V. A. Huck-Pezzei

Characterization of normal and malignant prostate tissue by Fourier transform infrared microspectroscopy

Prostate cancer has become one of the most common malignancies worldwide. Morphological and histomorphological evaluation of this disease is a well established technique for the cancer classification and has remained relatively unchanged since several decades, although it remains a time consuming and subjective technique, with unsatisfactory levels of inter- and intra-observer discrepancy. Novel approaches for histological recognition are necessary to identify and to investigate cancer in detail. Fourier transform infrared (FTIR) spectroscopic imaging has become an essential tool for the detection, identification and characterization of the molecular components of biological processes, such as those responsible for the dynamic properties of cancer progression. Major advantage of this new technique is the acquisition of local molecular expression profiles while maintaining the topographic integrity of the tissue and avoiding time-consuming extraction, purification and separation steps. By using this method it is possible to investigate the spatial distribution of proteins, lipids, carbohydrates, cholesterols, nucleic acids, phospholipids and small molecules within biological systems by in situ analysis of tissue sections. We applied this technique on prostate cancer patients radical prostatectomy specimens in order to develop new tools for histomorphological analysis and the characterization of snap frozen prostate cancer tissues. As a first step, an optimization of sample preparation, tissue section thickness and IR slide material was performed. Special preparation methods for FTIR imaging are the essential requirements to maintain the spatial arrangement of compounds and avoid delocalization and degradation of the analytes. Subsequently, selected cancer samples were characterized with the prior optimized parameters and analyzed by univariate and cluster analysis. For the interpretation and calibration of the system we correlated the FTIR-images with the histopathological information. With this method it is possible to distinguish between cancer and noncancer areas within a prostate cancer tissue with a resolution of 6.25 μm × 6.25 μm on frozen sections.

Fourier transform infrared imaging analysis in discrimination studies of St. John's wort (Hypericum perforatum)

In the present study, Fourier transform infrared (FTIR) imaging and data analysis methods were combined to study morphological and molecular patterns of St. Johns wort (Hypericum perforatum) in detail. For interpretation, FTIR imaging results were correlated with histological information gained from light microscopy (LM). Additionally, we tested several evaluation processes and optimized the methodology for use of complex FTIR microscopic images to monitor molecular patterns. It is demonstrated that the combination of the used spectroscopic method with LM enables a more distinct picture, concerning morphology and distribution of active ingredients, to be gained. We were able to obtain high-quality FTIR microscopic imaging results and to distinguish different tissue types with their chemical ingredients.

Near-Infrared Imaging Spectroscopy as a Tool to Discriminate Two Cryptic Tetramorium Ant Species

Correct species identification is a precondition for many ecological studies. Morphologically highly similar, i.e., cryptic, species are an important component of biodiversity but particularly difficult to discriminate and therefore understudied ecologically. To find new methods for their rapid identification, thus, is important. The cuticle’s chemical signature of insects often is unique for species. Near-infrared spectroscopy (NIRS) can capture such signatures. Imaging NIRS facilitates precise positioning of the measurement area on biological objects and high-resolution spatial capturing. Here, we tested the applicability of imaging NIRS to the discrimination of cryptic species by using the ants Tetramorium caespitum and T. impurum. The classification success of Partial Least Squares Regression was 98.8%. Principal Component Analysis grouped spectra of some T. impurum individuals with T. caespitum. Combined with molecular-genetic and morphological evidence, this result enabled us to pose testable hypotheses about the biology of these species. We conclude that discrimination of T. caespitum and T. impurum with imaging NIRS is possible, promising that imaging NIRS could become a time- and cost-efficient tool for the reliable discrimination of cryptic species. This and the direct facilitation of potential biological insight beyond species identification underscore the value of imaging NIRS to ecology.

