Microscopy confocal Raman

At the same time, the objective can collect the scattered light under a wide solid angle, increasing Q. Moreover, the use of a microscope allows to select any part of interest of a sample and, thus, to develop Raman mapping (see Section 7.7). 

In order to obtain a real confocal configuration, a very accurate optical alignment is required as well as a high degree of stability and reproducibility of the mechanical and optical alignment. 

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Figure Bl.2.12. Schematic diagram of apparatus for confocal Raman microscopy. From [3], used with penuission.

Figure 1 Principle of confocal Raman microscopy A laser spot in the focal plane passes through the pinhole P. A laser spot at a distance z from the focal plane is projected in the image plane with size P, and is largely blocked by the pinhole P. [L — lens M = beam splitter fi, f2 = focal length of lens L, and L2, respectively b2 — image distance of out-of-focus laser spot). Reproduced from Tabaksblat et al. [14], with permission of the Society for Applied Spectroscopy. 2000.

Yakovlev, V. V. 2007. Time-gated confocal Raman microscopy. Spectroscopy 22 34-41. 

Puppels, G. J., de Mul, E. E. M., Otto, C., Greve, J., Robert-Nicoud, M., Amdt-Jovin, D. J., and Jovin, T. M. 1990. Smdying single bving cells and chromosomes by confocal Raman microscopy. Nature 347 301-3. 

Abstract The utility of confocal Raman microscopy to study biological events in skin is demonstrated with three examples, (i) monitoring the spatial and structural differences between native and cultured skin, (ii) tracking the permeation and biochemical transformation in skin of a Vitamin E derivative and (iii) tracking the spatial distribution of three major skin proteins (keratin, collagen, and elastin) during wound healing in an explant skin model. 

Experimental Considerations Confocal Raman Microscopy for the Study of Skin... 

Applications of Confocal Raman Microscopy to Skin Pharmacology and Wound Healing... 

Another striking difference between normal and cultured skin is shown in Fig. 15.6. As discussed above (see Fig. 15.3c, factor 2), cholesterol-rich pockets containing highly ordered lipid chains are occasionally detected in human skin and are characterized by a Raman-active mode of cholesterol near 700 cm-1 and an intense lipid C-C stretch near 1130 cm-1 in Fig. 15.4a and b, respectively. The intensity of the cholesterol mode is normalized to a Phe vibration near 620 cm-1 and imaged in Fig. 15.6b. As is evident there are many such pockets in the cultured skin model, in contrast to human skin where they are only rarely observed (Fig. 15.3c, factor 2), and usually in the viable epidermis rather than in the SC (as in the cultured skin). These measurements illustrate the power of confocal Raman microscopy for combining spatial measurements with molecular structure characterization. 

Vitamin E Acetate Delivery and Metabolism in Skin by Confocal Raman Microscopy... 

The traditional methods for evaluation of the delivery and metabolism of exogenous materials in skin involve the use of diffusion cells and/or tape stripping followed by HPLC and mass spectrometry. These methods involve modification of the skin, provide no spatial information, and may alter skin transport properties. In this section, both the permeation and metabolism of a-TAc are monitored inside skin with confocal Raman microscopy. 

Tracking the formation of the bioactive material (a-TH in this case) from its acetate pre-cursor is an important demonstration of the power of confocal Raman microscopy. To develop an analytical approach to track the in situ hydrolysis, we observe (Fig. 15.7c) that a-TAc has a strong feature at 560 cm-1 and weaker bands at 582 and 591 cm-1, while a-TH exhibits a strong feature at 590 cm-1 and a weaker feature at 558cm 1. Thus the area ratio / (590) // (560), with the denominator metric including both the 558 (a-TH) and 560 (a-TAc) bands, provides a useful measure of the hydrolysis. The area ratio of the high-frequency half of the 590 cm-1 to the band at 558/560 cm-1 was used to track hydrolysis. This procedure reduces interference from the 582 cm-1 feature of a-TAc. 

Overall, confocal Raman microscopy demonstrates the ability to track changes in the spatial distribution of two major skin proteins (collagen and... 

Everall, N. H. (2000) Modeling and measuring the effect of refraction on the depth resolution of confocal Raman microscopy. Appl. Spectrosc. 54, 773-82. 

