Attenuated Total Reflection Mode Research Articles

FORENSIC DATING OF OFFICIAL DOCUMENTS: UTILIZING FTIR ANALYSIS OF STAMP PAD INK DEGRADATION FOR DOCUMENT AUTHENTICATION

This study delves into the deterioration of stamp pad ink found on official documents and explores its potential in dating these documents. The credibility of official documents relies on the application of an official seal or stamp using stamp pad ink. However, individuals with malicious intent may seek to manipulate the ink's appearance to deceive the document's perceived age. To counteract this, we propose a non-destructive method employing Fourier Transform Infrared (FTIR) analysis in Attenuated Total Reflection (ATR) mode to analyze the degradation of stamp pad ink. Through the application of FTIR analysis in ATR mode and the utilization of chemometric approaches like Principal Component Analysis (PCA), we have effectively established a connection between the degradation of stamp pad ink and the document's age. This methodology stands as a crucial tool in authenticating official documents and identifying fraudulent attempts to alter their purported preparation date. The implications of these findings extend to the realms of forensic document examination and legal contexts, offering a dependable means of dating documents through the analysis of ink degradation patterns.

Attenuated Total Reflection at THz Wavelengths: Prospective Use of Total Internal Reflection and Polariscopy

Capabilities of the attenuated total reflection (ATR) at THz wavelengths for increased sub-surface depth characterisation of (bio-)materials are presented. The penetration depth of a THz evanescent wave in biological samples is dependent on the wavelength and temperature and can reach 0.1–0.5 mm depth, due to the strong refractive index change ∼0.4 of the ice-water transition; this is quite significant and important when studying biological samples. Technical challenges are discussed when using ATR for uneven, heterogeneous, high refractive index samples with the possibility of frustrated total internal reflection (a breakdown of the ATR reflection mode into transmission mode). Local field enhancements at the interface are discussed with numerical/analytical examples. Maxwell’s scaling is used to model the behaviour of absorber–scatterer inside the materials at the interface with the ATR prism for realistic complex refractive indices of bio-materials. The modality of ATR with a polarisation analysis is proposed, and its principle is illustrated, opening an invitation for its experimental validation. The sensitivity of the polarised ATR mode to the refractive index between the sample and ATR prism is numerically modelled and experimentally verified for background (air) spectra. The design principles of polarisation active optical elements and spectral filters are outlined. The results and proposed concepts are based on experimental conditions at the THz beamline of the Australian Synchrotron.

Identification and composition of carbonate minerals of the calcite structure by Raman and infrared spectroscopies using portable devices

A portable Raman device with a 532 nm excitation laser and a portable infrared spectrometer with ATR (Attenuated Total Reflection) mode were used to analyse the spectral features associated with the identification and compositional variation of Ca-Mg-Fe-Mn natural carbonate minerals with a calcite structure (calcite, ankerite, dolomite, siderite, rhodochrosite, and magnesite). A systematic study of the variations of the peak positions with various compositional ratios was carried out. Most of the band positions were shifted to lower wavenumbers with increasing ionic radius or atomic mass of the divalent cations but the band of the translational lattice (T) mode in Raman and the symmetric bending (ν4) band in the mid-infrared were the most sensitive. Therefore, the elemental variation of the Ca-Mg-Fe-Mn ratio in this carbonate series can be estimated from Raman and infrared band positions from spectra acquired with portable spectrometers.

Using Attenuated Total Reflection (ATR) Apparatus to Investigate the Temperature Dependent Dielectric Properties of Water, Ice, and Tissue-Representative Fats

