Better understanding of the fundamental mechanisms behind metabolic diseases requires methods to monitor lipid stores on single-cell level in vivo. We have used Caenorhabditis elegans as a model organism to demonstrate the limitations of fluorescence microscopy for imaging of lipids compared with coherent antiStokes Raman scattering (CARS) microscopy, the latter allowing chemically specific and label-free imaging in living organisms. CARS microscopy was used to quantitatively monitor the impact of genetic variations in metabolic pathways on lipid storage in 60 specimens of C. elegans. We found that the feeding-defective mutant pha-3 contained a lipid volume fraction one-third of that found in control worms. In contrast, mutants (daf-2, daf-4 dauer) with deficiencies in the insulin and transforming growth factors (IGF and TGF-␤) signaling pathways had lipid volume fractions that were 1.4 and 2 times larger than controls, respectively. This was observed as an accumulation of small-sized lipid droplets in the hypodermal cells, hosting as much as 40% of the total lipid volume in contrast to the 9% for the wild-type larvae. Spectral CARS microscopy measurements indicated that this is accompanied by a shift in the ordering of the lipids from gel to liquid phase. We conclude that the degree of hypodermal lipid storage and the lipid phase can be used as a marker of lipid metabolism shift. This study shows that CARS microscopy has the potential to become a sensitive and important tool for studies of lipid storage mechanisms, improving our understanding of phenomena underlying metabolic disorders.lipid metabolism ͉ nonlinear microscopy ͉ obesity

In this work, we show how broad-bandwidth femtosecond pulses can be used to achieve high spectral resolution in nonlinear spectroscopy and microscopy. Our approach is based on chirping the excitation pulses in order to focus their entire bandwidth into a narrow spectral region. We show that spectral features which are 100 times narrower than the excitation light can be resolved with this simple spectral focusing. The gain in spectral selectivity and sensitivity makes its application to nonlinear microscopy very convenient. This is demonstrated with diffraction-limited coherent anti-Stokes Raman scattering microscopy.

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Electronically controlled coherent linear optical sampling for low coherence interferometry (LCI) and optical coherence tomography (OCT) is demonstrated, using two turn-key commercial mode-locked fiber lasers with synchronized repetition rates. This novel technique prevents repetition rate limitations present in previous implementations based on asynchronous optical sampling. Adjustable scanning ranges and scanning rates are realized within an interferometric setup by full electronic control of the mutual time delay of the two laser pulse trains. We implement this novel linear optical sampling scheme with broad spectral bandwidths for LCI, optical filter characterization and OCT imaging in two and three dimensions.

In this manuscript, we present a detailed investigation of the impact of dispersion on the spectral resolution achievable by the application of spectral focusing in coherent Raman imaging. Our results reveal the detrimental effect of third order dispersion that limits the resolution for group delay dispersion of 100 000 fs and more. Experimental examples for the exact determination of the described effects are given as well as a condensed presentation of the known equations. We introduce useful approximations to the latter, which serve to facilitate the straightforward integration of spectral focusing into any multimodal microscope.