Human neural stem were cultivated and characterized using infrared spectroscopic imaging. A classification algorithm
based on linear discriminate analysis was developed to distinguish the differentiation of the stem cells to neurons,
astrocytes and stem cells without labeling. The classification is based upon spectral features which mainly arise from
proteins, nucleic acids. A spectral training set was formed with spectra from cells which were identified by a
subsequently staining according to a standard histological protocol. Differentiated cells could be classified with a high
accuracy whereas not differentiated stem cells did exhibit some misclassifications
Polarization Modulation Infrared Reflection Absorption Spectroscopy (PM-IRRAS) is a very sensitive imaging
technique for the characterization of molecular films. In order to achieve a spatial resolution close to the diffraction limit
a very small pinhole which acts as a point-source has to be used. However, such a small pinhole, the typical diameter
would be app. 100 μm, may reduces dramatically the intensity of the infrared beam. Using a common FTIR spectrometer
the spatial resolution is mainly limited by the brilliance of the globar infrared source. Therefore, an improvement in
lateral resolution requires a more brilliant light source.
The free electron laser (FEL) is such a high brilliant infrared source. The combination of the FEL with the PM-IRRAS
imaging system is a new approach to capture spectroscopic images with an excellent spatial resolution close to the
diffraction limit. PM-IRRAS images of a self assembly monolayer of phosphonic acid molecules onto a microstructures
gold / aluminum oxide surface where characterized. The spectroscopic image exhibits a spatial resolution of app. 5 μm.
An evaluation of characteristic absorbance bands of the phosphate group reveals that phosphonic acid molecules bound
with a high degree of orientation but differently at the gold and aluminum oxide surfaces. However, the spectroscopic
image reveals also several domains of disordering across the surface. Such domains have a dimension of only few
micrometers and can be identified in a high resolved PM-IRRAS image.
Functionality of biosensor arrays based on SPR imaging is mainly defined by quality of patterned SAM, which is used for sensing of biological substances of interest. The monolayers are commonly not uniform in the micrometer scale. In the current work we describe experimental setups for PM-IRRAS mapping and SPR imaging of self
assembly monolayer (SAM) formed by octadecyl phosphonic acid (CH3(CH2)17PO(OH)2) on a patterned Au/Al2O3 surface. By combining highly resolved surface plasmon resonance (SPR) images of the surface with the corresponding PM-IRRAS maps we achieved enhancement of the resolution. In this work we show, that changes of molecular structure in the thin films also induce changes in the SPR signal. Contrary, changes in SPR have their origin in molecular structure within the film.
As a molecular probe of tissue composition, infrared spectroscopic imaging serves as an adjunct to histopathology
in detecting and diagnosing disease. In the past it was demonstrated that the IR spectra of brain tumors can be
discriminated from one another according to their grade of malignancy. Although classification success rates up
to 93% were observed one problem consists in the variation of the models depending on the number of samples
used for the development of the classification model. In order to open the path for clinical trials the classification
has to be validated. A series of classification models were built using a k-fold cross validation scheme and
the classification predictions from the various models were combined to provide an aggregated prediction. The
validation highlights instabilities in the models, error rates, sensitivity as well as specificity of the classification
and allows the determination of confidence intervals. Better classification models could be achieved by an
aggregated prediction. The validation shows that brain tumors can be classified by infrared spectroscopy and
the grade of malignancy corresponds reasonably to the histopathological assignment.
The determination of protein structure and function plays an important role in biomedical and biochemical research. Common techniques that give full structural information do not permit in-vivo measurements. Infrared spectroscopy has a sufficient sensitivity to examine the structure of proteins solution under in-situ conditions and even on surfaces. However, measurements at few spots on the surface are not suitable to find out the blood compatibility of the protein layer, because the changes in conformation occur often in small domains. Here we report on the investigations of adsorbed fibrinogen with FTIR imaging. FTIR imaging permits an identification
of coagulation spots in the micrometer range and the identification of coagulation spots on the implant material.
A decisive problem in biomedical or biomaterial research of the post-genomic era is the determination of protein structure and function. Common techniques that can give full structural information do not permit in-vivo measurements. Vibrational Proteomics, an innovative combination of biochemical techniques and infrared or Raman spectroscopy, can provide information which will help substantially to fill this gap. Infrared and Raman spectroscopy are well established as methods for qualitative and quantitative analysis of protein secondary structure, in solution and even when adsorbed to implant surfaces. Their singular advantage over other techniques is that spectra can be obtained for proteins in a wide range of environments, in solutions and on surfaces including polymers, metals and bioceramics. Here we report on structural changes in fibrinogen from the dissolved to the adsorbed state on implant material of different hydrophobicity. FTIR imaging permits the identification of coagulation spots on the implant.
