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.

<ol>
	<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 authors declare that they have no competing financial interests.

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><tbody><tr><td>Oscilloscope</td><td>Tektronix</td><td>TDS 520D</td><td>500 MHz&amp;nbsp;</td></tr><tr><td>Photodetector</td><td>Thorlabs</td><td>DET08C/M, T4290</td><td>5 GHz InGaAs, 800 - 1,700 nm</td></tr><tr><td>Ti:Sapphire laser Chameleon Ultra Family II</td><td>Coherent</td><td></td><td></td></tr><tr><td>Optical parametric oscillator OPO Compact Family</td><td>APE Berlin</td><td></td><td></td></tr><tr><td>Axio Examiner microscope LSM 7 MP</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Motorized periscope</td><td>Newport</td><td></td><td></td></tr><tr><td>Objective W Plan-Apochromat 20X/1.0</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Beam combiner</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Acousto-optic modulator</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>OPO power attenuator</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Photomultiplier tube</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>ZEN software</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Bandpass filters</td><td>Carl Zeiss</td><td>LSM BiG 1935-176</td><td>400 - 480 nm; 500 - 550&amp;nbsp;nm; 465 - 610&amp;nbsp;nm</td></tr><tr><td>Dichroic mirror</td><td>Carl Zeiss</td><td></td><td>Cutoff wavelength 760&amp;nbsp;nm</td></tr><tr><td>Silver mirrors</td><td>Newport</td><td>10D20ER.2&amp;nbsp;</td><td>&amp;lambda;/10, 480 - 20,000&amp;nbsp;nm, Quantity 4</td></tr><tr><td>Single-axis translation stage with standard micrometer</td><td>Thorlabs</td><td>PT1/M</td><td>Quantity 1</td></tr><tr><td>Aluminium breadboard</td><td>Thorlabs</td><td>MB1015/M&amp;nbsp;</td><td>Quantity 1</td></tr><tr><td>Mirror mount</td><td>Thorlabs</td><td>KMSS/M&amp;nbsp;</td><td>Quantity 4</td></tr><tr><td>Mirror holder for &amp;Oslash;1&quot; Optics&amp;nbsp;</td><td>Thorlabs</td><td>MH25</td><td>Quantity 4</td></tr><tr><td>Iris diaphragms&amp;nbsp;</td><td>Thorlabs</td><td>ID8/M</td><td>Quantity 3</td></tr><tr><td>Protective box</td><td>Thorlabs</td><td>TB4, XE25L900/M, T205-1.0, RM1S</td><td>Quantity 1</td></tr><tr><td>Optical posts</td><td>Thorlabs</td><td>TR40/M, PH50/M, PH75/M, BA2/M</td><td>Quantity 8 (lengths depending on the set-up)</td></tr><tr><td>661 - 690 nm bandpass filter</td><td>Semrock</td><td>676/29 nm BrightLine&amp;reg; single-band bandpass filter</td><td>Quantity 1</td></tr><tr><td>Fluorescent beads</td><td>ThermoFisher</td><td>TetraSpeck&amp;trade; Fluorescent Microspheres Size Kit</td><td></td></tr><tr><td>Laser viewing card</td><td>Thorlabs</td><td></td><td>IR laser viewing card</td></tr><tr><td>Laser safety glass</td><td>Newport</td><td>LV-F22.P5L07&amp;nbsp;</td><td></td></tr><tr><td>FluoroMyelin&amp;trade; Red Fluorescent Myelin Stain&amp;nbsp;</td><td>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 ...

implementation-coherent-anti-stokes-raman-scattering-cars-system-on

Research

JoVE Journal

Biology

11.2K Views.  INSERM U1051, Institut des Neurosciences de Montpellier (INM), Université de Montpellier.  Coherente anti-Stokes Raman scattering (CARS) microscopie gebaseerd op inherente trilling van molecuul obligaties maakt label-free chemisch selectieve live cell imaging. Dit werk toont de uitvoering van een aanvullende microscopietechniek op een standaard multifoton laser scanning microscoop gebaseerd op een femtoseconde Ti: saffier laser en een OPO laser. 

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

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. SRS imaging modality can be obtained using commercial laser-scanning microscopes by equipping with a non-descanned forward detector with proper bandpass filters and lock-in amplifier (LIA) detection scheme. A schematic layout of a typical SRS microscope includes the following: two pulsed laser beams, (i.e., the pump and probe directed in a scanning microscope), which must be overlapped in both space and time at the image plane, then focused by a microscope objective into the sample through two scanning mirrors (SMs), which raster the focal spot across an x-y plane. After interaction with the sample, transmitted output pulses are collected by an upper objective and measured by a forward detection system inserted in an inverted microscope. Pump pulses are removed by a stack of optical filters, whereas the probe pulses that are the result of the SRS process occurring in the focal volume of the specimen are measured by a photodiode (PD). The readout of the PD is demodulated by the LIA to extract the modulation depth. A two-dimensional (2D) image is obtained by synchronizing the forward detection unit with the microscope scanning unit. In this paper, the implementation of an SRS microscope is described and successfully demonstrated, as well as the reporting of label-free images of polystyrene beads with diameters of 3 &#181;m. It is worth noting that SRS microscopes are not commercially available, so in order to take advantage of these characteristics, the homemade construction is the only option. Since SRS microscopy is becoming popular in many fields, it is believed that this careful description of the SRS microscope implementation can be very useful for the scientific community.

