Name: implementation of a coherent anti-stokes raman scattering (cars) system on a ti:sapphire and opo laser based standard laser scanning microscope
Uploaded: 2016-07-17T14:00:00
Description: Watch this Scientific Journal Video about Implementation of a Coherent Anti-Stokes Raman Scattering CARS System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope at JoVE.com

The authors want to thank Dr. Philippe Combette (IES, UM, Montpellier, France) for the loan of the fast oscilloscope and acknowledge financial supports from Montpellier RIO Imaging (MRI). HR acknowledges ANR grants France Bio Imaging (ANR-10-INSB-04-01) and France Life Imaging (ANR-11-INSB-0006) infrastructure networks for coherent Raman imaging developments. This work was mainly supported by an European Research Council grant (FP7-IDEAS-ERC 311610) and an INSERM - AVENIR grant to NT.

<img alt="Figure 1" src="/files/ftp_upload/54262/54262fig1.jpg" /> 
Figure 1. Schematic view of the general set-up. It includes the Ti:sapphire (680 - 1,080 nm) and the OPO (1,050 - 1,300 nm) lasers, the delay line with the 4 mirrors (M1 to M4), the fast oscilloscope, the photodiode and two fixed iris diaphragms I1 and I2. Mirrors M2 and M3 are fixed on a linear translation stage enabling to change the...