A novel method of investigating the temperature dependent variation of aspects of the complex refractive index n* in samples in the THz range using continuous, non-polarised, synchrotron radiation is presented. The method relies on the use of ATR apparatus, and retains the advantage of minimal sample preparation, which is a feature of ATR techniques. The method demonstrates a “proof of concept” of monitoring temperature reflectance whilst continuously heating or cooling samples by using a temperature variable Thermal Sample Stage. The method remains useful when the refractive index of the sample precludes attenuated total reflection study. This is demonstrated with the water reflectance experiments. The temperature dependent ATR reflectance of tissue-representative fats (lard and Lurpak® butter) was investigated with the novel approach. Both are within the ATR range of the diamond crystal in a “true” ATR mode. Lard showed no clear temperature variation between −15 °C and 24 °C at 0.7 to 1.15 THz or 1.70 to 2.25 THz. Lard can be regarded as having invariable, constant, dielectric properties within mixtures when biological substances are being assessed for temperature dependent dielectric variation within the stated THz ranges. Lurpak® butter (water content 14.7%) displayed temperature dependent reflected signal intensity features with a steady decline in reflectivity with increasing temperature. This is in line with the temperature-dependent behaviour of liquid water. There is no rapid change in reflected signal intensity even at −20 °C, suggesting that emulsified water retains liquid-water-like THz properties at freezing temperatures.

Detection of Gadolinium with an Impedimetric Platform Based on Gold Electrodes Functionalized by 2-Methylpyridine-Substituted Cyclam.

Gadolinium is extensively used in pharmaceuticals and is very toxic, so its sensitive detection is mandatory. This work presents the elaboration of a gadolinium chemical sensor based on 2-methylpyridine-substituted cyclam thin films, deposited on gold electrodes, using electrochemical impedance spectroscopy (EIS). The 2-methylpyridine-substituted cyclam (bis-N-MPyC) was synthesized in three steps, including the protection of cyclam by the formation of its CH2-bridged aminal derivative; the product was characterized by liquid 1H and 13C NMR spectroscopy. Spin-coated thin films of bis-N-MPyC on gold wafers were characterized by means of infrared spectroscopy in ATR (Attenuated Total Reflectance) mode, contact angle measurements and atomic force microscopy. The impedimetric chemical sensor was studied in the presence of increasing concentrations of lanthanides (Gd3+, Eu3+, Tb3+, Dy3+). Nyquist plots were fitted with an equivalent electrical circuit including two RC circuits in series corresponding to the bis-N-MPyC film and its interface with the electrolyte. The main parameter that varies with gadolinium concentration is the resistance of the film/electrolyte interface (Rp), correlated to the rate of exchange between the proton and the lanthanide ion. Based on this parameter, the detection limit obtained is 35 pM. The bis-N-MPyC modified gold electrode was tested for the detection of gadolinium in spiked diluted negative urine control samples.

Engineering Electronic Structure of Single-Atom Pd Site on Ti 0.87 O 2 Nanosheet via Charge Transfer Enables C–Br Cleavage for Room-Temperature Suzuki Coupling

The palladium (Pd)-catalyzed Suzuki reaction is widely applied in the pharmaceutical industry, where constructing highly active and low-cost Pd sites are impendent. Here, we report the fabrication ...

Utilization of ATR-FTIR Spectroscopy-Multivariate Chemometric Analysis in the Examine of Food Quality Edible Sesame Oils

Attenuated total reflectance (ATR)-Fourier transform infrared spectroscopy (FTIR) and multivariate chemometric analysis was established to distinguish and classify edible sesame oils of various branded (refined) and unbranded (cold pressed) oils and principal components. For this determination, available branded and unbranded traditional cold pressed sesame oils from southern districts of Tamilnadu, India were qualitative and quantitatively analyzed and authenticated. The FTIR spectra were generated in absorption ranges from 599- 4000 cm-1 observed through ATR mode. The generated spectral data combined with multivariate chemometric analyses such as Principal components analysis (PCA), Principal coordinate analysis (PCoA), Agglomerative hierarchical clustering analysis (AHC), Discriminant analysis (DA) were analyzed. Further, observed qualitative analyses through colour, UV-light absorption, absolute viscosity, saponification and iodine values were well differentiated between branded and unbranded sesame oil samples. Based on the multivariate chemometric analysis, unbranded 2 and branded 1 sesame oil samples have shown essential phytocomponents like sesamin, sesaminol, and sesamolin. However, other oil samples did not show those essential phytocomponents and may be missing/eliminated during the refining/filtration processes. In addition, branded and unbranded sesame oil samples have been classified through discriminant analysis (DA) based on the major absorption frequencies. As per the experimental and statistical outcomes, it might be established that ATR-FTIR method has potential to detect and identify essential components of similar food products for classification and authentication, further; it is nondestructive, effective and simple alternative method.