In this manuscript, the implementation of a stimulated Raman scattering (SRS) microscope, obtained by the integration of an SRS experimental set-up with a laser scanning microscope, is described. The SRS microscope is based on two femtosecond (fs) laser sources, a Ti-Sapphire (Ti:Sa) and synchronized optical parametric oscillator (OPO).

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. ...

Engineering

Ultrafast Time-resolved Near-IR Stimulated Raman Measurements of Functional &#960;-conjugate Systems

Femtosecond time-resolved stimulated Raman spectroscopy is a promising method of observing the structural dynamics of short-lived transients with near infrared (near-IR) transitions, because it can overcome the low sensitivity of spontaneous Raman spectrometers in the near-IR region. Here, we describe technical details of a femtosecond time-resolved near-IR multiplex stimulated Raman spectrometer that we have recently developed. A description of signal generation and optimization, measurement, data acquisition, and calibration and correction of recorded data is provided as well. We present an application of our spectrometer to analyze the excited-state dynamics of &#946;-carotene in toluene solution. A C=C stretch band of &#946;-carotene in the second lowest excited singlet (S2) state and the lowest excited singlet (S1) state is clearly observed in the recorded time-resolved stimulated Raman spectra. The femtosecond time-resolved near-IR stimulated Raman spectrometer is applicable to the structural dynamics of &#960;-conjugate systems from simple molecules to complex materials.

Details of signal generation and optimization, measurement, data acquisition, and data handling for a femtosecond time-resolved near-IR stimulated Raman spectrometer are described. A near infrared stimulated Raman study on the excited-state dynamics of &#946;-carotene in toluene is shown as a representative application.

Femtosecond time-resolved stimulated Raman spectroscopy is a promising method of observing the structural dynamics of short-lived transients with near ...

Chemistry

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps at high speed, and high spatial resolution of thin samples by directly interrogating their molecular vibrations. In its standard implementation, SRS microscopy is narrowband and forms images with only a single vibrational frequency at a time. However, this approach not only hinders the chemical specificity of SRS but also neglects the wealth of information encoded within vibrational spectra. 
These limitations can be overcome by broadband SRS, an implementation capable of extracting a vibrational spectrum per pixel of the image in parallel. This delivers hyperspectral data that, when coupled with chemometric analysis, maximizes the amount of information retrieved from the specimen. Thus, broadband SRS improves the chemical specificity of the system, allowing the quantitative determination of the concentration of the different constituents of a sample. Here, we report a protocol for chemical imaging with broadband SRS microscopy, based on a home-built SRS microscope operating with a custom differential multichannel-lock-in amplifier detection. It discusses the sample preparation, alignment of the SRS apparatus, and chemometric analysis. By acquiring vibrational Raman spectra, the protocol illustrates how to identify different chemical species within a mixture, determining their relative concentrations.

We present a protocol to acquire chemical images with broadband stimulated Raman scattering (SRS) microscopy. Based on an SRS microscope that operates with differential multichannel-lock-in detection, the protocol describes the sample preparation, adjustment of the SRS apparatus, and chemometrics to disentangle different constituents of chemically heterogeneous samples.

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps ...

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging technologies. Hyperspectral SRS and CARS imaging offer Raman spectral information at every pixel, which enables better separation of different chemical compositions. Although both techniques require two excitation lasers, their signal detection schemes and spectral properties are quite different. The goal of this protocol is to perform both hyperspectral SRS and CARS imaging on a single platform and compare the two microscopy techniques for imaging different biological samples. The spectral focusing method is employed to acquire spectral information using femtosecond lasers. By using standard chemical samples, the sensitivity, spatial resolution, and spectral resolution of SRS and CARS in the same excitation conditions (i.e., power at the sample, pixel dwell time, objective lens, pulse energy) are compared. The imaging contrasts of CARS and SRS for biological samples are juxtaposed and compared. The direct comparison of CARS and SRS performances would allow for optimal selection of the modality for chemical imaging.

This paper directly compares the resolution, sensitivity, and imaging contrasts of stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) integrated into the same microscope platform. The results show that CARS has a better spatial resolution, SRS gives better contrasts and spectral resolution, and both methods have similar sensitivity.

