CARS and Confocal

A Successful Affair

November 02, 2010

Since the advent of confocal microscopy, scientists have gained many new insights that lead to a deeper understanding of how life works. The most important drawback of single-photon and multiphoton confocal microscopy is the need to label the specimen. CARS (Coherent Anti-Stokes Raman Spectroscopy) addresses this issue because it is non-toxic, non-destructive, and minimally invasive.

Staining processes have constraints: Dyes bleach with time, can be phototoxic, and can influence subsequent research methods and camouflage subsequent research information. Processes with stained specimens are not suitable for long term analysis:  Atmospheric conditions destroy dyes, and dyes alter when in contact with air or moisture – any change to environmental condition will influence the experimental result. Also, the staining process is time consuming. Some samples cannot be stained at all, because the attachment of a fluorophore or the presence of the dye changes the functionality of the molecule or the organism.

Coherent Anti-Stokes Raman Spectroscopy – the technique

CARS is a third-order, nonlinear process that involves a pump beam at a frequency of wp and a Stokes beam at a frequency of ws. The specimen is stimulated through a wave-mixing process. The anti-Stokes signal at ϖas=2ϖp–ϖs is generated in the phase matching direction as vibrational contrast at the frequency difference ∆ν=vsp–vs between the pump beam and the Stokes beam. This equals the frequency of the vibrational energy of a particular chemical bond.

Fig 1: CARS energy diagram
Energy diagram for the CARS process (Coherent Anti-Stokes Raman Spectroscopy). A pump-photon ‘excites’ the molecule into a virtual state, from which it is forced to return to a specific vibrational state of the ground-state by a stokes-photon. A secon
Fig. 2: Deviation of lipid droplets in cream, overlay image. The green background shows the water (acquired at 3,150 cm–1) while the red dots are fatty components (acquired at 2,850 cm–1).
Two color CARS image recorded with two different pump frequencies. Sample: cream. Green: Water at 3150 cm-1. Red: Fat-droplets at 2850 cm-1. To avoid thermal movement artefacts, resonant scanning with Leica TCS SP5II confocal microscope is recommended.

Live molecular profiling with CARS

The main applications of CARS microscopy are found in biological, pharmaceutical, and dermatological research, biomedical imaging, food processing, and materials science. Its potential has been demonstrated for various biomedical applications, such as the imaging of lipid transport, protein concentrations, DNA, RNA, tissue in a living organism, and order in liquid crystals. By integrating CARS technology into modern optical scanning microscope systems, the researcher has the latest technology in hand combined with an easy-to-use confocal system.

High resolution images

Fig. 3: Skin of a mouse ear (Maximum Projection), Raman shift at 2,849 cm–1 (which corresponds to CH2 stretching vibration). Courtesy of Prof. Sunney Xie, Department of Chemistry and Chemical Biology, Harvard University, Cambridge, USA
CARS image at Raman shift 2849 cm-1 which is specific for CH2 stretching vibration. Sample: mouse skin. Instrument: Leica TCS SP5II CARS.

A conventional scanner is optimized for morphological studies as in brain and skin, or for imaging subcellular features such as the cytoskeleton. It allows sampling up to 8,196 × 8,196 pixels per image, combining a large field of view with high resolution. Also, the speed of the scanner can be adapted from 400 Hz to 2,800 Hz in bidirectional mode.

At the skin surface, long filaments corresponding to the hairs with chromospheres can be seen with a very strong signal because hairs are covered with oil. At the skin surface, a bright polygonal pattern outlines the corneocytes forming the top layer of the skin, the corneum. The signal arises from the intercellular space rich in lipids, cholesterol, and ceramids. In a second layer, bright structures surrounding the roots of hair are detected, the sebaceous glands. They are multicellular compartments packed with sebum reservoirs containing triglycerides and wax esters. At 70–80 µm of the surface, adipocytes, rich in fat, are found in the dermis. Because the CARS signal is generated only at the focal point, the 3D imaging capability is shown with the maximum projection.

