Raman Spectromicroscopy

The advantage of Raman spectromicroscopy is that very small specimens can be studied while still allowing the determination of the second and fourth moments of the ODF. However, the expressions for the Raman intensities are more complex since the optical effects induced by the microscope objective have to be considered. Although the corrections may be small, they are not necessarily negligible [59]. This problem was first treated by Turrell [59-61] and later by Sourisseau and coworkers [5]. Turrell has mathematically quantified the depolarization of the incident electric field in the focal plane of the objective and the collection efficiency of the scattered light by high numerical aperture objectives. For brevity, only the main results of the calculations will be presented. Readers interested in more details are referred to book chapters and reviews of Turrell or Sourisseau [5,59,61]. The intensity in Raman spectromicroscopy is given by [59-61]... 

Silk fibers, a basic system with a uniaxial symmetry, have also been investigated by Raman spectromicroscopy [63] that is one of the rare techniques capable of providing molecular data on such small (3-10 pm diameter) single filaments. The amide I band of the silk proteins has been particularly studied to determine the molecular orientation using the cylindrical Raman tensor approximation. In this work, it was assumed that Co Ci, C2 and the a parameter was determined from an isotropic sample using the following expression of the depolarization ratio... 

Figure 10 shows polarized spectra of two types of silks recorded by Raman spectromicroscopy the dragline silk (the lifeline) of the spider Nephila edulis and the cocoon silk of a wild silkworm Sarnia cynthia ricini. The position of the amide I band at 1,668-1,669 cm-1 for both threads is characteristic of the /i-sheet... 

The biaxial orientation in photoaddressable azobenzene films has been observed recently by polarized Raman spectromicroscopy [64]. Here, IR spectroscopy has been advantageously used as a complementary technique to measure the order... 

Recent work using polarized Raman spectromicroscopy has elucidated the structural conformation of proteins in all of the various fibers produced by spiders.They can be divided into three categories (Figure 4) whereby major and minor... 

Finally, as in macro-Raman experiments, orientation-insensitive spectra can also be calculated for spectromicroscopy. A method has been developed recently for uniaxially oriented systems and successfully tested on high-density PE rods stretched to a draw ratio of 13 and on Bombyx mori cocoon silk fibers [65]. This method has been theoretically expanded to biaxial samples using the K2 Raman invariant and has proved to be useful to determine the molecular conformation in various polymer thin films [58]. 

Details on other complementary techniques, such as small and wide angle X-ray scattering, X-ray Raman scattering, and X-ray emission, and synchrotron infrared spectromicroscopy methods are not discussed here. However, these methods can provide complementary information on the macromolecular structure and functional group chemistry of organic molecules at different resolution in different sample environments. 

The analysis of MDA-MB-231 cells incubated with 53 was also carried out on a synchrotron-based multiple beam FT-IR imaging (IRENI) set up at the Synchrotron Radiation Center, Stroughton, Wisconsin, equipped with a FPA detector of 96 X 96 pixels, where each pixel corresponds to a 0.54 x 0.54 pm area of the sample. With the better resolution afforded by this technique, the complex appears heterogeneously distributed in the cell, but with a higher perinuclear concentration [79]. In addition, 53 was mapped in cell by SR-UV-SM (synchrotron radiation UV spectromicroscopy), confocal Raman microscopy, and AFM-IR (see Sections 11.3.3 and 11.3.4). Thus, this innovative family of complexes appeared to be a valuable multimodal (and not only bimodal) tool for cell imaging. 

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