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging ...

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity 1. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations 1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted 3. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals 4. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm-1 CH stretch vibration in lipids and the 2120 cm-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule's vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging ...

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

Bioengineering

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has been shown to be able to provide hematoxylin and eosin (H&amp;E)-equivalent images that allow fast and reliable diagnosis of brain cancer. Such capability has enabled exciting intraoperative cancer diagnosis applications. Two-color SRS imaging of tissue can be done with either a picosecond or femtosecond laser source. Femtosecond lasers have the advantage of enabling flexible imaging modes, including fast hyperspectral imaging and real-time, two-color SRS imaging. A spectral-focusing approach with chirped laser pulses is typically used with femtosecond lasers to achieve high spectral resolution. 
Two-color SRS acquisition can be realized with orthogonal modulation and lock-in detection. The complexity of pulse chirping, modulation, and characterization is a bottleneck for the widespread adoption of this method. This article provides a detailed protocol to demonstrate the implementation and optimization of spectral-focusing SRS and real-time, two-color imaging of mouse brain tissue in the epi-mode. This protocol can be used for a broad range of SRS imaging applications that leverage the high speed and spectroscopic imaging capability of SRS.

Stimulated Raman scattering (SRS) microscopy is a powerful, nondestructive, and label-free imaging technique. One emerging application is stimulated Raman histology, where two-color SRS imaging at the protein and lipid Raman transitions are used to generate pseudo-hematoxylin and eosin images. Here, we demonstrate a protocol for real-time, two-color SRS imaging for tissue diagnosis.

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has ...

Highly-Multiplexed Tissue Imaging with Raman Dyes

Visualizing a vast scope of specific biomarkers in tissues plays a vital role in exploring the intricate organizations of complex biological systems. Hence, highly multiplexed imaging technologies have been increasingly appreciated. Here, we describe an emerging platform of highly-multiplexed vibrational imaging of specific proteins with comparable sensitivity to standard immunofluorescence via electronic pre-resonance stimulated Raman scattering (epr-SRS) imaging of rainbow-like Raman dyes. This method circumvents the limit of spectrally-resolvable channels in conventional immunofluorescence and provides a one-shot optical approach to interrogate multiple markers in tissues with subcellular resolution. It is generally compatible with standard tissue preparations, including paraformaldehyde-fixed tissues, frozen tissues, and formalin-fixed paraffin-embedded (FFPE) human tissues. We envisage this platform will provide a more comprehensive picture of protein interactions of biological specimens, particularly for thick intact tissues. This protocol provides the workflow from antibody preparation to tissue sample staining, to SRS microscope assembly, to epr-SRS tissue imaging.

Electronic pre-resonance stimulated Raman scattering (epr-SRS) imaging of rainbow-like Raman dyes is a new platform for highly multiplexed epitope-based protein imaging. Here, we present a practical guide including antibody preparation, tissue sample staining, SRS microscope assembly, and epr-SRS tissue imaging.

Visualizing a vast scope of specific biomarkers in tissues plays a vital role in exploring the intricate organizations of complex biological systems. ...

Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development scientists to mechanistically understand topical bioavailability (BA). Semi-invasive techniques, such as tape-stripping, dermal microdialysis, or dermal open-flow microperfusion, all quantify macroscale cPK. While these techniques have provided vast cPK knowledge, the community lacks a mechanistic understanding of active pharmaceutical ingredient (API) penetration and permeation at the cellular level. 
One noninvasive approach to address microscale cPK is coherent Raman scattering imaging (CRI), which selectively targets intrinsic molecular vibrations without the need for extrinsic labels or chemical modification. CRI has two main methods-coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS)-that enable sensitive and selective quantification of APIs or inactive ingredients. CARS is typically utilized to derive structural skin information or visualize chemical contrast. In contrast, the SRS signal, which is linear with molecular concentration, is used to quantify APIs or inactive ingredients within skin stratifications. 
Although mouse tissue has commonly been utilized for cPK with CRI, topical BA and bioequivalence (BE) must ultimately be assessed in human tissue before regulatory approval. This paper presents a methodology to prepare and image ex vivo skin to be used in quantitative pharmacokinetic CRI studies in the evaluation of topical BA and BE. This methodology enables reliable and reproducible API quantification within human and mouse skin over time. The concentrations within lipid-rich and lipid-poor compartments, as well as total API concentration over time are quantified; these are utilized for estimates of micro- and macroscale BA and, potentially, BE.

A coherent Raman scattering imaging methodology to visualize and quantify pharmaceutical compounds within the skin is described. This paper describes skin tissue preparation (human and mouse) and topical formulation application, image acquisition to quantify spatiotemporal concentration profiles, and preliminary pharmacokinetic analysis to assess topical drug delivery.

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development ...

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

Samenvatting

Meer video's verkennen