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Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy.

ABSTRACT: Imaging living organisms with molecular selectivity typically requires the introduction of specific labels. Many applications in biology and medicine, however, would significantly benefit from a noninvasive imaging technique that circumvents such exogenous probes. In vivo microscopy based on vibrational spectroscopic contrast offers a unique approach for visualizing tissue architecture with molecular specificity. We have developed a sensitive technique for vibrational imaging of tissues by combining coherent anti-Stokes Raman scattering (CARS) with video-rate microscopy. Backscattering of the intense forward-propagating CARS radiation in tissue gives rise to a strong epi-CARS signal that makes in vivo imaging possible. This substantially large signal allows for real-time monitoring of dynamic processes, such as the diffusion of chemical compounds, in tissues. By tuning into the CH(2) stretching vibrational band, we demonstrate CARS imaging and spectroscopy of lipid-rich tissue structures in the skin of a live mouse, including sebaceous glands, corneocytes, and adipocytes, with unprecedented contrast at subcellular resolution.

SUBMITTER: Evans CL

PROVIDER: S-EPMC1283840 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy.

Evans Conor L CL Potma Eric O EO Puoris'haag Mehron M Côté Daniel D Lin Charles P CP Xie X Sunney XS

Proceedings of the National Academy of Sciences of the United States of America 20051101 46

Imaging living organisms with molecular selectivity typically requires the introduction of specific labels. Many applications in biology and medicine, however, would significantly benefit from a noninvasive imaging technique that circumvents such exogenous probes. In vivo microscopy based on vibrational spectroscopic contrast offers a unique approach for visualizing tissue architecture with molecular specificity. We have developed a sensitive technique for vibrational imaging of tissues by combi ...[more]

PMID: 16263923

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Project description:BackgroundCannabis possesses a rich spectrum of phytochemicals i.e. cannabinoids, terpenes and phenolic compounds of industrial and medicinal interests. Most of these high-value plant products are synthesised in the disk cells and stored in the secretory cavity in glandular trichomes. Conventional trichome analysis was so far based on optical microscopy, electron microscopy or extraction based methods that are either limited to spatial or chemical information. Here we combine both information to obtain the spatial distribution of distinct secondary metabolites on a single-trichome level by applying Coherent anti-Stokes Raman scattering (CARS), a microspectroscopic technique, to trichomes derived from sepals of a drug- and a fibre-type.ResultsHyperspectral CARS imaging in combination with a nonlinear unmixing method allows to identify and localise Δ9-tetrahydrocannabinolic acid (THCA) in the secretory cavity of drug-type trichomes and cannabidiolic acid (CBDA)/myrcene in the secretory cavity of fibre-type trichomes, thus enabling an easy discrimination between high-THCA and high-CBDA producers. A unique spectral fingerprint is found in the disk cells of drug-type trichomes, which is most similar to cannabigerolic acid (CBGA) and is not found in fibre-type trichomes. Furthermore, we differentiate between different cell types by a combination of CARS with simultaneously acquired two-photon fluorescence (TPF) of chlorophyll a from chloroplasts and organic fluorescence mainly arising from cell walls enabling 3D visualisation of the essential oil distribution and cellular structures.ConclusionHere we demonstrate a label-free and non-destructive method to analyse the distribution of secondary metabolites and distinguish between different cell and chemo-types with high spatial resolution on a single trichome. The record of chemical fingerprints of single trichomes offers the possibility to optimise growth conditions as well as guarantee a direct process control for industrially cultivated medicinal Cannabis plants. Moreover, this method is not limited to Cannabis related issues but can be widely implemented for optimising and monitoring all kinds of natural or biotechnological production processes with simultaneous spatial and chemical information.

Dataset Information

Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy.

Publications

Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy.

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