Structural changes in proteins may result in interactions between proteins themselves or between proteins and other biomolecules, which in turn may introduce deterioration of the health state of the system, might it be biological or biomimetic. The initial structural changes in proteins can not be observed by established methods of material analysis like X-ray diffraction or NMR spectroscopy, because proteins in biological cells have an extremely low abundance and are in the non-crystalline state. An obvious gap exists between sequencing techniques for amino acids and the molecular understanding of the functional properties of a particular protein. Vibrational spectroscopic methods offer the potential of fast and accurate characterization of essential supramolecular properties in the native state of the biomaterial. Imaging techniques recently became available for both FTIR and Raman spectroscopy. Here we report on the utilization of the enormous information content of FTIR and Raman spectra for health monitoring in engineering and medicine.
The composition of human brain tissue and brain tumors were studied by near infrared Raman spectroscopy with 785 nm excitation. The amounts of lipids, cholesterol, protein and water in fresh specimens were determined from Raman spectra by a combination of pure component spectra. Normal brain tissue was found to contain higher levels of lipids and cholesterol, brain tumors such as glioma and meningeoma displayed less lipids and cholesterol, but more proteins, in particular more hemoglobin-like molecules. These results demonstrate the applicability of Raman spectroscopy for real-time, in vivo, intraoperative diagnosis.
Ion channels are promising biomolecules for sensor applications. They combine high sensitivity with excellent selectivity. Presently the main problem consists in the integration of such ion channels into synthetic matrices but keeping them operative. In this study we report on the development of a microstructured polymer layer as matrix for an ion channel sensor array and an optical method for parallel detection. A thin PMMA layer was prepared by spin coating a gold surface. Small pores with diameter in the range of few micrometers were made by e-beam lithography. FTIR imaging spectroscopy and SPR imaging were used to characterize the quality of microstructured arrays.
Infrared (IR) optical fiber have aroused great interest in recent years because of their potential in in-vivo spectroscopy. This potential includes the ability to be flexible, small and to guide IR light in a very large range of wavelengths. Two types - silver halide and chalcogenide - infrared transmitting fibers are investigated in the detection of a malignant tumor. As a test sample for all types of fibers we used a thin section of an entire rat brain with glioblastoma. The fibers were connected with a common infrared microscope. Maps across the whole tissue section with more than 200 spectra were recorded by moving the sample with an XY stage. Data evaluation was performed using fuzzy c-means cluster analysis (FCM). The silver halide fibers provided excellent results. The tumor was clearly discernible from healthy tissue. Chalcogenide fibers are not suitable to distinguish tumor from normal tissue because the fiber has a very low transmittance in the important fingerprint region.
A new approach is presented to distinguish cancerous from normal brain tissue via linear discriminant analysis of Fourier transform infrared (FTIR) spectra. FTIR microspectroscopy was used to map various thin-section tumor samples with different malignancy grades (grades II-VI) and non-tumor samples obtained from various patients by surgical removal. Spectral analysis revealed features characteristic of tumors with increasing malignancy. A genetic region selection algorithm combined with linear discriminant analysis was used to derive classifiers distinguishing among spectra of control tissue, astrocytoma grade II, astrocytoma grade III and glioblastoma grade IV. Employing the World Health Organization histopathological diagnostic scheme as the gold standard, the spectra were classified with a success rate of approximately 85 percent. These results demonstrate the potential of the combination of FTIR spectroscopy and pattern recognition routines in providing a more objective method for brain tumour grading and diagnosis.
The use of several silver halide and chalcogenide infrared transmitting fibers in the detection of cancer is investigated. As a test sample for all types of fibers we used a thin section of an entire rat brain with glioblastoma. Moving the sample with an XY stage maps across the whole tissue section with more than 200 spectra were recorded. Data evaluation was performed using Principal Components Analysis (PCA). The silver halide fibers have provided excellent results. The tumor was clearly differentiable from the normal tissue. It wasn't possible to identify the tumor region using chalcogenide fibers because the fiber has a very low transmittance in the important fingerprint region.
Lipid bilayers containing the nicotinic acetylcholine receptor were investigated by attenuated total reflection (ATR) FTIR spectroscopy and by surface plasmon resonance (SPA). FTIR-ATR spectra reveal structural details of the transmembrane protein. Surface enhanced IR absorption provides access to structural details in the close vicinity of the surface of the ATR crystal. Information about the abundance of protein substructures as well as about the orientation of those substructures are obtained. SPR was applied in order to detect changes within the adsorbed biomembrane during admission of a neurotransmitter as acetylcholine. Switching of the ion channel was successfully by SPR. This result is a step forward towards highly selective biosensors and bioactuators.
