Dataset Information

Coherent anti-stokes Raman scattering (CARS) microscopy: a novel technique for imaging the retina.

ABSTRACT:

Purpose

To image the cellular and noncellular structures of the retina in an intact mouse eye without the application of exogenous fluorescent labels using noninvasive, nondestructive techniques.

Methods

Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Cross sectional transverse sections and sequential flat (en face) sagittal sections were collected from a region of sclera approximately midway between the limbus and optic nerve. Imaging proceeded from the surface of the sclera to a depth of ?60 ?m.

Results

The fluorescent signal from collagen fibers within the sclera was evident in the TPAF channel; the scleral collagen fibers showed no organization and appeared randomly packed. The sclera contained regions lacking TPAF and CARS fluorescence of ?3 to 15 ?m in diameter that could represent small vessels or scleral fibroblasts. Intense punctate CARS signals from the retinal pigment epithelial layer were of a size and shape of retinyl storage esters. Rod outer segments could be identified by the CARS signal from their lipid-rich plasma membranes.

Conclusions

CARS microscopy can be used to image the outer regions of the mammalian retina without the use of a fluorescent dye or exogenously expressed recombinant protein. With technical advancements, CARS/TPAF may represent a new avenue for noninvasively imaging the retina and might complement modalities currently used in clinical practice.

SUBMITTER: Masihzadeh O

PROVIDER: S-EPMC3643396 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Publications

Coherent anti-stokes Raman scattering (CARS) microscopy: a novel technique for imaging the retina.

Masihzadeh Omid O Ammar David A DA Kahook Malik Y MY Lei Tim C TC

Investigative ophthalmology & visual science 20130501 5

<h4>Purpose</h4>To image the cellular and noncellular structures of the retina in an intact mouse eye without the application of exogenous fluorescent labels using noninvasive, nondestructive techniques.<h4>Methods</h4>Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Cross sectional transverse sections and sequential flat (en face) sagittal sections were collected from a region o ...[more]

PMID: 23580484

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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 ?9-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

Coherent anti-stokes Raman scattering (CARS) microscopy: a novel technique for imaging the retina.

Purpose

Methods

Results

Conclusions

Publications

Coherent anti-stokes Raman scattering (CARS) microscopy: a novel technique for imaging the retina.

Similar Datasets

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