Raman spectroscopy gives detailed information about the molecular composition of materials. Combined with multivariate data regression methods, such as Partial Least Squares, it is possible to find a quantitative relation between the measured spectra and the concentration of a particular compound. In this study the feasibility of quantifying glycogen concentrations in rat liver by Raman spectroscopy and PLS analysis was investigated.

We used Raman microspectroscopy to study all major morphological structures from normal and atherosclerotic tissue expressing different stages of disease. Thin sections from coronary artery sample;s were studied with a Raman microspectrometer system. Raman spectra were collected from the internal and external elastic lamina, collagen fibers/fibrous cap, foam cells, smooth muscle cells, necrotic core, adventitial fat, (beta) -carotene containing crystal, cholesterol crystals and calcium mineralizations. To assess the chemical composition of the examined morphological structures each spectrum was modeled with a chemical algorithm containing the Raman spectra of the major chemicals present in artery wall. The results of this analysis show that the chemical composition of each morphological structure is well defined and shows little variation between similar structures from different samples.

We used Raman spectroscopy to assess the chemical composition and pathological states of atherosclerosis in in vitro intact human coronary artery tissue. Human coronary artery samples expressing different stages of atherosclerosis were mounted in an in vitro set-up and perfused with a salt solution. NIR laser light was delivered to the tissue through the central fiber of an optical fiber catheter that was inserted transluminally into a buffer- perfused arterial segment. Tissue Raman signal was collected by seven fibers surrounding the central fiber. The collected Raman light was launched into a spectrometer and imaged onto a CCD. High signal to noise, low background tissue Raman spectra were obtained in 10-60 s form artery samples. The spectral information from each collection fiber was linearly modeled with a Raman spectral model that quantifies the chemical composition of the arterial wall. The model results showed excellent fits to all Raman spectra. A diagnostic algorithm, that has proven to have excellent correlation with historilogical classification by a pathologist, classified the examined tissue into one of three pathological states. From these experiments we conclude that intravascular optical fiber Raman spectroscopy can provide in situ histopathology, which may be used to study vascular disease in vivo.

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.

Raman spectroscopy has been implemented into pharmaceutical analytical laboratories for identity testing. Raman spectroscopy meets the requirements for a specific identity test and provides several opportunities for the pharmaceutical scientist to improve testing systems. Identity testing applications are demonstrated form product development including the identification of TLC spots using microspectroscopy. Rapid identity verification of raw materials, drug products, and packaging components is demonstrated using examples from product development and quality assurance applications. Raman spectroscopy can provide significant time savings for high volume identity testing applications.

The characterization of pharmaceutical solids has always ben a concern for the pharmaceutical scientist, whether it be a formulator, analytical chemist or process chemist. Traditionally, characterization meant chemical purity, but today a more comprehensive characterization is crucial for a safe and consistent pharmaceutical product. Typically, FTIR and FT-Raman are the vibrational techniques used to physical characterize solids. Recent advances in imaging technology have made FT-Raman microscopy another powerful tool for obtaining information of pharmaceutical products. This advance has made it possible to not only obtain chemical information, but gives the ability to spatially resolve the obtained information. Descriptions of various experimental procedures for analyzing formulated tablets as well as other materials of pharmaceutical interest using imaging techniques will be given.

The manufacture of Nadalol at our facilities in Humacao, Puerto Rico, poses a difficult challenge for process analysis because the highly toxic epichlorohydrin makes routine analysis of the chemistry very hazardous. Raman spectroscopy enables us to gather potentially quantifiable and irrefutable data from samples without exposing manufacturing personnel to any hazard. The reaction of epichlorohydrin and sodium (CTA) phenolate monitored by Raman spectroscopy measures both the presence of CTA, epichlorohydrin and tert-butylamine. The Raman shifts of epichlorohydrin at 400-350 cm^-1 and sodium at 1630- 1560 cm^-1 were easily discernible and useful. On one occasion, the increase of moisture in this mixture alerted plant operators to verify the extent of this unexpected contamination. In a short time, optimization of these three aspects with one technique resulted in reliable performance for this stage of the process. The final stage of the process is isolation of the drug substance by crystallization and we learned that this step is strongly influenced by residual tert-butylamine. Using the Raman technique, the presence of this amine is easily detected and accommodated in real time prior to crystallization.

