Combined Coherent Anti-Stokes Raman Spectroscopy (CARS) and Two-Photon Confocal Microscope
Cuts from a three-dimensional data set, showing the structure of live macrophages stained with Rhodamine-123 (green fluorescence) that have ingested fine titania particles. The coherent anti-Stokes Raman scattering (CARS) signal from the ingested TiO2 particles is shown in red. TiO2 is nonfluorescent and transparent. Photo by Yuangang Zheng.
Pacific Northwest National Laboratory's (PNNL's) combined coherent anti-Stokes Raman spectroscopy (CARS) and two-photon confocal microscope is used to visualize molecules based on their vibrational and fluorescent properties. The capabilities of the individual microscopes in this combined instrument are merged to yield novel information about systems of study.
CARS - An anti-Stokes Raman scattering apparatus uses two lasers to create a resonance that matches the vibrational frequency of a molecule of interest, such as lipids and deuterated compounds. The detected visible signal shows the number of molecules present in the sample, and provides an image of chemically important, but non-fluorescent, species.
Left: CARS image showing concentrations of carbon-hydrogen bonds in live D2XRII murine bone marrow stromal cells (white is the most intense). This image was obtained with two ultrafast lasers; no stain was required. Right: Fluorescence of DNA and RNA stained with Syto 13 dye, obtained simultaneously as the CARS image. The nuclear region is shown very clearly and serves to identify known cell structures.
Two-photon - Traditional fluorescence microscopy uses dyes that require relatively high-energy photons for fluorescence, thus photobleaching and cell damage are problematic consequences. Two-photon microscopy is a more gentle approach. It uses dye molecules that fluorescence upon absorption of two relatively low-energy photons within a short period of time.
Confocal - Using confocal microscopy, computer processing of multiple scans in the x, y, and z directions produces a three-dimensional image of a living or fixed specimens. The CARS and fluorescence signals are produced simultaneously, so the volume images are perfectly registered.
