An FTIR image of an 8 µm section of de-paraffinised bronchial biopsy that shows a histological transition from normal to severe dysplasia/squamous cell carcinoma (SCC) insitu was obtained in transmission by stitching together images of 256 x 256 µm recorded using a 96 x 96 element FPA detector. Each pixel spectrum was calculated from 128 co-added interferograms at 4 cm−1 resolution. In order to improve the signal to noise ratio, blocks of 4x4 adjacent pixels were subsequently averaged. Analyses of this spectral image, after conversion of the spectra to their second derivatives, show that the epithelium and the lamina propria tissue types can be distinguished using the area of troughs at either 1591, 1334, 1275 or 1215 cm−1 or, more effectively, by separation into two groups by hierarchical clustering (HCA) of the 1614-1465 region. Due to an insufficient signal to noise ratio, disease stages within the image could not be distinguished with this extent of pixel averaging. However, after separation of the cell types, disease stages within either the epithelium or the lamina propria could be distinguished if spectra were averaged from larger, manually selected areas of the tissue. Both cell types reveal spectral differences that follow a transition from normal to cancerous histology. For example, spectral changes that occurred in the epithelium over the transition from normal to carcinoma insitu could be seen in the 1200-1000 cm−1 region, particularly as a decrease in the second derivative troughs at 1074 and 1036 cm−1 , consistent with changes in some form of carbohydrate. Spectral differences that indicate a disease transition from normal to carcinoma in the lamina propria could be seen in the 1350-1175 cm−1 and 1125-1030 cm−1 regions. Thus demonstrating that a progression from healthy to severe dysplasia/squamous cell carcinoma (SCC) insitu can be seen using FTIR spectroscopic imaging and multivariate analysis.
The cutting of bone is routinely required in medical procedures, especially in dental applications. In such cases, bone
regeneration and new bone quality can determine the success of the treatment. This study investigated the main spectral
differences of undamaged and healed bone using the ATR-FTIR spectroscopy technique. Three rabbits were submitted
to a surgical procedure; a small piece of bone (3x3 mm2) was removed from both sides of their jaws using a high speed
drill. After 15 days, the rabbits were euthanized and the jaws were removed. A bone slice was cut from each side of the
jaw containing regions of undamaged and newly formed bone, resulting in six samples which were polished for
spectroscopic comparison. The samples were analyzed by FTIR spectroscopy using a diamond ATR accessory. Spectral
characteristics were compared and particular attention was paid to the proportion of phosphate to amide I bands and the
width of the phosphate band. The results show that the ratio of phosphate to amide I is smaller in new bone tissue than in
the undamaged bone, indicating a higher organic content in the newly formed bone. The analysis of the width of the
phosphate band suggests a crystallinity difference between both tissues, since the width was higher in the new bone than
in the natural bone. These results suggest that the differences observed in bone aging processes by FTIR spectroscopic
can be applied to the study of healing processes.
Diffusion of two model drugs-benzyl nicotinate and ibuprofen-and the plasma macromolecule albumin across atherosclerotic rabbit aorta was studied ex vivo by attenuated total reflection-Fourier transform infrared (ATR-FTIR) imaging. Solutions of these molecules were applied to the endothelial surface of histological sections of the aortic wall that were sandwiched between two impermeable surfaces. An array of spectra, each corresponding to a specific location in the section, was obtained at various times during solute diffusion into the wall and revealed the distribution of the solutes within the tissue. Benzyl nicotinate in Ringer's solution showed higher affinity for atherosclerotic plaque than for apparently healthy tissue. Transmural concentration profiles for albumin demonstrated its permeation across the section and were consistent with a relatively low distribution volume for the macromolecule in the middle of the wall. The ability of albumin to act as a drug carrier for ibuprofen, otherwise undetected within the tissue, was demonstrated by multivariate subtraction image analysis. In conclusion, ATR-FTIR imaging can be used to study transport processes in tissue samples with high spatial and temporal resolution and without the need to label the solutes under study.
Infrared (IR) spectroscopy is one of the most chemically specific analytical methods that gives information about composition, structure, and interactions in a material. IR spectroscopy has been successfully applied to study the permeation of xenobiotics through the skin. Combining IR spectroscopy with an IR array detector led to the development of Fourier transform infrared (FTIR) spectroscopic imaging, which generates chemical information from different areas of a sample at the microscopic level. This is particularly important for heterogeneous samples, such as skin. Attenuated total reflectance (ATR)-FTIR imaging has been applied to measure, in situ, the diffusion of benzyl nicotinate (BN) through the outer layer of human skin [stratum corneum (SC)]. In vitro experiments have demonstrated the heterogeneous distribution of SC surface lipids before the penetration of a saturated solution of BN. Image analysis demonstrated a strong correlation between the distribution of lipids and drugs, while ethanol appeared to be homogenously distributed in the SC. These results show the ability of ATR-FTIR imaging to measure simultaneously the affinities of drug and solvent to the lipid-rich and lipid-poor skin domains, respectively, during permeation. This information may be useful in better understanding drug-diffusion pathways through the SC.