Alginates are biological polymers extracted from brown algae and well known for their gelling and viscosifying properties and biodegradability. Calcium-alginate gels are used for spinning fibers which are particularly interesting as wound dressings since exhibit antihemorrhagic and healing effects. Alginates are copolymers of (alpha) -guluronate (G) and (beta) -D-mannuronate (M). The M/G ratio and the monomer sequence are determinant for their physico-chemical properties but are difficult to control since they are variable depending on algae family, region of origin and season of their harvesting. For rapid and non-destructive structural characterization of single fibers of alginates we used confocal Raman spectral imaging with a micron-scale spatial resolution. The characteristic Raman features were assigned as correlated with the M/G ratio. For the fibers with a determined average M/G ratio, the spectral images appeared rather homogeneous. On the other hand, the Raman relative intensities were found to be dependent on relative orientation of the fiber and laser polarization. We concluded that, with a micron-scale resolution, the fiber samples are well homogeneous and the polymer chains are highly ordered with domination of parallel stacking of the M-M blocks. The approach is actually being applied to study the alginate fibers at the tissular level.

It is increasingly important to fully characterize solid state pharmaceutical systems at the bulk, particulate, and molecular levels. A thorough characterization of the bulk drug at the onset of drug development may save significant time and alleviate a myriad of problems during later stages of development. As with any solid state investigation, a multi-disciplinary approach must be adopted to fully characterize the system, whether it be polymorphic, pseudopolymorphic, or a salt selection process. Typically, techniques such as XRPD, thermal analysis, micromeritics, and spectroscopy are used. At the molecular level, solid- state spectroscopy techniques are being widely used, specifically IR, NMR, and more recently, Raman spectroscopy. This discussion will focus on the use of Raman spectroscopy for the physical characterization of pharmaceutical solids. Descriptions of various experimental procedures will be highlighted by referring to examples of specific solid state characterization problems. An overall approach to polymorphic characterization and quantitation will also be outlined including requirements for a regulatory filing of a quantitative assay.

FT-Raman spectroscopy has been used as a tool for investigating permeation into and diffusion through human skin membranes. Thermal studies showed that the lipid component of stratum corneum, the outermost layer of skin and the main barrier to diffusion of most drugs, is disrupted as the lipids melt. This measure for disruption provides a positive control against which penetration enhancer effects on the lipids can be judged. 1,8-Cineole, a model enhancer unexpectedly increases order in the lipid domains, probably as a result of phase separation within the tissue although permeation defects where the enhancer bounds the skin lipids may allow improved drug flux. Permeation through skin membranes was successfully followed for simple one component permeants but with more complex mixtures of permeants there were several technical problems, which require further attention. However, the result suggest that the technique may be valuable for examining permeation of complex mixtures through membranes.

The potential environmental risks associated with dental amalgams have forced many European countries to ban their use and turn to alternative materials, composite resins. The purpose of this study was to correlate morphologic characterization of the dentin/adhesive bond with chemical analyses using micro-FTIR and micro-Raman spectroscopy. A commercial dental adhesive was placed on dentin substrates cut from extracted, unerupted human third molars. Sections of the dentin/adhesive interface were investigated using IR radiation produced at the Aladdin synchrotron source; visible radiation from Kr⁺ laser was used for the micro-Raman spectroscopy and through the use of differentially staining in conjunction with light microscopy. Due to its limited spatial resolution and the unknown samples thickness the IR results cannot be used quantitatively in determining the extent of diffusion. The result from the micro-Raman spectroscopy and light microscopy indicate exposed protein at the dentin/adhesive interface. Using a laser that reduces background fluorescence, the micro-Raman spectroscopy provides quantitative chemical and morphologic information on the dentin/adhesive interface. The staining procedure is sensitive to sites of pure protein and complements the Raman results.

Camptothecin (CPT) derivatives are the well known inhibitors of the human DNA topoisomerase (topo) I. Two of them, irinotecan and topotecan, are just in the clinics; 9-amino- CPT is on the stage II of clinical trials, and the active search for new derivatives is now in progress. Stability of the CPT derivatives on their way to the target and resistance of cancer cells to these drugs present the crucial problem of the chemotherapy. Human serum albumin (HSA) is the mediator of transport and metabolism of numerous pharmaceuticals in the blood and P-glycoprotein (P- gp) plays a crucial role of the mediator of the multidrug resistance (MDR) of the cancer cells. This paper present the result of analysis of molecular interactions of some drugs of CPT family with the HSA and P-gp. Induced circular dichroism (CD) and Raman techniques have been applied for monitoring molecular interaction of drugs with HSA as well as to identify the conformational transition of the protein induced by the drug binding. Drug molecular determinants responsible for interaction have been identified and their binding sites within the HSA have been localized. New cancer cells lines exhibiting an extremely high level of MDR resistance have been established and were shown to contain the P-gp overproduced in the quantities of 35 percent from the all membrane proteins. The membrane fractions of these cells with the controls presented by the membranes of the parental membrane proteins. The membrane fractions of these cells with the controls presented by the membranes of the parental sensitive cells may be used as a model system for spectroscopic analysis of the specific pharmaceuticals/P-gp interactions.

