Coherent Anti-Stokes Raman Scattering Microscopy for Molecular Vibrational Imaging  
 
oherent anti-Stokes Raman scattering (CARS) microscopy is a novel nonlinear microscopic imaging technique that has received much interest for imaging living cells and tissues. This is due to its several outstanding

capabilities which include providing both the chemical composition and structural information of biological samples without using fluorescence probes, its high sensitivity, and its three-dimensional (3D) optical sectioning ability with high spatial and spectral resolution.

CARS is a third-order nonlinear optical process (Figure 1) that involves a pump beam at a frequency of ω p, a Stokes beam at a frequency of ω s, and a signal at the anti-Stokes frequency of 2ω p-ω s, generated in the phase matching direction (Figure 1(b)). A strong CARS signal is created when the frequency difference (ω p-ω s) between the pump and the Stokes beams is tuned to be resonant with a Raman-active molecular vibration of the tissue and cells. Therefore, the contrast of CARS signals is directly derived from molecular vibrations that are characteristic of the chemical composition and molecular structure of the sample.

CARS also possesses a higher sensitivity than Raman microscopy because the coherent CARS radiations produce a large and directional signal. This permits fast imaging by using relatively low average laser power which leads to less photodamage/ photobleaching of cells, and consequently, is superior to confocal fluorescence microscopy. The nonlinear excitation intensity dependence of CARS also provides an inherent 3D sectioning capability with submicron spatial resolution, which is ideally suited for spatially resolving and imaging small, physiologically important molecules in live cells. Furthermore, the wavelength of CARS is shorter than those of CARS excitation lasers, and thus the CARS emission is separable from tissue and cell autofluorescence. These advantages make CARS microscopy a very attractive tool to study biological samples.

However, CARS microscopy is not background free, as there exists a non-resonant signal from the electronic response of the medium (e.g, water in cells) which is virtually independent of the Raman shift. The interference of the strong non-resonant background arising from surrounding solvent and other media in cells degrades the image contrast and spectral selectivity.


 

To solve this problem, we have recently developed an interferometric polarization CARS (P-CARS) microscopy system that is capable of providing high contrast images of bio-samples while effectively suppressing background signals. Figure 2 shows an example of CARS images of 10 µm polystyrene beads in water under (a) non-polarization, and (b) polarization excitation conditions, respectively. It can be seen that the P-CARS imaging gives a much better image contrast as compared to the non-polarization case, as the nonresonant background from water is significantly reduced. Figure 3 (a) shows the CARS image acquired from 8 µm polystyrene beads at vibrational frequency of 3050 cm-1 with 0.2 µm resolution. The intensity distribution of the polystyrene bead image is highly contrasted compared to the surrounding water background as can be observed in Figure 3 (b). The results demonstrate that the novel interferometric P-CARS microscopy technique developed has the ability for imaging various molecular vibrational bands of biochemicals and biomaterials with submicron spatial resolution. We anticipate that this nonlinear microscopy technique will be particularly advantageous in imaging small biomolecules such as DNA/RNA, proteins and lipids, hormones, drug molecules, and small cellular organelles and structures for which fluorescence labeling is not well-suited due to its tendency to alter normal function in cells and tissues.

 


Contact person

Dr ZW Huang
Tel: 6516 8856,
Fax: 6872 3069
E-mail: biehzw@nus.edu.sg
 
 


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