Critical Review Upon the Role and Potential of Fluorescence and Near-Infrared Imaging and Absorption Spectroscopy in Cancer Related Cells, Serum, Saliva, Urine and Tissue Analysis

During the last years, non-invasive or minimally invasive diagnostic tools in cancer diagnostics have become more important. Many fluorescence spectroscopic methodologies have been established for nearly all different kinds of cancer. The reason therefore is its high sensitivity, low amount of sample required, short testing time, and the suitability for in situ testing. The potential influence factors for cancer diagnostics and the subsequent suitability of the method to different applications are well described. Near-Infrared spectroscopy (NIRS) is based on differences of endogenous chromophores between cancer and normal tissues using either oxyhaemoglobin or deoxy-haemoglobin, lipid or water bands, or a combination of two or more of these diagnostic markers. These marker bands are known to provide the fundamental for the diagnosis of several cancers and the spectroscopic setup can be applied for the analysis of cells, urine and tissue. For the preparation of this review the literature published during the last fifteen years has been taken into consideration. It will provide an overview on the importance of the fluorescence and NIRS tools in cancer analysis giving hints about how these techniques can play a crucial role in cancer diagnosis, treatment decisions and therapy. The two techniques, fluorescence and near-infrared spectroscopy (NIRS) are faced to each other and individual advantages and/or drawbacks are discussed. Finally, it will be taken into consideration; how the synergistic combination of different approaches can give additional information related to development and progression stages of cancer.

A chromatographic and spectroscopic analytical platform for the characterization of St John's wort extract adulterations

In the present study, a novel analytical platform is introduced, which enables both analysis and quality control of St Johns wort extracts and tissue. The synergistic combination of separation techniques (including thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC)) with mass spectrometry (MS) and vibrational spectroscopy is demonstrated to get deeper insights into the ingredients composition. TLC was successfully employed to identify some unknown ingredients being present in samples with Chinese provenience. On the one hand, the novel HPLC method described here allowed us to clearly differentiate between European and Chinese samples; on the other hand, this method could successfully be employed for the semi-preparative isolation of an unknown ingredient. Matrix-free laser desorption ionization time of flight mass spectrometry (mf-LDI-TOF/MS) using a specially designed titanium oxide layer was employed to identify the structure of the substance. The analytical knowledge generated so far was used to establish an infrared spectroscopic model allowing both quantitative analysis of ingredients as well as differentiating between European and Chinese provenience. Finally, infrared imaging spectroscopy was conducted to obtain knowledge about the highly resolved distribution of ingredients. The analytical platform established so far can be used for fast and non-destructive quantitation and quality control to identify adulteration being of interest according to the Deutsche Arzneimittel Codex (DAC).

Near infrared spectroscopy as a tool for quality control of food of the alpine region—A short report about a cross-border project

leader Christian Huck) in this cross-border project and is responsible for the NIR analyses of the regional food samples. The Agrarmarketing Tyrol, the SouthernTyrolean alpine dairy association and the Agrarian and Forestry Research Institute “Laimburg” (Pfatten) provided the milk, meat and apple samples. To obtain the reference values necessary for calibrating the NIR method, high-performance liquid chromatography (HPLC) and gas chromatography (GC) coupled with mass spectrometry (MS) measurements were performed by Laimburg, while the Free University of Bolzano produced isotopic fingerprints of the samples for origin determination (Table 1). The focus in the first phase of the project is on the analysis of quality parameters and origin determination of apples and milk.

Advances of Infrared Spectroscopic Imaging and Mapping Technologies of Plant Material

Abstract Fourier Transform Infrared (FTIR) spectroscopic imaging and mapping techniques have become essential tools for the detection and characterization of the molecular components of biological tissues and are modern analytical techniques enabling molecular imaging of complex botanical samples. These techniques are based on the absorption of IR radiation by vibrational transitions in covalent bonds and their major advantage is the acquisition of local molecular expression profiles, while maintaining the topographic integrity of the tissue by avoiding time-consuming extraction, purification and separation steps. These new techniques enable global analysis of biological samples with high spatial resolution and provide unique chemi-morphological information about the tissue status. With these nondestructive examination methods it is possible to get qualitative and quantitative information of heterogeneous samples. In this paper recent applications of infrared spectroscopic imaging and mapping technologies of plant material are described.

