Coherent anti-Stokes Raman spectroscopy
المؤلف:
Peter Atkins، Julio de Paula
المصدر:
ATKINS PHYSICAL CHEMISTRY
الجزء والصفحة:
ص465-466
2025-12-06
56
Coherent anti-Stokes Raman spectroscopy
The intensity of Raman transitions may be enhanced by coherent anti-Stokes Raman spectroscopy (CARS, Fig. 13.47). The technique relies on the fact that, if two laser beams of frequencies ν1 andν2pass through a sample, then they may mix together and give rise to coherent radiation of several different frequencies, one of which is , ν′=2ν1−ν2 ,
Suppose that ν2 is varied until it matches any Stokes line from the sample, such as the one with frequency ν1 −∆ν; then the coherent emission will have frequency , ν′=2ν1−(ν1−∆ν) =ν1+∆ν , which is the frequency of the corresponding anti-Stokes line. This coherent radiation forms a narrow beam of high intensity. An advantage of CARS is that it can be used to study Raman transitions in the presence of competing incoherent background radiation, and so can be used to observe the Raman spectra of species in flames. One example is the vibration–rotation CARS spectrum of N2 gas in a methane–air flame shown in Fig 13.48.
Fig. 13.47 The experimental arrangement for the CARS experiment.
Fig. 13.48 CARS spectrum of a methane–air f lame at 2104 K. The peaks correspond to the Q branch of the vibration–rotation spectrum of N2 gas. (Adapted from J.F. Verdieck et al., J. Chem. Educ. 59, 495 (1982).)
Optical microscopes can now be combined with infrared and Raman spectrometers and the vibrational spectra of specimens as small as single biological cells obtained. The techniques of vibrational microscopy provide details of cellular events that cannot be observed with traditional light or electron microscopy. The principles behind the operation of infrared and Raman microscopes are simple: radiation illuminates a small area of the sample, and the transmitted, reflected, or scattered light is first collected by a microscope and then analysed by a spectrometer. The sample is then moved by very small increments along a plane perpendicular to the direction of illumination and the process is repeated until vibrational spectra for all sections the sample are obtained. The size of a sample that can be studied by vibrational microscopy depends on a number of factors, such as the area of illumination and the excitance and wavelength of the incident radiation. Up to a point, the smaller the area that is illuminated, the smaller the area from which a spectrum can be obtained. High excitance is required to increase the rate of arrival of photons at the detector from small illuminated areas. For this reason, lasers and synchrotron radiation (see Further information 13.1) are the preferred radiation sources. In a conventional light microscope, an image is constructed from a pattern of diffracted light waves that emanate from the illuminated object. As a result, some information about the specimen is lost by destructive interference of scattered light waves. Ultimately, this diffraction limit prevents the study of samples that are much smaller than the wavelength of light used as a probe. In practice, two objects will appear as distinct images under a microscope if the distance between their centres is greater than the Airy radius, rAiry = 0.61λ/a, where λ is the wavelength of the incident beam of radiation and a is the numerical aperture of the objective lens, the lens that collects light scattered by the object. The numerical aperture of the objective lens is defined as a = nr sin α, where nr is the refractive index of the lens material (the greater the refractive index, the greater the bending of a ray of light by the lens) and the angle α is the half-angle of the widest cone of scattered light that can collected by the lens (so the lens collects light beams sweeping a cone with angle 2α). Use of the best equipment makes it possible to probe areas as small as 9 µm2 by vibrational microscopy.
Figure 13.49 shows the infrared spectra of a single mouse cell, living and dying. Both spectra have features at 1545 cm−1 and 1650 cm−1 that are due to the peptide carbonyl groups of proteins and a feature at 1240 cm−1 that is due to the phosphodiester (PO2 −) groups of lipids. The dying cell shows an additional absorption at 1730 cm−1, which is due to the ester carbonyl group from an unidentified compound. From a plot of the intensities of individual absorption features as a function of position in the cell, it has been possible to map the distribution of proteins and lipids during cell division and cell death. Vibrational microscopy has also been used in biomedical and pharmaceutical laboratories. Examples include the determination of the size and distribution of a drug in a tablet, the observation of conformational changes in proteins of cancerous cells upon administration of anti-tumour drugs, and the measurement of differences between diseased and normal tissue, such as diseased arteries and the white matter from brains of multiple sclerosis patients.
Fig. 13.49 Infrared absorption spectra of a single mouse cell: (purple) living cell, (blue) dying cell. Adapted from N. Jamin et al., Proc. Natl. Acad. Sci.USA 95, 4837 (1998).
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