Study of the Degradation and Conservation of Historical Leather Book Covers with Macro Attenuated Total Reflection-Fourier Transform Infrared Spectroscopic Imaging.

The analytical study of historical collagen-based materials such as leather book covers is a complex task for conservation scientists. Historical leather presents a heterogeneous composition of both organic and inorganic materials that show an evident reactivity, particularly when exposed to specific environments. Its correct preservation in archival documents remains challenging as some aspects of its chemical composition, degradation, and effectiveness of conservation treatments are still not fully understood. Here, we describe a novel application of attenuated total reflection (ATR)–Fourier transform infrared spectroscopic imaging coupled with a macro ATR accessory to study historical leather book covers. This nondestructive and high spatial resolution approach has allowed the visualization of degradation phenomena affecting this fragile material, particularly the gelatinization of collagen and, for the first time, the detection of the formation of calcium stearate (metal carboxylates or soaps). In addition, the distribution of modified soybean oil used as a treatment to maintain properties such as elasticity and hydrophobicity of the leather was studied. The effect of anomalous dispersion on the strong IR bands obtained in the ATR mode and the resulting changes to the band positions are also discussed. This research addresses issues that are relevant to the conservation of archival materials of cultural heritage for future generations.

Optical performance of ultra-thin silver films under the attenuated total reflection mode

Ultra-thin silver films were deposited by thermal evaporation, and the dielectric functions of samples were simulated using Drude-Lorentz oscillators. When s-polarized incident light from the BK7 glass into thin silver film at 45° angle using attenuated total reflection (ATR) mode, we experimental observed that the reflection reach a minimum of 1.87% at 520 nm for thickness of d~6.3 nm silver film, and it reach a minimum of 10.1% at 500 nm for thickness of d~4.1 nm. Moreover, we simulated the absorption changes with incident angles at 520 nm for both p-polarized (TM wave) and s-polarized (TE wave) light using transfer matrix theory, and calculated the electric field distributions. The absorption as a function of incident angles of TM wave and TE wave showed different characteristics under ATR mode, TE wave reached the maximum absorption around the critical angle θc~41.1°, while TM wave reached the minimum absorption.

Development of Visible Light Absorption Measurement for Concentrated Dye Solution based on Attenuated Total Reflection Technique and Improvement on its Analysis Method

An absorption measurement working in ATR (Attenuated Total Reflection) mode has been design for characterization of concentrate dye solution. The system consists of a visible light system as polychromatic wave source, an ATR plate made up from glass or polymer, a spectrometer with a CCD detector. The system is connected to a computer for measurement and data processing. Some commercial inks measured by this ATR system showed absorption spectra which are in agreement with the color of the inks. The absorption parameter will be determined from the incident and reflection beam intensities measured at incident angle larger than the critical angle. We have also carried out Poynting vector analysis and we will show that the energy flow in the normal direction of the interface is not zero. The energy flow, however, decreases along the normal direction of the interface, indicating energy loss due to light absorption in the sample medium. The amount of energy loss depends on the attenuation index or absorption parameters, which is important for formulating the relation between the measured intensities and absorption parameters.