We discuss the use of Raman microprobe spectroscopy and Raman imaging to study the chemical composition of fresh, unmounted bone at a microscopic level. A specimen of human cortical bone was analyzed and evidence for the presence of amorphous-type calcium phosphate, a theoretical precursor in the bone formation process, was found. In general the amorphous-type calcium phosphate appears away from osteons, in the interstitial tissue. This finding calls into question the role of amorphous-type calcium phosphate as a precursor to apatitic phosphate, since it was not found in the recently remodeled bone near the osteon center, but rather in older bone tissue. Some reasons for the presence of amorphous calcium phosphate are proposed. Possible relations of the amorphous mineral to bone damage and bone remodeling are discussed.

Near IR confocal Raman spectroscopy is a non-invasive and non-destructive technique that can provide information about molecular structure and composition in vivo. It is therefore of great interest for skin research. Neither samples preparation nor the use of markers and dyes are required. High quality spectra of the skin can be obtained in several seconds to minutes. Here we present in vivo Raman spectra of the outermost skin layer: stratum corneum. The spectra are interpreted in terms of differences between the molecular composition of the stratum corneum at two different body locations.

The resurgence of Raman spectroscopy, in the late 1980's has led to an increase in the use of the technique for the analysis of biological tissues. Consequently, Raman spectroscopy is now regarded to be a well-established non- invasive, non-destructive technique, which is used to obtain good quality spectra from biological tissues with minimal fluorescence. What is presently of interest to our group is to develop further and establish the technique for in vivo investigations of healthy and diseased skin. This presentation discusses some potentially valuable clinical applications of the technique, and also highlights some of the experimental difficulties that were encountered when examining patients who were receiving treatment for psoriasis.

We have developed a confocal Raman spectroscopy system in order to noninvasively characterize ocular tissue with both an in vitro nd in vivo capability. This systems consists of a long working distance optical probe, which focuses the incident laser light on the tissue and collects the backscattered Raman signal, a single grating spectrometer with CCD camera, and an optical fiber which couples the optical probe to the spectrometer. Essential to the confocal design is that the sample volume is limited, preventing detection of interference signals and fluorescence from adjacent ocular tissues. This sample volume is adjustable by changing the diameter of the collection fiber which acts as the pinhole in the system. Potential applications of this technique such as assessing corneal hydration and quantifying pharmacokinetic drug transport across the cornea will be discussed.

Imaging methodologies present some of the most exciting new frontiers in the biological and medical sciences. Raman spectroscopic imaging combines the power of chemical imaging with the spatial resolution for translating microscopic spectroscopic information into statements relevant to biological and medical function. Imaging results will be presented using mapping, dielectric filters, and liquid- crystalline tunable filters at different excitation wavelengths for selectively determining the spatial distribution of biomaterials in a variety of biological systems.

We discuss the use of Raman microprobe spectroscopy and Raman imaging to study the chemical composition of fresh, unmounted bone at a microscopic level. A specimen of human cortical bone was analyzed and evidence for the presence of amorphous-type calcium phosphate, a theoretical precursor in the bone formation process, was found. In general the amorphous4ype calcium phosphate appears away from osteons, in the interstitial tissue. This finding calls into question the role of amorphous-type calcium phosphate as a precursor to apatitic phosphate, since it was not found in the recently remodeled bone near the osteon center, but rather in older bone tissue. Some reasons for the presence of amorphous calcium phosphate are proposed. Possible relations ofthe amorphous mineral to bone damage and bone remodeling are discussed.

The utility of Raman microscopy and imaging for the characterization of a variety of chemical and biological systems is discussed. Measurements have been carried out with an optical microscope coupled to a Raman spectrometer that contains light paths for both single point and imaging measurements. Laser irradiation and signal collection are implemented using epi-illumination through a single microscope objective. For point Raman microspectroscopy. In our arrangement, the laser is defocused to provide wide- field illumination. The Raman signal from within the irradiated sample area is directed through a narrow-band liquid crystal tunable filter (LCTF) and imaged onto the CCD. Spectroscopic information is obtained by recording Raman images through the LCTF over successively tuned frequencies. Raman spectra for various point within the sample thus are obtained in parallel by each pixel in the detector array. Microspectra were recorded within various sample, including bacteria. Spectroscopic features of interest were then investigated in greater spatial detail using the LCTF imaging methodology.