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	<li>Valeur, B., Berberan-Santos, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Fluorescence: Principles and Applications. , (2012).</li><li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Science. 248 (4951), 73-76 (1990).</li><li>Moreaux, L., Sandre, O., Mertz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+imaging+by+second-harmonic+generation+microscopy.">Membrane imaging by second-harmonic generation microscopy.</a> JOSA B. 17 (10), 1685-1694 (2000).</li><li>Zoumi, A., Yeh, A., Tromberg, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cells+and+extracellular+matrix+in+vivo+by+using+second-harmonic+generation+and+two-photon+excited+fluorescence.">Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence.</a> Proc. Natl. Acad. Sci. USA. 99 (17), 11014-11019 (2002).</li><li>Yelin, D., Silberberg, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+scanning+third-harmonic-generation+microscopy+in+biology.">Laser scanning third-harmonic-generation microscopy in biology.</a> Opt. Express. 5 (8), 169-175 (1999).</li><li>Campagnola, P. J., Millard, A. C., Terasaki, M., Hoppe, P. E., Malone, C. J., Mohler, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+high-resolution+Second-Harmonic+Generation+imaging+of+endogenous+structural+proteins+in+biological+tissues.">Three-dimensional high-resolution Second-Harmonic Generation imaging of endogenous structural proteins in biological tissues.</a> Biophys. J. 81 (1), 493-508 (2002).</li><li>Olivier, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+reconstruction+of+early+zebrafish+embryos+using+label-free+nonlinear+microscopy.">Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy.</a> Science. 329 (5994), 967-971 (2010).</li><li>Farrar, M. J., Wise, F. W., Fetcho, J. R., Schaffer, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+myelin+in+the+vertebrate+central+nervous+system+using+third+harmonic+generation+microscopy.">In vivo imaging of myelin in the vertebrate central nervous system using third harmonic generation microscopy.</a> Biophys. J. 100 (5), 1362-1371 (2011).</li><li>Lim, H., Sharoukhov, D., Kassim, L., Zhang, Y., Salzer, J. L., Melendez-Vasquez, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+imaging+of+Schwann+cell+myelination+by+third+harmonic+generation+microscopy.">Label-free imaging of Schwann cell myelination by third harmonic generation microscopy.</a> Proc. Natl. Acad. Sci. U.S.A. 111 (50), 18025-18030 (2014).</li><li>Strupler, M., Pena, A. M., Hernest, M., Tharaux, P. L., Martin, J. L., Beaurepaire, E., Schanne-Klein, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second+harmonic+imaging+and+scoring+of+collagen+in+fibrotic+tissues.">Second harmonic imaging and scoring of collagen in fibrotic tissues.</a> Opt. Express. 15 (7), 4054-4065 (2007).</li><li>Cheng, J. X., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coherent+anti-Stokes+Raman+scattering+microscopy:+Instrumentation,+theory,+and+applications.">Coherent anti-Stokes Raman scattering microscopy: Instrumentation, theory, and applications.</a> J. Phys. Chem. B. 108 (3), 827-840 (2004).</li><li>Volkmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrational+imaging+and+microspectroscopies+based+on+coherent+anti-Stokes+scattering+microscopy.">Vibrational imaging and microspectroscopies based on coherent anti-Stokes scattering microscopy.</a> J. Phys. D: Appl. Phys. 38, R59-R81 (2005).</li><li>Evans, C. L., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coherent+anti-Stokes+Raman+scattering+microscopy:+chemical+imaging+for+biology+and+medicine.">Coherent anti-Stokes Raman scattering microscopy: chemical imaging for biology and medicine.</a> Annu. Rev. Anal. Chem. 1, 883-909 (2008).</li><li>Mukamel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of nonlinear optical spectroscopy. , (1995).</li><li>Duncan, M. D., Reintjes, J., Manuccia, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+coherent+anti-Stokes+Raman+microscope.">Scanning coherent anti-Stokes Raman microscope.</a> Opt. Lett. 7 (8), 350-352 (1982).</li><li>Zumbusch, A., Holtom, G. R., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+vibrational+imaging+by+coherent+anti-Stokes+Raman+scattering.">Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering.</a> Phys. Rev. Lett. 82 (20), 4142-4145 (1999).</li><li>Folick, A., Min, W., Wang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+imaging+of+lipid+dynamics+using+Coherent+Anti-Stokes+Raman+Scattering+(CARS)+and+Stimulated+Raman+Scattering+(SRS)+microscopy.">Label-free imaging of lipid dynamics using Coherent Anti-Stokes Raman Scattering (CARS) and Stimulated Raman Scattering (SRS) microscopy.</a> Curr. Opin. Genet. Dev. 21 (5), 585-590 (2011).</li><li>Wang, P., Liu, B., Zhang, D., Belew, M. Y., Tissenbaum, H. A., Cheng, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+lipid+metabolism+in+live+Caenorhabditis+elegans+using+fingerprint+vibrations.">Imaging lipid metabolism in live Caenorhabditis elegans using fingerprint vibrations.</a> Angew. Chem. Int. Ed. Engl. 53 (44), 11787-11792 (2014).</li><li>Min, W., Freudiger, C. W., Lu, S., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coherent+nonlinear+optical+imaging:+beyond+fluorescence+microscopy.">Coherent nonlinear optical imaging: beyond fluorescence microscopy.</a> Annu. Rev. Phys. Chem. 62, 507-530 (2011).</li><li>Cheng, J. X., Jia, Y. K., Zheng, G., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-scanning+coherent+anti-Stokes+Raman+scattering+microscopy+and+applications+to+cell+biology.">Laser-scanning coherent anti-Stokes Raman scattering microscopy and applications to cell biology.</a> Biophys J. 83 (1), 502-509 (2002).