Evaluation of Pre-Fixed Biological Tissues Preparation Methods for ATR-FTIR Biospectroscopy

Evaluation of Pre-Fixed Biological Tissues Preparation Methods for ATR-FTIR Biospectroscopy Fourier transform infrared (FTIR) spectroscopy in ATR (attenuated total reflection) mode is a powerful tool for studying biomedical samples, which can provide important structural information on the molecular composition. Currently, formalin fixation and paraffin preservation (FFPP) is the preferred source for the histological examination of tissue sections. There is lack of consensus with regard to a standard protocol for de-paraffinization of embedded sections in the field of FTIR spectroscopy for which several approaches have been used. The aim of present study is to optimize the de-paraffinization procedure for biological samples FTIR spectroscopy. To this aim , Rat’s lung tissue samples were paraffinazed in blocks according to standard procedures. Different exposure, duration, and dewaxing timing protocols using any or combinations of n-hexane, xylene, acetone and absolute ethanol have been applied on embedded sections. Results were evaluated with the comparison of the spectroscopic outcome from these methods with comparison to fresh tissues dried with other methods, as well as pure paraffin spectra. As a result , Although n-hexane is an effective dewaxing agent for biological samples after a 24 hours exposure, xylene is a better choice with higher efficiency in less time (6_8 minutes). However sections that was immersed in xylene for 8 minutes and then rinsed in acetone for 5 minutes showed amide Ӏ and П bands and DNA contents in FTIR spectra better than other strategies, visualization of the sections has shown that the paraffin is not removed completely. The disappearance of peaks at 1426 & 2850_2950 cm-1 of the FTIR spectrum was used to ensure complete deparaffinization that happened with 15 min xylene embedding, which effects on other cellular structures and subsequently on the spectrums. However it is important to note that, these processes is not instantaneous and two important properties of the dewaxing agents are its penetration rate and binding time which obey diffusion law, whereby the depth of penetration was proportional to the square root of time. According to this, we have also demonstrated that using pressure, sample proper thickness and higher surface in ATR spectroscopy play an important role in optimization of spectra & decreasing wave disturbances.

In-Line Monitoring of Bacteria in Drinking Water By Infrared Spectroscopy and Micro-Flow Cytometry

Water from a municipal network water supply is stored in tanks and then pumped to tanks in tall buildings, which is so called “second water supply”. However, the water stored in tanks provides the potential environment for bacteria to grow and multiply. It is labor and time-consuming to routinely and frequently sample water from the water tanks distributed in a city, and to test all samples by biological methods in labs, which thus restricts the detection and prevention of water-borne diseases. Therefore, a rapid and low-cost method1,2 is urgently needed to routinely and automatically monitor and report the bacterial level in water tanks, allowing for taking appropriate actions in time. Infrared (IR) spectroscopy is a powerful tool for characterization and identification of micro-organisms because of its sensitivity and specificity, speed, and potential for automation. Microorganisms have unique “fingerprint” spectral features in the mid-infrared (MIR) range. Therefore, IR spectroscopy is a feasible technique for the identification of unknown bacterial samples based on the pre-established bacterial spectrum libraries. There are mainly two modes in IR spectroscopy to obtain the IR spectrum of bacteria in water - attenuated total reflection (ATR) and transmission modes. The first mode has been used by many researchers previously because it could avoid the influence of water on the bacterial spectrum, which has three strong absorption bands in the range of 400 to 4000 cm-1. However, the ATR technique lacks the ability to provide spectral data for quantitative analysis because the spectrum acquired in ATR mode is dependent on the thickness and density of the bacterial film formed on the outer surfaces of the ATR crystal. Therefore the transmission mode of FTIR is used in this research. Three strains of two species, E.coli K-12 MG1655 and E.coli K-12 HS2210 from Ecoli and Staphylococcus aureus (ATCC 25923) were placed directly into double distilled water. Then, the CaF2 cuvette containing bacteria suspention with different concentrations were used to obtain the IR spectra using a Vertex v80 FTIR spectrometer. Ultra-high concentrations of bacteria over 1010 cells/ml are used to guarantee enough changes in absorption between double distilled water and samples. Time-resoved IR spectra of E.coli K-12 MG1655 shows that its spectrum varies slightly within one hour. Obvious differences were observed in the spectra of E.coli K-12 MG1655 and Staphylococcus aureus (ATCC 25923), which shows the specificity on the IR spectrum of different species. However, similar differences were not found between E.coli K-12 MG1655 and HS2210. In addition, the intensity of the absorption peaks in the spectra of all the three strains is proportional to the concentration of the samples.Because of limitation of FTIR spectroscopy on quantifying bacteria accurately, a photonic-microfluidic integrated device based flow cytometer3-5 is incorporated into the monitoring system to ensure reliable estimation of bacterial concentration. The functionality layer of the device including input and output waveguides, lens systems and microfluidic channels is fabricated by patterning SU-8 photoresist using photolithography, which is actually supported by a pyrex glass substrate and covered by a PDMS covering layer with holes aligned with all the inlets and outlet. Light from a laser is directed into the input waveguide of the device through an optic fiber, then focused to the center of the microfluidic channel by a lens system, where the bacteria will be interrogated. Bacterial suspension in a syringe is injected to the sample inlet by a syringe pump and hydro-focused into the center of microfluidic channel by the sheath flows at both sides. Scattered light produced by bacteria passing through the focused light beam will be collected by the objective and filtered by an optic filter with passband of 525-535 nm. A photomultiplier tube is used to detect, convert and amplify the light signals into current signals, which will be further amplified into votage singals by a current to voltage amplifier. Voltages signals will be digitized by a data acqusition card and recorded by a custom LabView program for data analysis.The performance of flow cytometer based on the integrated devices with different beam shaping lens systems was calibrated by detecting scattered light from 1µm-diameter blank polystyrene beads running through the device. Due to uniform geometry, size and material, the device yielding minimal coefficient of variation (CV) will be the best for applications.Enumeration of E.coli in phosphate-buffered saline was carried out to validate the accuracy of counting bacteria using this micro-chip based flow cytometer. A 92% of detection efficiency was achieved. Expanded population of distribution of E. coli counting than beads counting was found because of the more flexibility of scattering light by rod-shape of E.coli than sphere-shape of beads as well as the natural size variation of E. coli. Tests on mixture of E.coli and 2µm, 4 µm beads show distinct separation of the population distribution, confirming the ability to differentiate E.coli from larger size inorganic particles by scattering intensity.