The analytical potential for routine Raman analyses has promoted the development of class 1 instruments configured for analytical laboratory use. A particular topic of interest regarding these systems for the biomedical and pharmaceutical fields is calibration standardization. Widespread acceptance of Raman spectroscopy in regulated industries requires automated, reliable, traceable instrument calibration. Key dispersive Raman analyzer elements that require calibration include excitation laser wavelength, Raman emission wavelengths, and the spectral response profile of the instrument. In this paper we will detail recent developments in fiber optically coupled Raman instruments. Hardware approaches to calibration issues will be the primary focus of this discussion. Candidate wavelength and intensity calibration references are evaluated. Potential system calibration/qualification protocols are discussed.

UV resonance Raman spectroscopy (UVRRS) is becoming a very popular spectroscopic method for bioanalytical investigations due to its high sensitivity, lack of fluorescence, and suitability for use in aqueous solutions. We have made a number of technological advances, especially the development of fiber-optic-based technologies, which permit the performance of remote/in-situ UVRRS measurements. We will be reporting on improved optical fiber probes and demonstrate their benefits in performing UVRRS on neurotransmitters, saliva, and urine.

Bacterial hydrolysis of triglycerides is followed in a sebum probe phantom by microprobe surface-enhanced Raman scattering (SERS) spectroscopy. The phantom consists of a purpose-built syringe pump operating at physiological flow rates connected to a 300 micron i.d. capillary. We employ silicon substrate SERS microprobes to monitor the hydrolysis products. The silicon support allows some tip flexibility that makes these probes ideal for insertion into small structures. Propionibacterium acnes are immobilized on the inner surface of the capillary. These bacteria hydrolyze the triglycerides in a model sebum emulsion flowing through the capillary. The transformation is followed in vitro as changes in the SERS caused by hydrolysis of triglyceride to fatty acid. The breakdown products consists of a mixture of mono- and diglycerides and their parent long chain fatty acids. The fatty acids adsorb as their carboxylates and can be readily identified by their characteristic spectra. The technique can also confirm the presence of bacteria by detection of short chain carboxylic acids released as products of glucose fermentation during the growth cycle of these cells. Co-adsorption of propionate is observed. Spatial localization of the bacteria is obtained by ex-situ line imaging of the probe.

Raman spectroscopy has been sued to differentiate between sensitive and MDR-resistant cells using Raman spectral imaging with a 632.8 nm excitation wavelength. The comparison between two spectral images allowed to quantify the differences between sensitive and resistant cell lines in term of proteins, lipids when MDR phenotype is expressed. SER spectroscopy has become a powerful and non-invasive probe for investigating the molecular and cellular interaction of drugs with their targets. The comparison between these models allow to elucidate the biological effect of the drugs. The development of new types of SERS- active substrates has extended the applicability of this technique to medical diagnosis. Two kinds of SERS active substrates, characterized as 'bio-compatible' systems, can be used for investigation on single living cells: colloid suspensions and microelectrodes and island films. This methodology is used for the study of cell membrane components in interaction with the SERS substrates with the aim to understand the resistance mechanism. The constitution of a data bank will allow the follow-up of cancer and future monitoring of therapeutic intervention.

Fast and exact identification of a great number of microorganisms is becoming a serious challenge. Differentiation and identification of microorganisms is today mainly achieved by the use of a variety of distinct techniques based on morphological, serological aspects and a set of biochemical test. Vibrational spectroscopic techniques can be complementary and useful methods in this field due to their rapidity, 'fingerprinting' capabilities, and the molecular information that they can provide. Using SERS at Ag colloids, we have conducted pilot studies to rapidly detect and identify bacterial clinical strains. Using a Raman microspectrometer equipped with a He/Ne laser, a first attempt to record SERS spectra was made on colloidal solutions. Spectra were of good quality but not very reproducible due to the movement of the microorganisms. Strains were then put in presence of Ag colloids and direct on-plate analysis was performed. Spectra were more reproducible, with diminished fluorescence, and reveal characteristic cellular-level information. Different growth conditions and colloid preparations have been tested. Pseudomonas aeruginosa and Escherichia coli clinical strains, responsible for nosocomial infections, have been our first test samples. An attempt has also been made to record SERS data from gold colloids in view of future measurement in the near-IR. Spectroscopic data are compared with ATR-FTIR results.

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.

