82 : 4142-5.
- 4. Volkmer A, Cheng JX, Xie XS. Vibrational imaging with high sensitivity via epidetected coherent anti-Stokes Raman scattering microscopy. Phys Rev Lett 2001 ; 87 : 23901-4.
- 5. Cheng JX, Jia YK, Zheng GF, Xie XS. Laser-scanning coherent anti-stokes Raman scattering microscopy and applications to cell biology. Biophysical J 2002 ; 83 : 502-9.
- 6. Cheng JX, Volkmer A, Book LD, Xie XS. An epi-detected coherent anti-stokes raman scattering (E-CARS) microscope with high spectral resolution and high sensitivity. J Phys Chem B 2001 ; 105 : 1277-80.
- 7. Nan X, Cheng JX, Xie XS. Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy. J Lipid Res 2003 ; 44 : 2202-8.
- 8. Potma EO, Boeij WP, Haastert PJM, Wiersma DA. Real-time visualization of intracellular hydrodynamics in single living cells. Proc Natl Acad Sci USA 2001 ; 98 : 1577-82.
- 9. Pautot S, Frisken BJ, Cheng JX, et al. Spontaneous formation of lipid structures at oil/water/lipid interfaces. Langmuir 2003 ; 19 : 10281-7.
- 10. Cheng JX, Pautot S, Weitz DA, Xie XS. Ordering of water molecules between phospholipid bilayers visualized by coherent anti-Stokes Raman scattering microscopy. Proc Natl Acad Sci USA 2003 ; 100 : 9826-30.
- 11. Cheng JX, Volkmer A, Book LD, Xie XS. Multiplex coherent anti-stokes Raman scattering microspectroscopy and study of lipid vesicles. J Phys Chem B 2002 ; 106 : 8493-8.
- 12. Muller M, Schins JM. Imaging the thermodynamic state of lipid membranes with multiplex CARS microscopy. J Phys Chem B 2002 ; 106 : 3715-23.
- 13. Potma EO, Xie XS. Detection of single lipid bilayers with coherent anti-Stokes Raman scattering (CARS) microscopy. J Raman Spectroscopy 2003 ; 34 : 642-50.
- 14. Wang H, Fu Y, Zickmund P, et al. Coherent anti-Stokes Raman scattering imaging of axonal myelin in live spinal tissues. Biophys J 2005 ; 89 : 581-91.
- 15. Nan X, Yang WY, Xie XS. CARS Microscopy light up lipids in living cells. Biophotonics Int 2004 ; 11 : 44-7.
- 16. Potma EO, Xie XS. Direct visualization of lipid phase segregation in single lipid bilayers with coherent anti-stokes Raman scattering microscopy. Chem Phys Chem 2005 ; 6 : 77-9.
- 17. Holtom GR, Thrall BD, Chin BY, et al. Achieving molecular selectivity in imaging using multiphoton Raman spectroscopy techniques. Traffic 2001 ; 2 : 781-8.
- 18. Potma EO, Xie XS. Direct visualization of lipid phase segregation in single lipid bilayers with coherent anti-stokes Raman scattering microscopy. Chem Phys Chem 2005 ; 6 : 77-9.
- 19. Li L, Wang HF, Cheng JX. Quantitative coherent anti-Stokes Raman scattering imaging of lipid distribution in coexisting domains. Biophysical J 2005 ; 89 : 3480-90.
- 20. Dimitrov DS, Angelova MI. Lipid swelling and liposome formation mediated by electric-field. Bioelectrochem Bioenergetics 1988 ; 19 : 323-36.
- 21. Muller M, Schins JM, Nastase N, et al. Imaging the chemical composition and thermodynamic state of lipid membranes with multiplex CARS microscopy. Biophysical J 2002 ; 82 : 175A.
- 22. Wurpel GWH, Schins JM, Müller M. Direct measurement of chain order in single phospholipids mono- and bilayers with multiplex CARS. J Phys Chem 2004 ; 108 : 3400-3.
- 23. Muller M, Schins JM. Imaging the thermodynamic state of lipid membranes with multiplex CARS microscopy. J Phys Chem 2002 ; 106 : 3715-23.