A combination of FTIR spectroscopy and positron emission tomography (PET) is shown to provide new information on tissue. Here we give a first demonstration on the potential of this combination in discriminating tumor tissue from healthy tissue. Examples are taken of cancer grown in muscle tissue in mice. Immediately before thin sections of the cancer tissue were prepared, a radiotracer was injected in the living mouse. Subsequently a native section was immobilized on a CaF2 window and an autoradiographic image was recorded from that immobilized section. FTIR maps of the thin sections were obtained by using an infrared microscopy equipped with computerized XY stage and MCT detector. Principal component analysis was chosen for chemometric evaluation of the spectra. Evaluated data were reassembled into 2D maps and compared with the corresponding PET image.
The potential of surface enhanced spectroscopy consists in the detection of very small but specific structures of biomaterials. Surface enhanced infrared absorption (SEIRA) yields fingerprint information on the biomaterials. Electronic interactions between individual groups in the molecule are detected by means of surface enhanced fluorescence (SEF). During the investigation of biomembranes with SEIRA a tenfold intensity enhancement could be obtained. Enhancement factors greater than 100 can be achieved with SEF. Enhancement is considerably influenced by the properties of the metal cluster structure. Biomembranes formed from vesicles containing the nicotinic acetylcholine receptor were spectroscopically characterized. The adsorption of the vesicles on Ge- and Ag surfaces was investigated. The metal cluster structure was optimized in order to obtain high intensity enhancement factors.
Native vesicles containing the nicotinic acetylcholine receptor (nAChR) prepared from the electric organ of the ray Torpedo marmorata were used to obtain fluorescence signal sin dependence of different concentrations of the local anesthetics procaine. Nonlinear concentration dependent spectral changes are found using ethidium bromide as a fluorescence marker. Structural changes are found for the proteins including the nAChR in the vesicles during immobilization onto surfaces such as IR transparent germanium (GE) crystal, Ge crystal coated with silver (Ag) cluster to use the SEIRA effect and/or crystals covered with a lipid subphase. A comparison between Ge and Ge coated with Ag (Ge/Ag) clusters reveals increased structural changes in the spectral regions around 1670 cm-1 upon adsorption of the vesicles on the latter surface. Is the Ge/Ag crystal precoated with a lipid subphase an almost similar spectral contour for the amide I band envelope as in the spectra recorded on a neat Ge crystal is found.
Surface enhanced FTIR spectroscopy with polarized light can be used to investigate the structure of functionalized membranes. We have used this method to characterize lipid membranes containing integral membrane proteins. The Ge ATR crystal was coated by a 9 nm layer of Ag clusters before adsorbing the membrane. The dipoles of the clusters get excited by the evanescent field. The observed spectra show an enhanced absorption of different functional groups. The enhancement factor depends upon the polarization of the incident light and depends upon the orientation of this groups towards the cluster surface. This allows the detection of the structure and orientation of the membrane fragments at the interface of the clusters.
The hydrogen spillover was investigated by FT-IR microscopy using two-component samples of Pt/NaY-HNaY zeolite. The evaluation of the intensities of the OH and OD stretching bands as a function of time lead to the conclusion that the diffusion as well as the H-D exchange of the hydroxyls are the rate determining steps in the spillover process. The H-D exchange occurred only in the presence of a platinum-containing component in the sample in contact with the platinum-free HNaY component. The exchange behavior could only be explained by deuterium spillover, i.e., the adsorption and activation of deuterium on the remote platinum catalyst, the transfer of the activated particles onto the zeolitic support, and the diffusion on the solid surface to the OH groups. All zeolitic OH groups are involved in the exchange process. The exchange kinetics indicate a storage capacity of the zeolite framework for activated spilt-over deuterium species.
A new sample stage with minimal thermal expansion was used to investigate the activation process of zeolites. The results were displayed quasi-continuously by mapping the reflectance changes across the investigated spectral range. Such contour plots provide a more detailed insight into the sample behavior than individual difference spectra do. Long-term investigations were performed in the mid-infrared on glass-sealed activated samples. The solid state ion exchange between ZnO and H-ZSM-5 zeolite was studied at 450 degree(s)C. In erionites, the relation between the desorption of different ammonia species and the formation of OH groups during the activation has been studied up to 600 degree(s)C. The diffusion of water and the formation of hydroxonium ions was observed. In activated Y zeolite the intercrystalline diffusion of protons has been analyzed.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.