Attenuated total reflection Fourier transform infrared spectroscopic imaging combined with tape-stripping is an advantageous approach to map the depth penetration and lateral distribution of topically applied chemicals in Stratum corneum (SC) and the conformational order of SC lipids. Tape-stripping progressively removes layers of SC, and chemical imaging provides spatially resolved information on the chemical composition of both the newly exposed SC surface and of the tapes used for stripping. The procedure is rapid, minimally invasive, and does not necessitate cross-sectioning of the skin. This approach offers a simple and direct way to determine the distribution of exogenous volatile and non-volatile chemicals in SC as a function of the chemical composition of the formulation and time, and the conformational order of SC lipids in native and topically treated skin. The procedure described here is well suited to address questions of relevance for the areas of drug delivery, dermatology, and skin care.
Attenuated total reflection Fourier transform infrared spectroscopic imaging was applied to study human stratum corneum (SC) tissue, the outermost layer of the skin. This imaging approach was combined with a controlled environment cell to demonstrate the possibility of obtaining chemical images of SC exposed to a wide range of relative humidities and diffusion of ethanol through the SC tissue with a specially designed liquid cell. The effect of water vapor sorbed into the SC on the distribution of other components in the SC was studied. Principal component analysis was applied in conjunction with univariate analysis to differentiate the distribution of different components in the SC. Swelling of the SC, a heterogeneous distribution of natural moisturizing factor and water, was detected upon the increase of relative humidity. The approach to image the penetration of liquid ethanol into the SC was also demonstrated and showed good potential and implications for studying transdermal drug delivery.
Conventional FTIR spectroscopy and microscopy has been widely used in forensic science. New opportunities exist to
obtain rapid chemical images and to enhance the sensitivity of detection of trace materials using attenuated total
reflection (ATR) Fourier transform infrared (FTIR) spectroscopy coupled with a focal-plane array (FPA) detector. In this
work, the sensitivity of ATR-FTIR spectroscopic imaging using three different kinds of ATR crystals (Ge coupled with
an infrared microscope, ZnSe and diamond) and resulting in three different optical arrangements for the detection of
model drug particles is discussed. Model systems of ibuprofen and paracetamol particles having a size below 32
micrometers have been prepared by sieving. The sensitivity level in the three different approaches has been compared
and it has been found that both micro and macro-ATR imaging methods have proven to be a promising techniques for
the identification of concealed drug particles. To demonstrate the power and applicability of FTIR chemical imaging to
forensic research, various examples are discussed. This includes investigation of the changes of chemical nature of latent
fingerprint residue under controlled conditions of humidity and temperature studied by ATR-FTIR imaging. This study
demonstrates the potential of spectroscopic imaging for visualizing the chemical changes of fingerprints.
This presentation outlines the research we are developing in the area of Fourier Transform Infrared (FTIR) spectroscopic
imaging with the focus on materials of forensic interest. FTIR spectroscopic imaging has recently emerged as a powerful
tool for characterisation of heterogeneous materials. FTIR imaging relies on the ability of the military-developed infrared
array detector to simultaneously measure spectra from thousands of different locations in a sample. Recently developed
application of FTIR imaging using an ATR (Attenuated Total Reflection) mode has demonstrated the ability of this
method to achieve spatial resolution beyond the diffraction limit of infrared light in air. Chemical visualisation with
enhanced spatial resolution in micro-ATR mode broadens the range of materials studied with FTIR imaging with
applications to pharmaceutical formulations or biological samples. Macro-ATR imaging has also been developed for
chemical imaging analysis of large surface area samples and was applied to analyse the surface of human skin (e.g.
finger), counterfeit tablets, textile materials (clothing), etc. This approach demonstrated the ability of this imaging
method to detect trace materials attached to the surface of the skin. This may also prove as a valuable tool in detection of
traces of explosives left or trapped on the surfaces of different materials. This FTIR imaging method is substantially
superior to many of the other imaging methods due to inherent chemical specificity of infrared spectroscopy and fast
acquisition times of this technique. Our preliminary data demonstrated that this methodology will provide the means to
non-destructive detection method that could relate evidence to its source. This will be important in a wider crime
prevention programme. In summary, intrinsic chemical specificity and enhanced visualising capability of FTIR
spectroscopic imaging open a window of opportunities for counter-terrorism and crime-fighting, with applications
ranging from analysis of trace evidence (e.g. in soil), tablets, drugs, fibres, tape explosives, biological samples to
detection of gunshot residues and imaging of fingerprints.
Supercritical CO2 can induce crystallization of amorphous polymers. Molecular level insight into the microstructures of CO2-processed polymers is needed to form a basis for utilization and optimization of supercritical fluid processing of polymeric materials. Spectroscopy is an economic tool to probe interactions at a molecular level. This understanding will help engineers to utilize molecular level information for improving macroscopic properties of polymeric materials by supercritical fluid processing. FT-IR spectroscopy has been applied to elucidate the morphology and microstructure of polymers processed with supercritical CO2. FTIR spectra of syndiotactic polystyrene show an increased degree of crystallinity after being subjected to scCO2. The various crystalline forms induced by CO2 in syndiotactic polystyrene were characterized via FTIR spectra. FTIR spectroscopy has been also used to measure the kinetics of CO2-induced crystallization in these polymers.
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