Evaluation of the effect of different paint cross section preparation methods on the performances of Fourier transformed infrared microscopy in total reflection mode

Abstract The purpose of the present research was to compare the effect of different embedding and polishing procedures on the performances achievable with Fourier Transformed Infrared (FTIR) microscopy in Attenuated Total Reflection (ATR) mode. The attention was specifically focused on this technique since it is generally considered one of the most appropriate methods to analyze cross sections, thanks to the fact that the recorded spectra are similar to those collected in transmission mode. The research work was structured as a round robin among six different conservation and research laboratories. They were asked to embed fragments derived from the same area of a real sample following each one a different procedure. The performances of FTIR microscopy in ATR mode were evaluated on the different cross sections, considering the cross section morphology and the possible source of contamination. The cross sections were previously observed with optical microscopy, Environmental Scanning Electron Microscope (ESEM) and confocal microscopy. Three procedures were eventually selected, because they provided the best results in terms of both limiting the contamination of the embedding medium and achieving good contact with the ATR crystal. They were: a) embedding in KBr; b) cyclododecane pre-treatment before embedding in an organic resin and cutting of the sample; c) embedding in an organic resin and polishing with argon ion milling. These three procedures were finally compared and the weak points outlined, indicating where improvements could be made for further researches.

Discrimination of single‐porin Escherichia (E.) coli mutants by ATR and transmission mode FTIR spectroscopy

Vibrational spectroscopy has long been used in bacterial identification with different levels of taxonomic discrimination but its true potential for intra-species differentiation remains poorly explored. Herein, both transmission Fourier-transform infrared (FTIR) and attenuated total reflectance (ATR)-FTIR spectroscopy are used to analyse E. coli strains that differ solely in their porin expression profile. In this previously unreported approach, the applicability of both FTIR-spectroscopy techniques is compared with the same collection of unique strains. ATR-FTIR spectroscopy proved to reliably distinguish between several E. coli porin mutants with an accuracy not replicated by FTIR in transmission mode (using previously optimized procedures). Further studies should allow the identification of the individual contribution of the single porin channel to the overall bacterial infrared spectrum and of molecular predictive patterns of porin alterations.

