Selective Chemical Microscopic Imaging Using Femtosecond Coherent Anti-Stokes Raman Scattering (CARS) Techniques

Bernhard von Vacano, Christoph Pohling, Lars Meyer, and Marcus Motzkus

Faculty of Chemistry, University of Marburg, Hans-Meerwein-Straße, 35032 Marburg, Germany




Coherent Anti-Stokes Raman Scattering (CARS) allows rapid three-dimensional microscopic imaging of native samples.[1] Image contrast is based on the intrinsic vibrational quantum level structure of the sample, and thus gives microscopic maps of the chemical composition. As no labelling is needed and fast imaging can be achieved, CARS is a very promising tool for the study of living biological cells or complex composite materials.

Technical challenges for the implementation of CARS are the necessity of high peak intensities and the availability of different coherent laser colours: Both issues can elegantly be addressed employing broadband femtosecond laser sources. The tailoring of the femtosecond pulses, e.g. by spectral broadening in a photonic crystal fibre and/or femtosecond pulse shaping, can dramatically improve spectral resolution, suppress unwanted background and therefore maximize the desired chemical contrast. These strategies are presented and open up exciting new perspectives for very versatile nonlinear microscopy systems in the emerging field of biophotonics.

We show examples of vibrational CARS microspectroscopy in both the low-frequency and fingerprint spectral regions. The low-frequency regime allows access to an intriguing spectroscopic window for the spatially resolved study of conformational vibrations of biomolecules. With high laser bandwidths, as created by photonic fibre technology,[2] the fingerprint region becomes accessible and allows discriminating even minor chemical changes on prototype systems, such as a proton exchange. We also show applications of CARS three-dimenstional chemical mapping to polymer blends,[3] and first results on the study of bacteriorhodopsin layers.