Methods utilizing relatively simple mathematical operations during physical analyses to enable the visualization of otherwise invisible correlations and effects are of particular appeal to researchers and students in pedagogical settings. At the same time, discerning the amorphous phase from its crystalline counterpart in materials is challenging with the use of vibrational spectroscopy and is nowhere as straightforward as in phase composition analytical methods such as X-ray diffraction. A method is demonstrated for the use of first- and second-order differentiation of Fourier transform infrared spectra of calcium phosphates to distinguish their amorphous states from the crystalline ones based on the exact line positioning rather than on comparatively vaguer band broadening and splitting effects. The study utilizes a kinetic approach, focusing on the comparison of spectral features of amorphous precursors annealed in air at different temperatures and aged for different periods of time in an aqueous solution until transforming to one or a mixture of crystalline phases, including hydroxyapatite and α- and β-tricalcium phosphate. One of the findings challenges the concept of the nucleation lag time preceding the crystallization from amorphous precursors as a "dead" period and derives a finite degree of constructive changes occurring at the atomic scale in its course. The differential method for highlighting spectral differences depending on the sample crystallinity allows for monitoring the process of conversion of the amorphous calcium phosphate phase to its crystalline analogue(s). One such method can be of practical significance for synthetic solid state chemists testing for the chemical stability and/or concentration of the reactive amorphous phase in these materials, but also for biologists measuring the maturity of bone and medical researchers evaluating its phase composition and, thus, the state of metabolic and mechanical stability.

Raman spectroscopy is a powerful non-invasive tool for detection and classification of chemical composition of materials including biological tissues. In this work, we report an Raman study on animal skin samples with a focus on high-frequency vibrations such as symmetric CH stretching mode at 2934 cm, and the symmetric CH vibration mode at 2854 cm, OH stretching modes near 3412 cm, and bounded OH mode near 3284 cm. Raman data was acquired with a customized InGaAs based Raman spectrometer that consolidates the NIR (866 nm) light and the InGaAs detector and is particularly suitable for probing high-frequency vibrations. The Raman spectra of fat, tendon, and muscle tissues are also analyzed to determine the spectroscopic identities of CH and OH groups in skin. Our results suggest that the protein is beneficial for the maintenance of skin hydration, as it has higher water capacity and greater capability to retain water than lipids. This conclusion is consistent with the additional discovery that water exists in fat mainly as unbound type, while part of water exists as bound type in muscle.

Colloidal silver (Ag) nanoparticles (AgNP) have been widely used for surface-enhanced Raman spectroscopy (SERS) applications. We report a simple, rapid and effective method to prepare AgNP colloids for SERS using the classic organic chemistry Ag mirror reaction with Tollens' reagent. The AgNP colloid prepared with this process was characterized using SEM, and the reaction conditions further optimized using SERS measurements. It was found that Ag mirror reaction conditions that included 20 mM AgNO3, 5 min reaction time, and 0.5 M glucose produced AgNP colloids with an average size of 319.1 nm (s.d ±128.1). These AgNP colloids exhibited a significant SERS response when adenine was used as the reporter molecule. The usefulness of these new AgNP colloids was demonstrated by detecting the nucleotides adenosine 5'-monophosphate (AMP), guanosine 5'-monophosphate (GMP), cytidine 5'-monophosphate (CMP), and uridine 5'-monophosphate (UMP). A detection limit of 500 nM for AMP was achieved with the as-prepared AgNP colloid. The bacterium Mycoplasma pneumoniae was also easily detected in laboratory culture with these SERS substrates. These findings attest to the applicability of this AgNP colloid for the sensitive and specific detection of both small biomolecules and microorganisms.

Estrogens are a group of steroid compounds found in the human body that are eventually discharged and ultimately end up in sewer effluents. Since these compounds can potentially affect the endocrine system its detection and quantification in sewer water is important. In this study, estrogens such as estrone (E1), estradiol (E2), estriol (E3), and ethynylestradiol (EE2) were discriminated and quantitated using Raman spectroscopy. Simulated Raman spectra were correlated with experimental data to identify unique marker peaks, which proved to be useful in differentiating each estrogen molecules. Among these marker peaks are Raman modes arising from hydroxyl groups of the estrogen molecules in the spectral region 3200-3700 cm. Other Raman modes unique to each of the estrogen samples were also identified, including peaks at 1722 cm for E1 and 2109 cm for EE2, which corresponds to their distinctive structures each containing a different set of functional groups. To quantify the components of estrogen mixtures, the intensities of each identifying Raman bands, at 581 cm for E1, 546 cm for E2, 762 cm for E3 and 597 cm for EE2, were compared and normalized against the intensity of a common peak at 783 cm. Quantitative analysis yielded most results within an acceptable 20% error.

Fourier transform infrared (FT-IR) microscopy was used to image tissue samples from twenty patients diagnosed with thyroid carcinoma. The spectral data were then used to differentiate between follicular thyroid carcinoma and follicular variant of papillary thyroid carcinoma using principle component analysis coupled with linear discriminant analysis and a Naïve Bayesian classifier operating on a set of computed spectral metrics. Classification of patients' disease type was accomplished by using average spectra from a wide region containing follicular cells, colloid, and fibrosis; however, classification of disease state at the pixel level was only possible when the extracted spectra were limited to follicular epithelial cells in the samples, excluding the relatively uninformative areas of fibrosis. The results demonstrate the potential of FT-IR microscopy as a tool to assist in the difficult diagnosis of these subtypes of thyroid cancer, and also highlights the importance of selectively and separately analyzing spectral information from different features of a tissue of interest.

