CARS Microscopy: Imaging Characteristic Vibrational Contrast of Molecules

The chemical bonds in molecules can shake, bend and rattle. They make these motions at particular rates or frequencies. These frequencies are so particular that we can identify what kind of chemical bond is rattling by tuning into their particular vibrational frequencies. For instance, many organic molecules contain bonds between carbon and hydrogen atoms, and thus they have C-H vibrational motions. More importantly, the C–H vibrational frequency is very different from the oscillatory motions of other chemical bonds, such as O–H, the oxygen-hydrogen bond. In other words, by examining the vibrational frequencies of a molecule, we can say something significant about chemical structure of the molecule. A vibrational analysis thus corresponds to a chemical analysis.

Some of these vibrational motions can be addressed by examining molecules with light. Unfortunately, the frequencies of molecular vibrations are much lower than the oscillation frequencies of visible light waves. This means that we cannot directly tune into these molecular motions with common light sources, including the type of lasers that are part of optical microscopes. However, the molecules can be inspected in an indirect way. Raman spectroscopy is an example of such an indirect inspection [1]. In Raman spectroscopy and microscopy, a conventional laser beam addresses the sample and the light that is scattered off the molecules is analyzed. The scattered light that is of a different color than the excitation light may contain information about the molecular vibration. This approach looks very similar to a fluorescence measurement, but the information collected is very different. In Raman spectroscopy, the difference between the frequency of the laser light and the Raman scattered light corresponds to the frequency of the chemical bonds. Using appropriate filters and spectrometers, it is possible to confidently collect the Raman-scattered light in an optical microscope, and thus gather chemical information about the sample. This offers unique possibilities. Since virtually every molecule exhibits particular chemical bond vibrations that are Raman active, it is possible to generate chemical maps of samples without the need of any extrinsic labels. Not surprisingly, this capability has sparked the interest of many research disciplines, including material synthesis, forensic research, mineralogy, toxicology, and the chemical analysis of artwork [2].

Raman spectroscopy and microscopy has also had an impact in biology. Raman microscopy enables the study of cells and tissues in a label-free manner, which opens opportunities towards studying biological samples that are difficult to stain or prepare for standard optical inspection. For instance, Raman spectroscopy has been shown to be sensitive enough to pick up subtle differences in the chemical makeup of healthy and cancerous tissues. Despite its enormous potential for biological research, Raman microscopy has not fully matured into a routine imaging technique in the biology laboratory. The reason for the limited impact of Raman microscopy in tissue and cell research is the intrinsic weakness of the Raman signal. The signal is weak because the Raman effect probes the molecules indirectly: the laser light is not in resonance with the molecular vibrations. The Raman effect is measurable, but, compared to fluorescence, it is a very weak effect. This implies that recording a Raman image takes much longer than taking a fluorescence image. Whereas most fluorescence images can be taken within a second, a Raman image of the same dimensions would require an hour or more. Clearly, such image acquisition times are not very attractive for biological imaging applications.

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