</li><li>Chiu, W. S., Belsey, N. A. N., Garrett, L., Moger, J., Delgado-Charro, M. B., Guy, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+diffusion+in+the+human+nail+measured+by+stimulated+Raman+scattering+microscopy.">Molecular diffusion in the human nail measured by stimulated Raman scattering microscopy.</a> Proc Natl. Acad. Sci. U.S.A. 112, 7725-7730 (2015).</li><li>Chen, X., Gr&#233;goire, S., Formanek, F., Galey, J. -. B., Rigneault, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+3D+molecular+cutaneous+absorption+in+human+skin+using+label+free+nonlinear+microscopy.">Quantitative 3D molecular cutaneous absorption in human skin using label free nonlinear microscopy.</a> J. of Control. Release. 200, 78-86 (2015).</li><li>Kano, H., Hamaguchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+multi-nonlinear+optical+imaging+of+a+living+cell+using+a+supercontinuum+light+source+generated+from+a+photonic+crystal+fiber.">In vivo multi-nonlinear optical imaging of a living cell using a supercontinuum light source generated from a photonic crystal fiber.</a> Opt. Express. 14 (7), 2798-2804 (2006).</li><li>Brustlein, S., Ferrand, P., Walther, N., Brasselet, S., Billaudeau, C., Marguet, D., Rigneault, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+parametric+oscillator-based+light+source+for+coherent+Raman+scattering+microscopy:+practical+overview.">Optical parametric oscillator-based light source for coherent Raman scattering microscopy: practical overview.</a> J. Biomed. Opt. 16 (2), 021106 (2011).</li><li>Chen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multimodal+platform+for+nonlinear+optical+microscopy+and+microspectroscopy.">A multimodal platform for nonlinear optical microscopy and microspectroscopy.</a> Opt. Express. 17 (3), 1282-1290 (2009).</li><li>Yue, S., Slipchenko, M. N., Cheng, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+nonlinear+optical+microscopy.">Multimodal nonlinear optical microscopy.</a> Laser Photonics Rev. 5 (4), 496-512 (2011).</li><li>Sun, Q., Li, Y., He, S., Situ, C., Wu, Z., Qu, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+multimodal+nonlinear+optical+microscopy+reveals+fundamental+insights+of+skeletal+muscle+development.">Label-free multimodal nonlinear optical microscopy reveals fundamental insights of skeletal muscle development.</a> Biomed Opt Express. 5 (1), 158-166 (2013).</li><li>Le, T. T., Langohr, I. M., Locker, M. J., Sturek, M., Cheng, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+molecular+imaging+of+atherosclerotic+lesions+using+multimodal+nonlinear+optical+microscopy.">Label-free molecular imaging of atherosclerotic lesions using multimodal nonlinear optical microscopy.</a> J. Biomed. Opt. 12 (5), 054007 (2007).</li><li>Ellis, D. I., Cowcher, D. P., Ashton, L., O'Hagana, S., Goodacre, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+disease+and+enlightening+biomedicine:+Raman+spectroscopy+as+a+diagnostic+tool.">Illuminating disease and enlightening biomedicine: Raman spectroscopy as a diagnostic tool.</a> Analyst. 138, 3871-3884 (2013).</li><li>Oz&#231;elik, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pals1+is+a+major+regulator+of+the+epithelial-like+polarization+and+the+extension+of+the+myelin+sheath+in+peripheral+nerves.">Pals1 is a major regulator of the epithelial-like polarization and the extension of the myelin sheath in peripheral nerves.</a> J Neurosci. 30 (11), 4120-4131 (2010).</li><li>Heinrich, C., Hofer, A., Ritsch, A., Ciardi, C., Bernet, S., Ritsch-Marte, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+imaging+of+saturated+and+unsaturated+lipids+by+wide-field+CARS-microscopy.">Selective imaging of saturated and unsaturated lipids by wide-field CARS-microscopy.</a> Opt. Express. 16 (4), 2699-2708 (2008).</li><li>Kyriakidis, N. B., Skarkalis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+spectra+measurement+of+olive+oil+and+other+vegetable+oils.">Fluorescence spectra measurement of olive oil and other vegetable oils.</a> J. AOAC Int. 83 (6), 1435-1439 (2000).</li><li>King, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+anatomy:+normal+structure.">Microscopic anatomy: normal structure.</a> Handb. Clin. Neurol. 115, 7-27 (2013).</li><li>Monsma, P. C., Brown, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FluoroMyelin+Red+is+a+bright,+photostable+and+non-toxic+fluorescent+stain+for+live+imaging+of+myelin.">FluoroMyelin Red is a bright, photostable and non-toxic fluorescent stain for live imaging of myelin.</a> J. Neurosci. Methods. 209 (2), 344-350 (2012).</li><li>Wang, H., Fu, Y., Zickmund, P., Shi, R., Cheng, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coherent+anti-stokes+Raman+scattering+imaging+of+axonal+myelin+in+live+spinal+tissues.">Coherent anti-stokes Raman scattering imaging of axonal myelin in live spinal tissues.</a> Biophys. J. 89 (1), 581-591 (2005).</li><li>Wang, H. W., Fu, Y., Huff, T. B., Le, T. T., Wang, H., Cheng, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chasing+lipids+in+health+and+diseases+by+coherent+anti-Stokes+Raman+scattering+microscopy.">Chasing lipids in health and diseases by coherent anti-Stokes Raman scattering microscopy.</a> Vib. Spectrosc. 50 (1), 160-167 (2009).</li><li>Jung, Y., Tam, J., Jalian, H. R., Anderson, R. R., Evans, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal,+3D+in+vivo+imaging+of+sebaceous+glands+by+coherent+anti-stokes+Raman+scattering+microscopy:+normal+function+and+response+to+cryotherapy.">Longitudinal, 3D in vivo imaging of sebaceous glands by coherent anti-stokes Raman scattering microscopy: normal function and response to cryotherapy.</a> J. Invest. Dermatol. 135 (1), 39-44 (2015).</li></ol>