Information about DNA sequence variation is increasingly being recognized as an important tool in the analysis of many diseases and in the development of diagnostic, therapeutic, and preventive strategies. Surface enhanced Raman scattering (SERS) is suggested as a new, potentially powerful method for detection and identification of single base differences in double stranded DNA fragments. Enhanced Raman signal is originated from nucleotides that are in direct contact with SERS active metal surface. Aromatic rings in double stranded perfectly matched DNA are hydrogen bonded and do not interact with the metal. In the case of an insertion, deletion or base mismatch, hydrogen bonding is disrupted and an open region is formed. Raman scattering from base pairs in this region undergoes an enhancement resulting in SERS spectrum. Model experiments were performed with 209 base pairs DNA fragment containing one mismatch and adsorbed on electrochemically roughened silver surface. A fragment of the same length but without mismatch was used as a control. No spectra were obtained from the control adsorbed on the SERS substrate, whereas the sample with one mismatch yielded a distinct SERS spectrum. It is believed that all bands in the spectrum correspond to the mismatched base pair and bases from the closest environment around the mismatch.

Traditionally methods for the detection of excitatory amino acids, which have been linked to secondary injury following head trauma, can be excessively time consuming clinically. A near real-time measurement system could provide clinical information in anticipation of pharmaceutical intervention for head injured patients. Our studies have shown that surface-enhanced Raman spectroscopy (SERS) with silver colloids has the ability to measure physiological concentrations of in vitro excitatory amino acids using short scan times. Employing a damage model for ischemia, preliminary ex vivo rat extracellular grain fluid analysis shows an intriguing correlation between SERS spectral features and expected Glutamate concentration fluctuations following head injuries.

The surface-enhanced Raman scattering spectra of insect virus virion protein of Granulosis Virus of Cabbage Batterfly Preris rapae in different pH silver hydrosols have been investigated and compared.

The surface-enhanced Raman Scattering (SERS) spectra of insect virus: the Polyhedrin Protein and Trabala Vishnou Nuclear Polyhedrosis Virus (TvNPV-PP) adsorbed on the surface of silver colloid particles and their Ordinary Raman Scattering (ORS) spectrum were investigated. The SERS spectra and the ORS spectrum of TvNPV-PP show their correlation. In ORS spectrum of TvNPV-PP. The intense amide I at 1656 nm^-1, the medium-intense amide I 1673 cm^-1 and a week amide III at 1280 cm^-1 show that TvNPV-PP has predominantly (alpha) -helical and (beta) - sheet structure. In SERS spectra of TvNPV-PP the intense bands related to carboxylate groups vibrations suggest that TvNPV-PP are adsorbed on the silver colloid surface through the carboxylate groups. The bands of amide I and amide III could not be observed in SERS residues increased their distance from Ag surface and weaken the enhanced effect of the corresponding vibrations. Chemisorption is a main mechanism in the SERS spectra on silver hydrosols for TvNPV- PP and its mechanism of enhancement has a short-range character. The SERS enhancement factor is approximately 10².

Raman microspectroscopy is a very well appropriate technique for the characterization of the molecules responsible of the wheat grain cohesion, since it is non-destructive and can be readily applied in-situ. The cohesion of the kernel or starchy endosperm depends on a protein content located at the interstices of starch granules. The separation between the kernel and the envelope depends on the composition of the aleurone cells layer, in phenolic acids and pentosans. Confocal Raman microscopy has been performed on kernel sections of various Triticum aestivum samples. Raman spectra recorded at different parts of such sections are very specific, such as spectra of the starchy endosperm protein. The technique has been also used to study the effect of chemical treatment on the binding of the constituents of the aleurone cells walls. In addition, certain marker bands of starch and proteins have been used to construct spectral images.

Detection of nonenzymatic glycated proteins is a very significant feature in diabetes, aging and related diseases, therefore we have carried out an FTIR spectroscopic study for glycated and native proteins such as (gamma) -globulin, human serum albumin. For this purpose, commercially available proteins were glycated by a usual procedure and their FTIR spectra were recorded together with that of the native ones. In order to follow the changes in time, (gamma) -globulin was glycated during 1, 2, 3, 5 and 8 weeks and their spectra were recorded. Direct verification was obtained by examining a model unit where the -NH₂ group was attached to glucose. The spectrum shows a strong peak at 3500 cm^-1 confirming the observed variation in time dependent spectra. The general features of the spectra are very similar and there was no additional structure or change in the peaks. This is understandable as not all the lysine residues are glycated, only a small fraction. Glucose is attached to the (epsilon) -amino group of lysine to form Amadori products, and therefore, the vibrational modes corresponding to the (epsilon) -NH₂ unit of lysine are expected to be altered. This region exactly lies in the Amide I region of protein structure. Careful investigation of this part, indeed, shows a complex structure originated from alternations of -NH₂ group. Thus, the present investigation indicates that an optical approach could be a rapid and effective method to identify the nonenzymatic glycation process.