Label free nonlinear imaging in microscopy and endoscopy

Abstract

Summary form only given. To get insights into the molecular interactions that take place in cells and tissues, fluorescence microscopy appears nowadays as the most powerful and common way to proceed. Advances in fluorescent fusion proteins such as green fluorescent protein (GFP), chemical dyes and nanoparticles provide the biologist with a rich labeling kit that is at the very basis of fluorescence microscopy. Although extremely powerful, fluorescence comes with a number of questions that cannot always be answered, the most dramatic one being to which extent a fluorescent label - often bigger than the biomolecule of interest - will affect the complex molecular interaction at work in cells and tissues? Furthermore labeling or staining often relies on complex bio-chemistry and is not always possible. Clearly, developing label free microscopy techniques as alternatives to fluorescence is important. Coherent Raman scattering (CRS) can address the molecular vibrations ubiquitous in any molecule. While possible to probe them directly by IR absorption, the strong absorbance of water in the IR range usually prohibits this strategy. Spontaneous Raman scattering, on the other hand, can do the job as it uses visible or near IR light that is not absorbed by water. Nevertheless it does not permit imaging because of the very low spontaneous Raman cross section that requires integration times on the order of seconds to minutes per pixel. In order to access imaging modalities, CRS is necessary. CRS addresses the molecular vibrations as spontaneous Raman would do, however it uses two lasers whose beating will imprint a coherent vibrational excitation on the targeted molecules and generate signals orders of magnitude greater than in spontaneous Raman. Two CRS mechanisms can be used to performed label free imaging, namely CARS (Coherent Anti Stokes Raman Scattering) and SRS (Stimulated Raman Scattering) both of them presenting specific features. The first part of the talk will be to review the assets and constrains of the two CARS and SRS mechanisms, both from fundamental and technical point of view. CARS probes the real and the imaginary part of the third order nonlinear tensor and this intermixing results in a so called `non resonant background' that degrades the image quality. On the contrary SRS probes the imaginary part only of third order nonlinear tensor but is also subjected to artifacts that are annoying when probing weak Raman band. We will present recent strategies that can remove CARS non resonant background and the SRS artifacts. The second part of the talk will address the implementation of CRS in endoscope. Such development would be extremely beneficial for deep tissue investigations and imaging. Nonlinear optics through fiber optics comes with a number of problems arising from the variety of nonlinear effects that take place when ultra-short pulses propagates in fiber. We will present various modalities using photonic crystal fibers that permit to build Raman and CRS endoscope.