The most challenging part of the work is the temporal synchronization of the laser beams. It requires a fast photodiode combined with a fast oscilloscope, but only a rough overlapping in time can be performed at first. Then a further adjustment of few cm is required. Finally, micrometer moves by a linear translation stage allows performing the final fine adjustment of the delay line length in order to trigger the CARS signal. This signal is maintained in a narrow range of around 20 micrometers, as observed by tuning the ...

Optical microscopy has become a major technique for nondestructive visualization of dynamic processes in living biological systems with a subcellular resolution. Fluorescence microscopy is currently the most popular imaging contrast used in live cells due to its high specificity and sensitivity1. A large palette of fluorescent probes has emerged (exogenous dyes, genetically encoded proteins, semiconductor nanoparticles). Various sample illumination fluorescent-based techniques have flourished (such as confocal or two-photon microscopy) to perform 3D imaging and to reduce a main drawback of this technique which is photobleaching2. Other limitations include the requirement of fluorophore labeling because most of molecular species are not intrinsically fluorescent and therefore these fluorophores have to be artificially introduced in the imaged sample. This artificial manipulation may be disruptive especially for small molecules or induces potential photo-toxicity. These reasons make fluorescence microscopy not well suited for in-vivo observations. Hence, the use of optical imaging techniques with high sensitivity and specific molecular contrasts without the use of fluorescent molecules is highly desirable in biomedical science.
Several nonlinear optical imaging techniques without labeling or staining have emerged, including second-harmonic generation (SHG)3,4 and third-harmonic generation (THG)5. SHG microscopy has been used to image structural arrangements at the supramolecular level such as microtubules or collagen6. THG is generated from optical heterogeneities such as interface between an aqueous medium and lipids7. THG was also demonstrated to image myelin8,9. Both techniques can be implemented on a two-photon fluorescence microscope and require only one laser beam. However they require high power laser intensity (typically 50 mW at 860 nm for SHG10, 25 - 50 mW at 1,180 nm for THG9), which is deleterious in living samples, and do not provide the chemical specificity that is required to unambiguously image specific biological structures.
Chemically selective imaging can be obtained from inherent molecular vibration signals based on Raman scattering. When a beam of light hits matter, photons can be absorbed and scattered by atoms or molecules. Most of the scattered photons will have the same energy, i.e., frequency, as the incident photons. This process is called Rayleigh scattering. However, a small number of photons will be scattered at an optical frequency different from the frequency of the incident photons, i.e., with an inelastic scattering process called Raman scattering. The difference in energy originates from excitation of vibrational modes depending on molecular structure and environment. Therefore, spontaneous Raman scattering provides chemically selective imaging as different molecules have specific vibrational frequencies. However it is limited because of its extremely weak signal. Confocal Raman microscopy has been developed and provides 3D spatial resolution, but it requires high average power and long acquisition time11. To overcome these difficulties, recent advances in laser technology have allowed the rise of nonlinear optical vibrational microscopy, in particular coherent anti-Stokes Raman scattering (CARS)11,12,13.
CARS is a third-order nonlinear optical process. Three laser beams, composed of a pump beam at frequency &#969;P, a Stokes beam at frequency &#969;S and a probe beam (most often being the pump) are focused in a sample and generate an anti-Stokes beam at frequency &#969;AS= (2&#969;P - &#969;S)14. The anti-Stokes signal can be significantly enhanced when the frequency difference between the pump and the Stokes beams is tuned to a Raman molecular vibration &#937;R=(&#969;P - &#969;S). CARS signal is based on multiple photon interaction. It generates therefore a coherent signal orders of magnitude stronger than spontaneous Raman scattering.
CARS microscopy was first experimentally demonstrated by Duncan et al.15. Zumbusch et al. improved then the technique, by using two focused near-infrared femtosecond laser beams with an objective lens of high numerical aperture, allowing the phase matching condition of CARS and avoiding the two-photon non-resonant background16. CARS microscopy has therefore emerged as a powerful tool for live cell and tissues imaging, by chemically detecting molecules such as lipids (via C-H stretch vibration)17,18, water (via O-H stretch vibrations), proteins, DNA in live cells19,20 but also deuterated chemical compounds for pharmaceutical21 and cosmetic applications22.
The major limitation of nonlinear microscopy originates from the complexity and the cost of the optical sources. A CARS system requires two wavelength tunable lasers with short pulse durations and with temporally and spatially synchronized pulse trains. Early CARS microscopes were based on two synchronized picosecond Ti:sapphire lasers20. CARS imaging was also obtained from a single femtosecond Ti:sapphire laser generating a supercontinuum light source23. Recently, laser sources composed of a single femtosecond Ti:sapphire laser pumping a tunable optical parametric oscillators (OPO) have been used for CARS microscopy. This set-up allows intrinsically temporally synchronized beams with a difference of frequency between the pump and the Stokes beam covering the full molecular vibrational spectrum24. In addition, laser scanning microscopes based on a turn-key fs laser and an OPO, primarily used for two-photon fluorescence (TPF) are now available for non-physicists. The potential of such set-ups can be greatly enhanced without requiring supplementary investment by the incorporation of other nonlinear optical imaging, since each nonlinear (NLO) imaging modality is sensitive to specific structures or molecules. Multimodal NLO imaging therefore capitalizes the potential of NLO microscopy for complex biological samples25. The coupling of these techniques has allowed the investigation of many biological questions, in particular on lipid metabolism, skin or cancer development26, skeletal muscle development27, atherosclerotic lesions28. Moreover, the implementation of laser beam scanning with CARS gives the capability of high-rate imaging, i.e., an appealing tool to study dynamical processes in vivo.
The aim of this work is to show each step to implement the CARS technique on a standard multiphoton laser scanning microscope. The microscope is based on a fsec Ti:sapphire laser and an OPO (pumped by the Ti:sapphire laser) operated by a software for biologists. The integration was performed by adjusting the length of one of the laser beam path in order to synchronize in time the two beams. We describe the step-by-step implementation of this technique which requires only basic background in experimental optics. We also illustrate CARS imaging obtained on myelin sheaths of sciatic nerve of rodents, and we show this imaging can be performed simultaneously with other nonlinear optical imaging, such as standard two-photon fluorescence technique and second-harmonic generation.

