SRS and CARS microscopy are rapidly developing techniques that are constantly finding new and exciting applications.

Applications to Cell Biology

Omega-3 fatty acid uptake by A549 human lung cancer cells monitored with SRL microscopy and microspectroscopy. (A) Spontaneous Raman spectra of docosahexaenoic acid (DHA, with six C=C bonds), eicosapentaenoic acid (EPA, with five C=C bonds), arachidonic acid (AA, with four C=C bonds), and oleic acid (OA, with a single C=C bond). The strong Raman peak around 3015 cm−1 is characteristic of unsaturated fatty acids. (B) SRL spectra of a lipid droplet (LD, red line) and a region inside the nucleus (blue line). Unlike the nuclear region, the SRL spectrum of the LD shows good correspondence with the spectra from the pure EPA shown in (A). (C) SRL image of a cell at 2920 cm−1. (D) SRL image of the same cell at 3015 cm−1. These findings indicate that EPA is absorbed by the cells and more strongly enriched in the LDs compared to other cellular organelles.

Biomedical applications

SRS imaging of fresh mouse skin. Depth stack shows a variety of lipid-richt tissue structures: stratum corneum, sebaceous gland, and subcutaneous fat layer.

Monitoring pharmaco-kinetics

Understanding transdermal drug delivery pathways is much needed in developing topically applied drugs. Fluorescent labels are usually larger than the drug molecule and can therefore alter the transport properties of molecules drastically. Although confocal spontaneous Raman microspectroscopy has been used to obtain longitudinal penetration profiles, lateral distribution is often compromised due to the long pixel dwell times in spontaneous Raman scattering. Three-dimensional information is, however, critical for unique identification of penetration pathways. As a label-free and sensitive imaging modality, SRS microscopy allows mapping of drugs in 3D and follows their dynamics in living cells and tissues (Freudiger, Min et al., Science, 2008).

 

 

 

 

Monitoring drug delivery in fresh mouse skin by SRS microscopy. (top) Raman spectra of dimethyl sulfoxide (DMSO, green), retinoic acid (RA, blue), and typical lipids in mouse skin (red). (mid left) Top view of the penetrated DMSO (green) in the stratum corneum imaged at 670 cm−1 and depth penetration profile. (mid right) Top view of the penetrated RA (blue) in the stratum corneum imaged at 1570 cm−1 and depth profile. (video) Depth-stack showing penetrated DMSO in fresh mouse skin. Images were recorded with simultaneous two-color SRS imaging.

Imaging of Brain Tumors

CARS microscopy provides an interesting new contrast mechanism for imaging tumors in brain tissue. In particular, by making use of the differences in lipid density between tumor tissue and healthy tissue, brain tumor margins can be seen with subcellular spatial resolution in fresh, unstained tissue. For this purpose, CARS microscopy is an optimal tool as any area of brain tissue of about one square millimeter can be acquired in about one second without any fixation, staining or freezing of the specimen. Comparison of histological data and Raman spectroscopy has established the chemical selectivity of the technique, and efforts to establish the use of CARS as a tool for virtual histology during brain tumor resectioning surgery are underway in collaboration with Prof. Geoffrey Young (Harvard Medical School and Brigham & Women's Hospital, Boston, MA), Dr. Santosh Kesari (Dana Farber Cancer Institute, Boston, MA), Xiaoyin Xu (Brigham Women's Hospital, Boston, MA) and Anita Hüttner (Yale University, New Haven, CT).

Related publications:

[Evans, et al. Opt. Exp., 2007]

[Légaré, Evans, et al., Opt. Express, 2006]

CARS images of astrocytoma in a SCID mouse sacrificed 4 weeks after inoculation of tumor cells. The pump and Stokes wavelengths were 924.0 and 1254.2 nm, respectively. (a) A low resolution, large field of view mosaic CARS microscopy image provides chemically selective anatomical information. Part (b) illustrates the ability of CARS to produce higher resolution images, in this case corresponding to the area enclosed by the rectangle in (a). This 80X, 175 x 175 μm image demonstrates the microscopic infiltration at the boundary between the tumor and normal tissue with a precision comparable to the conventional fixed tissue H&E.