Fourier Transform Infrared Spectroscopy of Low-k Dielectric Material on Patterned Wafers

With many of research on Fourier transform IR (FTIR) on low-k materials, our experiments extended the FTIR spectroscopy application to characterization and analysis of the low-k dielectric thin film properties on patterned wafers. FTIR spectra on low-k materials were successfully captured under three sampling modes: reflection, attenuated total reflectance (ATR), and mapping mode. ATR mode is more suitable for CHx band than reflection mode due to its higher sensitivity in this range. FTIR spectroscopy signal analysis on mixed structures (metal and low-k dielectric material) on patterned wafers was also investigated with mapping mode. Based on our investigation, FTIR can be used for low-k material studies on patterned wafer.

Evaluation of the effect of six different paint cross section preparation methods on the performances of Fourier Transformed Infrared microscopy in attenuated total reflection mode

The purpose of the present research was to compare the effect of different embedding and polishing procedures on the performances achievable with Fourier Transformed Infrared (FTIR) microscopy in Attenuated Total Reflection (ATR) mode. The attention was specifically focused on this technique since it is generally considered one of the most appropriate methods to analyze cross sections, thanks to the fact that the recorded spectra are similar to those collected in transmission mode.The research work was structured as a round robin among six different conservation and research laboratories. They were asked to embed fragments derived from the same area of a real sample following each one a different procedure. The performances of FTIR microscopy in ATR mode were evaluated on the different cross sections, considering the cross section morphology and the possible source of contamination. The cross sections were previously observed with optical microscopy, Environmental Scanning Electron Microscope (ESEM) and confocal microscopy.Three procedures were eventually selected, because they provided the best results in terms of both limiting the contamination of the embedding medium and achieving good contact with the ATR crystal. They were: a) embedding in KBr; b) cyclododecane pre-treatment before embedding in an organic resin and cutting of the sample; c) embedding in an organic resin and polishing with argon ion milling.These three procedures were finally compared and the weak points outlined, indicating where improvements could be made for further researches.

Differences Among Epilobium and Hypericum Species Revealed by four IR Spectroscopy Modes: Transmission, KBr Tablet, Diffuse Reflectance and ATR

Quality control of dried plant material in assessments of suitability of herbal medicinal products is of extreme importance. Commonly used procedures for identification of species are time consuming and expensive. The development of multivariate statistical methods has enabled application of vibrational spectroscopy for establishing plant species membership. To determine which infrared spectroscopy mode gives most informative spectra for plant species identification. Different modes of infrared spectroscopy were applied for investigation of differences among Epilobium and Hypericum species: diffuse reflectance (DR), attenuated total reflectance (ATR), direct transmission of whole leaves and KBr tablet transmission with comminuted leaves. The same chemometrical methods were applied to all spectra. The informative wave numbers were chosen by one-way analysis of variance. Afterwards the colinearity was reduced with principal component analysis. In the final step the species identification was determined with discriminant analysis. Transmission and diffuse reflectance mode did not give satisfactory results. Best results for discrimination among Epilobium species were obtained with ATR mode (98%), while best results for Hypericum species were obtained with KBr transmission mode (97%). This might be explained by the morphological properties of the two genera. Epilobium species differ in distribution and morphology of trichomes on the surface of the leaves. Hypericum species differ in structures and secondary metabolites in the interior of the leaves. For best results in using infrared spectroscopy for plant species identification in quality control, the morphological properties of plant material should be taken into consideration.