Near infrared spectroscopy compared to liquid chromatography coupled to mass spectrometry and capillary electrophoresis as a detection tool for peptide reaction monitoring

Peptide interaction is normally monitored by liquid chromatography (LC), liquid chromatography coupled to mass spectrometry (LC-MS), mass spectrometric (MS) methods such as MALDI-TOF/MS or capillary electrophoresis (CE). These analytical techniques need to apply either high pressure or high voltages, which can cause cleavage of newly formed bondages. Therefore, near infrared reflectance spectroscopy (NIRS) is presented as a rapid alternative to monitor the interaction of glutathione and oxytocin, simulating physiological conditions. Thereby, glutathione can act as a nucleophile with oxytocin forming four new conjugates via a disulphide bondage. Liquid chromatography coupled to UV (LC-UV) and mass spectrometry via an electrospray ionisation interface (LC-ESI-MS) resulted in a 82% and a 78% degradation of oxytocin at pH 3 and a 5% and a 7% degradation at pH 6.5. Capillary electrophoresis employing UV-detection (CE-UV) showed a 44% degradation of oxytocin. LC and CE in addition to the NIRS are found to be authentic tools for quantitative analysis. Nevertheless, NIRS proved to be highly suitable for the detection of newly formed conjugates after separating them on a thin layer chromatography (TLC) plate. The recorded fingerprint in the near infrared region allows for a selective distinct qualitative identification of conjugates without the need for expensive instrumentation such as quadrupole or MALDI-TOF mass spectrometers. The performance of the established NIRS method is compared to LC and CE; its advantages are discussed in detail.

Application of Near-Infrared Spectroscopy (NIRS) as a Tool for Quality Control in Traditional Chinese Medicine (TCM)

Traditional Chinese Medicine (TCM) is becoming more and more popular all over the world. Novel analytical tools for quality control are highly demanded enabling analysis starting at breeding and ending at biological fluids including urine or serum. Compared to analytical separation methods (chromatography, electrophoresis) near-infrared spectroscopy (NIRS) allows analyzing matter of interest non-invasively, fast and physical/chemical parameters simultaneously. It can be used for the quantitative control of certain (active) ingredients. In many cases identification can only be achieved by pattern recognition. Therefore, NIRS combined with cluster analysis offers huge potential to identify e.g. species, geographic origin, special medicinal formula etc. In the present contribution the fundamentals, possibilities of NIR applied in quality control of TCM are pointed out and its adand disadvantages are discussed in detail by several practical examples.

Application of benchtop and portable near-infrared spectrometers for predicting the optimum harvest time of Verbena officinalis

This study examined the applicability of near-infrared (NIR) spectroscopy coupled with multivariate data analysis (MVA) to determine the ideal harvest time of Verbena officinalis. NIR analyses were performed non-invasively on the fresh plant material based on the quantification of the key constituents verbenalin and verbascoside. Vibrational spectroscopic measurements were performed applying a conventional NIR benchtop device as well as a laboratory independent handheld NIR spectrometer. A novel high performance liquid chromatography (HPLC) method was applied as a reference method. For both instruments partial least squares (PLS) regression models were established performing cross validations (CV) and test-set validations (TSV). Quality parameters obtained for the benchtop device revealed that the newly established NIR method enabled reliable quantifications of the main compounds verbenalin and verbascoside related to the dried and fresh plant material. The results of the miniaturised spectrometer revealed that accurate quantitative calibration models could be developed for verbascoside achieving a comparable prediction power to the benchtop device. PLS models for verbenalin were less precise suggesting the application of portable devices including a different spectral range and resolution. The work demonstrated the feasibility of NIR vibrational spectroscopy performing direct measurements on pharmaceutically relevant fresh plant material enabling a quick and simple determination of the ideal harvest time of Verbena officinalis.