<table border="0" cellpadding="0" cellspacing="0" width="1157">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:419px;">Oscilloscope</td>
			<td style="width:120px;">Tektronix</td>
			<td style="width:317px;">TDS 520D&#160;</td>
			<td style="width:301px;">500 MHz&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:419px;">Photodetector</td>
			<td style="width:120px;">Thorlabs</td>
			<td>DET08C/M, T4290</td>
			<td>5 GHz InGaAs, 800-1700 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Ti:Sapphire laser Chameleon Ultra Family II</td>
			<td style="width:120px;">Coherent</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Optical parametric oscillator OPO Compact Family</td>
			<td style="width:120px;">APE Berlin</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Axio Examiner microscope LSM 7 MP</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Motorized periscope</td>
			<td style="width:120px;">Newport</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:419px;">Objective W Plan-Apochromat 20x/1.0</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Beam combiner</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Acousto-optic modulator</td>
			<td style="width:120px;">Carl Zeiss</td>
			<td style="width:317px;"></td>
			<td></td>
		</tr>
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			<td>LSM BiG 1935-176</td>
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			<td style="width:120px;">Carl Zeiss</td>
			<td></td>
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			<td height="21" style="height:21px;width:419px;">Silver mirrors</td>
			<td style="width:120px;">Newport</td>
			<td>10D20ER.2&#160;</td>
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			<td style="width:120px;">Thorlabs</td>
			<td style="width:317px;">PT1/M</td>
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			<td height="21" style="height:21px;width:419px;">Mirror mount</td>
			<td style="width:120px;">Thorlabs</td>
			<td>KMSS/M&#160;</td>
			<td>Quantity 4</td>
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			<td height="21" style="height:21px;">Mirror holder for &#216;1&#34; Optics&#160;</td>
			<td style="width:120px;">Thorlabs</td>
			<td>MH25</td>
			<td>Quantity 4</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Iris diaphragms&#160;</td>
			<td style="width:120px;">Thorlabs</td>
			<td>ID8/M</td>
			<td>Quantity 3</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Protective box</td>
			<td style="width:120px;">Thorlabs</td>
			<td>TB4, XE25L900/M, T205-1.0, RM1S</td>
			<td>Quantity 1</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Optical posts</td>
			<td style="width:120px;">Thorlabs</td>
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			<td style="width:120px;">Thorlabs</td>
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			<td>IR laser viewing card</td>
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			<td style="width:120px;">Newport</td>
			<td style="width:317px;">LV-F22.P5L07&#160;</td>
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			<td height="21" style="height:21px;width:419px;">FluoroMyelin&#8482; Red Fluorescent Myelin Stain&#160;</td>
			<td style="width:120px;">ThermoFisher</td>
			<td>F34652</td>
			<td></td>
		</tr>
	</tbody>
</table>