Most recently Puppels and co-workers to determine the concentration of defined NMF component non-invasively in vivo in the SC have pioneered the use of confocal Raman microscopy.84 Figure 18.3 shows depth profiles for the major filaggrin derived components, urea and lactate obtained using this technique. Evidence of leaching from the skin surface is characteristically seen in most profiles and the precipitous drop off in levels of filaggrin derived components deeper in the SC indicates the boundary at which filaggrin hydrolysis is rapidly initiated. 

Due to recent advancements in instrumentation, Raman spectroscopy has become one of the most powerful tools in medical research. Such advancements include development of new lasers, FT-Raman spectroscopy, CCD detectors, confocal Raman microscopy, Raman imaging, fiber optic probes, and computer software. In the following, the utility of Raman spectroscopy in medical science is demonstrated by using selected examples. A more complete coverage of the field is found in review articles by Ozaki (32) and Levin et al. (33). 

In the following, it will be demonstrated that confocal surface-enhanced Raman microscopy is a powerful tool for exploring optical and spectroscopic properties of molecules adsorbed on silver and gold nanoparticles. It is shown that confocal Raman microscopy coupled with high-resolution electron microscopy has the potential to deliver detailed information about the structure of points of high Raman intensity the so-called hot spots. 

Instead of SNOM, in many cases, particularly if the sample is to be screened for Raman active spots and their spatial distance is more than half of the wavelength of the laser, it is also confocal Raman microscopy that delivers enough morphological and spectral information on both nanoparticle structure and SERS activity, respectively. Thus, confocal Raman microscopy is interesting for a wide variety of applications in biology, medicine, and technological materials research. 

Combining confocal Raman microscopy with scanning electron microscopy, the SERS active spots can be retrieved in the SEM image, if the active sample region has been labeled before investigating. 

Silver and gold nanoparticles were generated electrochemically and investigated with confocal Raman microscopy. The combination of surface-enhanced Raman spectroscopy with confocal microscopy accompanied by subsequent scanning electron microscopy provided an image of the geometrical stmcture of the Raman spots. 

Recently, it has been demonstrated that confocal Raman microscopy can be employed to obtain information on the phase, growth direction and radial crystallographic orientation of GaN nanowires [46, 47]. Fan et al. have demonstrated the orientation-dependent micro-Raman measurements on single wurtzite CdS nano wires with an average diameter of 60 nm [48]. The Raman spectra of nanowires were compared with those of bulk ribbon. As opposed to the Raman spectra fi om the bulk samples, the intensity of most Raman bands of the nanowires exhibited significant dependence on the polarization of the excitation with respect to the nanowire axis. The Raman bands were found to be polarized along the longitudinal axis because of the shape-induced depolarization effect, similar to... 

Fig. 15.8 Confocal Raman microscopy and SNOM images of a locally stressed SiC crystal (a) Rayleigh intensity map. (b), (c) Spectral position maps of fitted TO and LO phonon lines obtained by fitting a Lorentzian peak, (d) Topography of the indent, (e) SNOM amplitude for cOiR = 924 cm. (f) SNOM amplitude for (0 = 944 cm (Reprinted from [68])...

Eleming OS, Kazarian SG (2004) Confocal Raman microscopy of morphological changes in poly(ethylene terepthalate) film induced by supercritical CO2. Appl Spectrosc 58 390-394... 

Gigler A, Huber AJ, Bauer M, Ziegler A, Hillenbrand R, Stark R (2009) Nanoscale residual stress-field mapping around nanoindents in SiC by IR s-SNOM and confocal Raman microscopy. Opt Express 17 22351-22357... 

Chemey DP, Harris JM (2010) Confocal Raman microscopy of optical-rrapped particles in liquids. In Annual review of analytical chemistry, vol 3, Annual Reviews, Palo Alto, pp 277-297... 

The application of confocal Raman microscopy to surface Raman has led to some promising results for transition-metal surfaces that yield weak or negligible field enhancement. High-performance CCD detection provided high SNR, and the confocal sampling reduced the background from the solution or gas over the surface (22, 23). Metals such as platinum, rhodium, and ruthenium... 

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