CARS at video rate

A resonant scanner provides the benefits of compact design and fast frame recording. When based on a true confocal concept of point-illumination and point-observation, the resonant scanner allows a speed of 16,000 Hz frequency in bidirectional mode. At a frame size of 512 × 512 pixels, the system acquires 29 images per second. With lower sampling, the speed can be increased up to 290 frames per second at a resolution of 512 × 32 pixels. Dynamic processes with high time resolution can be imaged and measured or a linescan can be taken at full rate.

Applying averaging improves the signal-to-noise ratio, which is especially useful in the case of the resonant scanner. It is possible to find the right compromise between image quality and acquisition speed, depending on the imaging require-ments. In professional designs the amplitude of the confocal resonant scanner is tunable, which allows it to zoom in by applying smaller amplitudes. With this feature it is possible to focus into regions of interest while acquiring images at video-rate. The pan function – another helpful device to quickly move into interesting areas, which are not necessarily in the center of the microscopic field – should also be available with the resonant scanner.

Subsequently CARS microscopy opens new ways to visualize  structures based on intrinsic vibrational properties without staining or labeling the specimen. The specimen does not suffer from perturbation by the dye or photo-bleaching. CARS opens new methods of research, especially in cell biology, neurosciences, pharmacology, dermatology, and medical imaging.

Fig. 4: These CARS pictures show lipid-rich adipocytes of the subcutaneous fat layer of mouse skin. The left image is taken at full speed of the resonant scanner, acquiring images at a rate of 29 images/second in bi-directional mode, i.e., at video rate. Within the pixel dwell time of 120 ns only a few CARS photons are detected, resulting in a noisy image. The image on the right corresponds to averaging over 30 images taken with the resonant scanner, i.e., corresponds to the image quality of the non-resonant scanner. Courtesy of Prof. Sunney Xie, Department of Chemistry and Chemical Biology, Harvard University, Cambridge, USA
Subcutanous fat layer of mouse skin, imaged with Leica TCS SP5 CARS. Left: resonant scanning at 30 frames per second, corresponding to 120ns per pixel still offers a good illustration of the structures. For comparison on the right: image taken with 30 ave

Making CARS microscopy accessible

Prof. Andreas Zumbusch, Department of Chemistry, University of Konstanz, Germany, heads a research group for Physical Chemistry. He focuses on single molecule fluorescence spectroscopy and microscopy, as well as on the development and application of non-linear optical microscopy: "Many research areas, whether from a life science or materials science background, require fast, non-invasive imaging with high spatial resolution, high molecular specificity, and high sensitivity. As an optical method by which contrast is generated on the basis of spectroscopic properties intrinsic to the sample, CARS microscopy can offer all of this. In neuroscience, for example, CARS microscopy can have an impact comparable to two-photon microscopy, except that it does not rely on the introduction of fluorescent labels.

The advent of commercial CARS microscopy systems is certainly a major step toward making the technique accessible to researchers interested in its application. Attractive system concepts offer hands-off operation while allowing the full potential of CARS microscopy to be used."

Fig. 5: Unstained skin of a mouse ear. Sebacouce glands are multicellular compartments, which contain triglycerides and wax ester. Due to their CH<sub>2</sub> bondings, fatty components can be perfectly imaged with CARS and lead to sharp, crisp imaging results. Courtesy of Prof. Sunney Xie, Department of Chemistry and Chemical Biology, Harvard University, Cambridge, USA
Sebaceous glands, CARS image of fatty components by exciting CH2 bondings. Image taken with Leica TCS SP5 CARS.
Fig. 6: The cross-section of a mouse tail imaged labelfree with epi-CARS (reflected light, red areas) and F-SHG (transmitted light, green areas) combining information about lipids and structural properties of the specimen.
Two parameter imaging in mouse tail. Color coding green for F-SHG (forward-Second Harmonic Generation, an MP-technique) and red for epi-CARS (backscattered Coherent Anti-Stokes Raman Spectroscopy). F-SHG reveals structural properties, especially from coll