implementation of a coherent anti-stokes raman scattering (cars) system on a ti:sapphire and opo laser based standard laser scanning microscope

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become available for biologists. These systems are primarily designed for multi-channel two-photon fluorescence microscopy. However, without any modification, complementary non-linear optical microscopy such as second-harmonic generation (SHG) or third harmonic generation (THG) can also be performed with this set-up, allowing label-free imaging of structured molecules or aqueous medium-lipid interfaces. These techniques are well suited for in-vivo observation, but are limited in chemical specificity. Chemically selective imaging can be obtained from inherent vibration signals based on Raman scattering. Confocal Raman microscopy provides 3D spatial resolution, but it requires high average power and long acquisition time. To overcome these difficulties, recent advances in laser technology have permitted the development of nonlinear optical vibrational microscopy, in particular coherent anti-Stokes Raman scattering (CARS). CARS microscopy has therefore emerged as a powerful tool for biological and live cell imaging, by chemically mapping lipids (via C-H stretch vibration), water (via O-H stretch vibrations), proteins or DNA. In this work, we describe the implementation of the CARS technique on a standard OPO-coupled multiphoton laser scanning microscope. It is based on the in-time synchronization of the two laser lines by adjusting the length of one of the laser beam path. We present a step-by-step implementation of this technique on an existing multiphoton system. A basic background in experimental optics is helpful and the presented system does not require expensive supplementary equipment. We also illustrate CARS imaging obtained on myelin sheaths of sciatic nerve of rodent, and we show that this imaging can be performed simultaneously with other nonlinear optical imaging, such as standard two-photon fluorescence technique and second-harmonic generation.

The pulse train frequency of standard Ti:sapphire laser is typically around 80 MHz. The OPO has the same frequency since it is pumped by the Ti:sapphire laser. A fast oscilloscope of at least 200 MHz is therefore required. A fast photodiode in the range 600 to 1,100 nm is also required. The maximal temporal shift occurs when the Ti:sapphire and the OPO signals are shifted of 1/(2&#215;80&#215;106) = 6.2 nanoseconds. It corresponds to a maximum beam path shift of 1.9 m. ...

Watch this Scientific Journal Video about Implementation of a Coherent Anti-Stokes Raman Scattering CARS System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope at JoVE.com

Implementation of a Coherent Anti-Stokes Raman Scattering CARS System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Coherent anti-Stokes Raman scattering (CARS) microscopy based on inherent vibration of molecule bonds permits label-free chemically selective live cell imaging. This work presents the implementation of a complementary microscopy technique on a standard multiphoton laser scanning microscope based on a femtosecond Ti:sapphire laser and an OPO laser.

Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become ...

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