Dual-CARS microscopy

We present a new Coherent Anti-Stokes Raman Scattering (CARS) microscopy technique for label-free imaging of bio-molecules in living cells; dual-CARS microscopy. The use of three synchronized laser pulses in a dual-pump/dual-detection configuration enables imaging of two species with different molecular vibrations simultaneously, as well as acquisition of images free of non-resonant background. We show the power of the method by imaging deuterated nonadecane slowly diffusing into a suspension of living yeast cells in medium, clearly distinguishing the medium and the lipid droplets in the cells by probing the CH2 vibration from the D-nonadecane by probing the CD vibration. In addition, images of lipid stores in living C. elegans nematodes free of non-resonant background are shown. This results in a significant enhancement of the image contrast, allowing the visualization of emerging, low-density lipid stores in a dauer larva, difficult to distinguish in conventional CARS microscopy. The separation of the non-resonant background is shown to be beneficial also when monitoring molecules with weak vibrational modes. The improved sensitivity obtained is illustrated by probing the C=C vibration in polyunsaturated lipids extracted from fish. This enables the monitoring of the degree of unsaturation of lipids, a high value of which is reported in foods known to have positive effects on human health.