Histopathology forms the gold standard for the diagnosis of breast cancer. Fourier Transform Infrared (FT-IR) spectroscopic imaging has been proposed to be a potentially powerful adjunct to current histopathological techniques. Most studies using FT-IR imaging for breast tissue analysis have been in the transmission or transmission-reflection mode, in which the wavelength and optics limit the data to a relatively coarse spatial resolution (typically, coarser than 5 μm × 5 μm per pixel). This resolution is insufficient to examine many histologic structures. Attenuated Total Reflectance (ATR) FT-IR imaging incorporating a Germanium optic can allow for a four-fold increase in spatial resolution due to the material's high refractive index in the mid-IR. Here, we employ ATR FT-IR imaging towards examining cellular and tissue structures that constitute and important component of breast cancer diagnosis. In particular, we resolve and chemically characterize endothelial cells, myoepithelial cells and terminal ductal lobular units. Further extending the ability of IR imaging to examine sub-cellular structures, we report the extraction of intact chromosomes from a breast cancer cells and their spatially localized analysis as a novel approach to understand changes associated with the molecular structure of DNA in breast cancer.

Fourier transform infrared microscopic imaging (FTIRI) was used to quantitatively examine the anisotropies of proteoglycan (PG) and collagen in articular cartilage. Dried 6 μm thick sections of canine humeral cartilage were imaged at 6.25 μm pixel-size in FTIRI with an infrared analyzer set at 26 different angles between 0° and 180° polarization. Like the amide II and amide III peaks, the 1338 cm(-1) band confirms the anisotropy of collagen fibrils in cartilage. The absorption profile of the sugar band shows an anisotropic flipping at the deeper part in the radial zone, just above the tidemark. Together with the reduction in the PG concentration and subsequent increase in tissue calcification in this region, this anisotropy flipping of sugar might be caused by the orientational change in the collagen-attaching PG from orthogonal to parallel when the fibrils are entering the calcified zone.

The integration of near IR picosecond pulse excitation, collinear beam geometry, epi-detection, and laser-scanning has produced a coherent anti-Stokes Raman scattering (CARS) microscope with a detection sensitivity of 10(5) vibrational oscillators, sub-micron 3D resolution, and video-rate acquisition speed. The incorporation of spectral detection and other imaging modalities has added versatility to the CARS microscope. These advances allowed sensitive interrogation of biological samples, particularly lipids that have a high density of CH(2) groups. With initial applications to membrane domains, lipid bodies, demyelinating diseases, obesity, and cardiovascular diseases, CARS microscopy is poised to become a powerful bio-imaging tool with the availability of a multifunctional, affordable, easy-to-operate CARS microscope, and the development of CARS endoscopy for in vivo diagnosis.

The influence of cholesterol (CHO) on the phase behavior of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) planar supported lipid bilayers (PSLBs) was investigated by sum-frequency vibrational spectroscopy (SFVS). The intrinsic symmetry constraints of SFVS were exploited to measure the asymmetric distribution of phase segregated phospholipid domains in the proximal and distal layers of DSPC + CHO binary mixtures as a function of CHO content and temperature. The SFVS results suggest that cholesterol significantly affects the phase segregation and domain distribution in PSLBs of DSPC in a concentration dependent manner, similar to that found in bulk suspensions. The SFVS spectroscopic measurements of phase segregation and structure change in the binary mixture indicate that membrane asymmetry must be present in order for the changes in SFVS signal to be observed. These results therefore provide important evidence for the delocalization and segregation of different phase domain structures in PSLBs due to the interaction of cholesterol and phospholipids.

We report microscopically collected infrared spectra of cells found in human urine in an effort to develop automatic methods for bladder cancer screening. Unsupervised multivariate analysis of the observed spectral patterns reveals distinct spectral classes, which correlated very well with visual cytology. Therefore, we believe that spectral analysis of individual cells can aid cytology in rendering reliable diagnoses based on objective measurements and discriminant algorithms.

We discuss the causes contributing to the variance of the spectra of individual human epithelial cells. This aspect has largely been ignored in previous studies, but needs to be understood for diagnostic applications of infrared micro-spectroscopy. We attribute the spectral variance to Mie scattering, and to variations of nuclear contributions to the overall spectra caused by different nuclear size.

In this contribution, we discuss state-of-the-art methodology for the collection and analysis of hyperspectral images of tissue that will become useful in complementing classical histopathology. In particular, we discuss sampling strategies, data collection methods, and computational approaches to produce pseudo-color maps of large tissue sections of lymph nodes, up to about 100 mm(2) in size. The latter efforts include methods to reduce the presence of dispersion artifacts in IR transflection micro-spectra which can greatly impact the statistical analyzes performed on the data, such as hierarchical cluster analysis and principal components analysis.

Vibrational spectroscopy (Infrared and Raman), and in particular micro-spectroscopy and micro-spectroscopic imaging has been used to characterize developmental changes in bone and other mineralized tissues, to monitor these changes in cell cultures, and to detect disease and drug-induced modifications. Examples of the use of infrared micro-spectroscopy and micro-spectroscopic imaging are discussed in this review.