Two-channel microfluidic CARS: experimental quantification of pure vibrational contrast in CARS images

The combination of linear and nonlinear Raman microspectroscopy has been established to be a powerful tool for biomedical diagnostics. In this contribution we discuss our recent approaches towards CARS (coherent anti-Stokes Raman scattering) based quantification of analytes, which is generally complicated by the CARS-signal strength dependence on the square of the molecular concentration and on the interplay between a molecular-specific vibrational signal and a nonresonant contribution in the signal generation. Due to these complications the quantification of analytes presents a major challenge in CARS microscopy. Here we discuss two recently developed approaches, i.e. on the one hand a simplified setup for coherent anti-Stokes Raman scattering (CARS) microscopy, which allows for recording CARS images with 30 cm-1excitation bandwidth for probing Raman bands between 500 and 900 cm-1with minimal requirements for alignment. This experimental arrangement is based on electronic switching between CARS images recorded at different Raman resonances by combining a photonic crystal fiber (PCF) as broad-band light source and an acoustooptical programmable dispersive filter (AOPDF) as tunable wavelength filter. On the other hand, we discuss how the introduction of carbon-deuterium (C-D) bonds into drug compounds constitutes a non-invasive labeling approach that allows for higher intrinsic CARS contrast to be obtained. The quantitative detection of C-deuterated drugs by Raman microspectroscopy and CARS microscopy is examined. Concentration-dependent studies on drugs with aliphatic and aromatic C-D moieties were performed in a two-channel microfluidic chip, using the corresponding non-deuterated (C-H) isotopomers as an internal reference.