In spite of the fact that the original Raman microscope was designed in the early 1970's for Raman imaging,
wide-spread practical use of the technology did not appear until the last 5 years. The instruments are smaller,
faster, easier-to-use, promoting reports of a variety of interesting applications in fields as diverse as
nanomaterials, pharmaceuticals, composites, semiconductors, bio-clinical studies, polymers, ceramics and
glasses. While the information content in Raman analysis is quite high, the time to acquire an image has been
a deterrent to its application. Recent innovations including Swift and DUO Scan have addressed and are
addressing these issues. SWIFT (Scanning with Incredibly Fast Times) is a rapid CCD read-out technique
that is based on the synchronization between the XY motion of the motorized or piezo stage and the CCD
readout. DUO scanning uses a set of scanning mirrors above the microscope objective to raster rapidly the
laser beam across a sample area. This can be used to create a "giant pixel" in the map without compromising
the NA of the light collection, or to create a map with step sizes as small as 10nm. Swift, in combination with
DUO scan, as been used to produce full spectral maps of pharmaceutical tablets in times as short as 10
minutes, something that was previously believed to be near impossible. Off-line analysis of such a map using
multivariate techniques produces Raman images indicating the quality of component mixing, and also the
presence of minor, difficult-to-detect components (such as Mgstearate in pharmaceutical tablets).
Raman and FTIR microprobe spectroscopy have been used to characterize the atherosclerotic process in Apo E and wild type mice. The Apo E null mouse is being studied in parallel with a healthy strain as a model of the human atherosclerotic disease. Preliminary Raman microprobe spectra have been recorded from the lumen of the aorta vessels from a normal black mouse (C57BL/6J) and the apo E null mouse fed on a normal chow diet. Spectra were also recorded from another normal mouse fed breeder chow containing a much higher content of fats. In the Raman spectra the fat cells exhibited spectra typical of esterified triglycerides while the wall tissue had spectra dominated by Amide I and III modes and the phenylalanine stretch at 1003 cm-1 of protein. The FTIR spectra showed the typical Amide I and II bands of protein and the strong >C=O stretch of the triglycerides. In addition, there were morphologically distinct regions of the specimens indicating a surprising form of calcification in one very old mouse (wild type), and free fatty acid inclusions in the knock out mouse. The observation of these chemistries provide new information for elucidation of the molecular mechanisms of the development of atherosclerosis.
Implementation of higher spectral and spatial resolution in dispersive Raman microscopes, including access to a variety of excitation wavelengths, has proven beneficial in the semiconductor industry. UV adaptations accommodate measurement of smaller defects, higher sensitivity to thin films (to the exclusion of the substrate) and access to enhancement conditions for materials such as GaN-based photodiodes and lasers, and diamond. The availability of a high dispersion spectrograph, especially for UV wavelengths, avoids compromising spectral resolution. Examples of successful analysis requiring longer focal length, mirror-based spectrographs are shown; these include stress in silicon-based devices, Raman and PL of InGaN (which provides information on composition) and carbon nanotube studies.
Lasers can induce subtle and not so subtle changes in material structure. We have found that certain pigments can undergo chemical and crystallographic changes and concomitant color shifts. Minerals and the related pigments may experience a loss of hydroxyl groups or other chemical reordering. The organic component of skeletal, keratinaceous, and cellulosic materials can be pyrolized, ablated, or etched. Polymers can discolor, undergo structural weakening, or be volatilized. A few of these processes have been investigated with regards to changes on ivory and bone, selected pigments and the removal of dye-based pen ink from porous substrates.
