title: Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope.
abstract: 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.
authors: INSERM U1051, Institut des Neurosciences de Montpellier (INM), Université de Montpellier.
authors: Vasyl
authors: V
authors: Mytskaniuk
authors: Université de Nîmes; CNRS, IES, UMR 5214; fabrice.bardin@unimes.fr.
authors: Fabrice
authors: F
authors: Bardin
authors: INSERM U1051, Institut des Neurosciences de Montpellier (INM), Université de Montpellier; Montpellier RIO Imaging (MRI).
authors: Hassan
authors: H
authors: Boukhaddaoui
authors: Aix-Marseille Université, CNRS, École Centrale Marseille, Institut Fresnel, UMR 7249.
authors: Herve
authors: H
authors: Rigneault
authors: INSERM U1051, Institut des Neurosciences de Montpellier (INM), Université de Montpellier.
authors: Nicolas
authors: N
authors: Tricaud
mesh_terms: D000537:Aluminum Oxide; D000818:Animals; D006801:Humans; D007834:Lasers; D051379:Mice; D018613:Microscopy, Confocal; D009186:Myelin Sheath; D012584:Sciatic Nerve / Q000000981:diagnostic imaging; D013059:Spectrum Analysis, Raman; D014025:Titanium
chemical_list: D014025:Titanium; D000537:Aluminum Oxide