Rapid prototyping of microfluidic devices for integrating with FT-IR spectroscopic imaging

A versatile approach for the rapid prototyping of microfluidic devices suitable for use with FT-IR spectroscopic imaging is introduced. Device manufacture is based on the direct printing of paraffin onto the surface of an infrared transparent substrate, followed by encapsulation. Key features of this approach are low running costs, rapid production times, simplicity of design modifications and suitability for integration with FT-IR spectroscopic measurements. In the current experiments, the minimum width of channel walls was found to be approximately 120 mum and approximately 200 when a 25 mum and 12 mum spacer is used, respectively. Water and poly(ethylene glycol) are used as model fluids in a laminar flow regime, and are imaged in both transmission and attenuated total reflection (ATR) modes. It is established that adoption of transmission mode measurements yields superior sensitivity whilst the ATR mode is more suitable for quantitative analysis using strong spectral absorption bands. Results indicate that devices manufactured using this approach are suitable for use with in situ FT-IR spectroscopic imaging.

Abstracts of The 29th Annual Meeting of the United Kingdom Environmental Mutagen Society, 19-22 March 2006. Held jointly with The British Toxicology Society at the University of Warwick, UK

The aetiology of cervical cancer is strongly associated with viral infection (i.e. human papillomavirus (HPV)); other risk factors include low socio-economic class, multiple sexual partners, smoking and poor diet. Cervical cancer screening employing smears of exfoliative cytology for the visual identification of atypical cells has successfully led to a fall in mortality from this disease. However, false negatives occur when a smear is determined normal although atypical cells (be they pre-malignant or malignant) are present. The use of attenuated total reflection (ATR)-FTIR microspectroscopy to interrogate samples of exfoliative cytology is a novel approach to screening that may allow the rapid profiling of a pathological status. Changes in derived vibrational spectra may be associated with a biochemical biomarker of dyskaryosis. To investigate this possibility, we interrogated eight cervical samples of exfoliative cytology using ATR-FTIR microspectroscopy. Two were normal, two were low-grade cervical intraepithelial neoplasia (CIN)1, two were high-grade (CIN2/3) and two were invasive carcinoma samples. Cellular material was applied to an ATR crystal and vibrational spectra were obtained using a Bruker Vector 22 FTIR spectrometer with Helios ATR attachment. At least 10 spectra per sample of exfoliative cytology were acquired in the ATR mode using Bruker OPUS software; these spectra were then individually baseline corrected. These derived spectra were subsequently analysed using principal component analysis (PCA). Cluster plots allowed the identification of any spatial separation (pointing to increasing dissimilarity) as a function of spectral differences between different samples in a 3-D ‘‘hyperspace’’. Apparently independent of oncogenic HPV genotype, successful discrimination between normal, CIN1, CIN2/3 and invasive carcinoma samples was obtained. Normal and invasive carcinoma samples exhibited 100% segregation in ‘‘hyperspace’’ and the samples classified as low- or high-grade clustered variously between, pointing towards a model of progression. Retrospective histological analysis of smears confirmed the diagnostic potential of this approach. ATR-FTIR microspectroscopy potentially could be used in routine cervical cancer screening in an objective approach less prone to the current limitation of false-negative occurrence. The combined application of ATR-FTIR microspectroscopy and PCA may also allow for the tracking of biochemical markers of progression from normal to invasive carcinoma. We are currently interrogating larger cohorts to determine the applicability of IR microspectroscopy for routine screening.

Optical Contact in ATR/FT-IR Spectroscopy

Optical contact between an internal reflection element (IRE) and a sample is crucial for obtaining a good spectrum in an attenuated total reflection (ATR) experiment. When an air gap is present between the IRE and the sample, the spectrum severely deteriorates. Two techniques for determining the goodness of contact are proposed—first, by making a comparison between polarized ATR spectra and, second, by making a comparison between ATR and transmission spectra. When the optical contact is achieved, the normalized p-polarized ATR spectrum is exactly the same as the normalized s-polarized ATR spectrum. Moreover, the ratio between the normalized absorbance measured in the ATR mode and the normalized absorbance measured in the transmission mode equals the ratio between the frequency being considered and the normalization frequency. Theoretical and experimental investigations have been performed in order to verify the proposed techniques.