A transparent organic/inorganic hybrid, which is simple to prepare by the sol-gel process, is poly(ethylene oxide) (PEO)/silica. The reason for its simplicity is that PEO is soluble in water and mixes easily with bonds with silanols. Hybrids of PEO/silica were prepared with PEO molecular weights 200, 1000, 2000, 3400, and 8000. Raman microscopy was used look at environmental effects of the polymer in the silica spectra. Both the hydrogen-bonding between the organic and inorganic components and the steric effects of the organic component resulted in changes in the Raman spectra. With low molecular weight PEO, the spectra showed large shifts in CH bands. For high molecular weight PEO, the spectra showed broadening, indicative of more fluid behavior of the PEO. The interaction between the ether oxygens and the silica network varied with molecular weight and the ratio of PEO to silica.
Prostaglandin H synthase (PGHS), commonly known as cyclooxygenase (COX), is the enzyme involved in the synthesis of biologically active prostanoids from arachidonic acid. There are two PGHS isozymes with identical functions, similar sizes and similar structures. PGHS-1 is constitutively expressed in most mammalian cells where as PGHS-2 is induced by various agents. PGHS-2 expression was increased with inflammation, mitogenesis, and some types of cancer. We have developed a method to simultaneously detect PGHS-1 and PGHS-2 in single cancer cells by using specific antibodies, surface-enhanced Raman scattering, and confocal Raman microspectroscopy. Cells were plated, cultured, incubated for immunocomplexation, and detected directly in the wells of a multiwell plate. The expression and localization of PGHS-1 and -2 in single malignant human hepatocytes was detected and compared to normal cells. Furthermore, the correlation between the expression of PGHS and the invasiveness of cancer cells was investigated in PC- 3 cell sublines.
We developed a confocal Raman microspectroscopic technique to study ligand-receptor bindings in single cells using Raman-labeled ligands and surface-enhanced Raman scattering (SERS). The adrenal zona glomerulosa (ZG) cells were used as a model in this study. ZG cells have a high density of angiotensin II (AII) receptors on the cellular membrane. There are two identified subtypes of AII receptors,namely AT1 and AT2 receptors. AII is a peptidic hormone, which upon binding to its receptors, stimulates the release of aldosterone from ZG cells. The cellular localization of these receptors subtypes was detected in single ZG cells by using immunocomplexation of receptors with specific antibodies and confocal Raman microspectroscopy. In the binding study, we used biotin-labeled AII to bind to its receptors in ZG cells. Then, avidin and Raman-labeled AII. The binding was measure directly on the single ZG cells. The results showed that the binding was displaced with unlabeled AII and specific AII antagonists. This is a rapid and sensitive technique for detection of cellular ligand bindings as well as antagonists screening in drug discovery.
Recent developments in Raman instrumentation have produced sensitive compact rugged systems capable of monitoring industrial processes in-situ using fiber optic probes. Raman spectroscopy's ability to give definitive molecular information in aqueous and organic matrices makes it exceedingly powerful in monitoring and troubleshooting chemical processing.
Polarized Raman spectra of spin-oriented and drawn fibers of high-tenacity polyethylene and polyethylene terephthalate have been measured in order to correlate bulk properties with microscopic structural order. Band-fitting is pivotal to separating components of closely spaced lines. In the case of polyethylene terephthalate, glycol bands just below ~1100 cm-i correlate in intensity with orientation as derived front optical birefringence. Separate bands can be correlated with the trans crystalline and trans amorphous phases. Polarized spectra of a fiber of polyethylene with high orientation and crystallinity can be used as a basis with which to compare partially crystalline materials. Since the amorphous phase of oriented partially crystalline polymers determines the practical strength of these fibers, the Raman data that provides information otherwise unobtainable can be useful in their characterization.
Polarized Raman micrbprobe spectra of spin-oriented, as well as spun
and drawn, single filaments of polyethylene terephthalate have demonstrated
the development of both orientation and crystallization with take-up speed
(TUS) and conditions of drawing1-. Careful examination of those spectra
indicated the presence of overlapping bands in the carbonyl and glycol
regions. By using commercially available band-fitting software2 we have
succeeded in separating carbonyl band components and correlating them with
different atomic arrangements of the polymer.
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