Unrealistic expectations for IR microspectroscopic imaging.
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Diagnosis, Differential↗
Biomedical subjects
Christoph Krafft
Publications and source records attributed to Christoph Krafft.
Classification of malignant gliomas by infrared spectroscopic imaging and linear discriminant analysis.
Infrared (IR) spectroscopy provides a sensitive molecular fingerprint for tissue without external markers. Supervised classification models can be trained to identify the tissue type based on the spectroscopic fingerprint. Infrared imaging spectrometers equipped with multi-channel detectors combine the spectral and spatial information. Tissue areas of 4 x 4 mm(2) can be analyzed within a few minutes in the macroscopic imaging mode. An approach is described to apply this methodology to human astrocytic gliomas, which are graded according to their malignancy from one to four. Multiple IR images of three tissue sections from one patient with a malignant glioma are acquired and assigned to the six classes normal brain tissue, astrocytoma grade II, astrocytoma grade III, glioblastoma multiforme grade IV, hemorrhage, and other tissue by a linear discriminant analysis model which was trained by data from a single-channel detector. Before the model is applied here, the spectra are shown to be virtually identical. The first specimen contained approximately 95% malignant glioma regions, that means astrocytoma grade III or glioblastoma. The smaller percentage of 12-34% malignant glioma in the second specimen is consistent with its location at the tumor periphery. The detection of less than 0.2% malignant glioma in the third specimen points to a location outside the tumor. The results were correlated with the cellularity of the tissue which was obtained from the histopathologic gold standard. Potential applications of IR spectroscopic imaging as a rapid tool to complement established diagnostic methods are discussed.
Artificial Intelligence↗
Studies on stress-induced changes at the subcellular level by Raman microspectroscopic mapping.
Raman microspectroscopic mapping enables one to study the chemical composition and molecular structure of subcellular components in individual cells without the need for labeling. Lung fibroblast cells were prepared under normal conditions and under stress, which was induced by 24 h of exposure to glyoxal. Raman microspectroscopic maps were recorded from fixed cells with 785-nm excitation and with 1-microm step width. Cluster analysis was applied to generate pseudocolor images of the cell morphology. Raman maps revealed that the cell nucleus shrinks in stressed cells, called pyknosis, which refers to an early stage of apoptosis. The intensity of nucleic acid bands decreased in cluster-averaged Raman spectra of the nucleus and cytoplasm, which is consistent with degradation and conformational changes of DNA and RNA. During a later stage of apoptosis, Raman maps indicate a rounding of cells, a further intensity decrease of nucleic acids bands, fragmentation of the nucleus, disappearance of lipid bodies, and formation of blisters at the cell surface. Whereas the peripheral membrane of the undisturbed cell is composed of lipids and cholesterol, the blisters have a higher protein content with nucleic acids incorporated. The results demonstrate that Raman spectroscopic mapping might become a powerful tool in cell biology for single cell analysis.
Cells, Cultured↗
Identification of primary tumors of brain metastases by SIMCA classification of IR spectroscopic images.
Brain metastases are secondary intracranial lesions which occur more frequently than primary brain tumors. The four most abundant types of brain metastasis originate from primary tumors of lung cancer, colorectal cancer, breast cancer and renal cell carcinoma. As metastatic cells contain the molecular information of the primary tissue cells and IR spectroscopy probes the molecular fingerprint of cells, IR spectroscopy based methods constitute a new approach to determine the origin of brain metastases. IR spectroscopic images of 4 by 4 mm2 tissue areas were recorded in transmission mode by a FTIR imaging spectrometer coupled to a focal plane array detector. Unsupervised cluster analysis revealed variances within each cryosection. Selected clusters of five IR images with known diagnoses trained a supervised classification model based on the algorithm soft independent modeling of class analogies (SIMCA). This model was applied to distinguish normal brain tissue from brain metastases and to identify the primary tumor of brain metastases in 15 independent IR images. All specimens were assigned to the correct tissue class. This proof-of-concept study demonstrates that IR spectroscopy can complement established methods such as histopathology or immunohistochemistry for diagnosis.
Adult↗
Classification of malignant gliomas by infrared spectroscopy and linear discriminant analysis.
Gliomas are the most frequent primary brain tumors. Their malignancies are graded from 1 to 4. Malignant gliomas are astrocytoma grade 3 and glioblastoma grade 4. An IR spectroscopic approach is presented to diagnose brain tissue at the molecular level probing chemical and structural properties without external markers. IR spectroscopic maps were recorded in transmission mode by sequential acquisition of IR spectra. Training spectra of various tissue types are selected from IR spectroscopic maps in accordance with histological assessment of hematoxylin and eosin stained parallel tissue sections. A decrease of the lipid-to-protein ratio in IR spectra is correlated with the malignancy of gliomas. This chemical property is described by the band intensity ratio 2850 to 1655 cm(-1). Two additional molecular descriptors are identified at 1545 cm(-1)/1655 cm(-1) and (1231 + 1450) cm(-1)/1655 cm(-1), which are associated with hemoglobin and collagen, respectively. This metric is used to train a classification model based on linear discriminant analysis. The model is applied to classify normal brain tissue, astrocytoma grade 2, astrocytoma grade 3, glioblastoma, hemorrhage, and leptomeninges in IR spectroscopic maps of cryosections from two glioma patients. As independent test samples, single IR spectra from cryosections of 51 patients are subjected to the classification model. Normal brain tissue is assigned with 100% accuracy; malignant gliomas are assigned with 93% accuracy. The high success rate demonstrates that IR spectroscopy can complement established methods such as histopathology or immunohistochemistry to characterize dried cryosections.
Astrocytoma↗
Identification of primary tumors of brain metastases by infrared spectroscopic imaging and linear discriminant analysis.
This study applies infrared (IR) spectroscopy to distinguish normal brain tissue from brain metastases and to determine the primary tumor of four frequent brain metastases such as lung cancer, colorectal cancer, breast cancer, and renal cell carcinoma. Standard methods sometimes fail to identify the origin of brain metastases. As metastatic cells contain the molecular information of the primary tissue cells and IR spectroscopy probes the molecular fingerprint of cells, IR spectroscopy based methods constitute a new approach to determine the primary tumor of a brain metastasis. IR spectroscopic images were recorded by a FTIR spectrometer equipped with a macro sample chamber and coupled to a focal plane array detector. Unsupervised cluster analysis of IR images revealed variances within each sample and between samples of the same tissue type. Cluster averaged IR spectra of tissue classes with known diagnoses were selected to develop a metric with eight variables. These data trained a supervised classification model based on linear discriminant analysis that was used to identify the origin of 20 cryosections including one brain metastasis with an unknown primary tumor.
Brain Neoplasms↗
Delimitation of squamous cell cervical carcinoma using infrared microspectroscopic imaging.
Infrared (IR) spectroscopic imaging coupled with microscopy has been used to investigate thin sections of cervix uteri encompassing normal tissue, precancerous structures, and squamous cell carcinoma. Methods for unsupervised distinction of tissue types based on IR spectroscopy were developed. One-hundred and twenty-two images of cervical tissue were recorded by an FTIR spectrometer with a 64x64 focal plane array detector. The 499,712 IR spectra obtained were grouped by an approach which used fuzzy C-means clustering followed by hierarchical cluster analysis. The resulting false color maps were correlated with the morphological characteristics of an adjacent section of hematoxylin and eosin-stained tissue. In the first step, cervical stroma, epithelium, inflammation, blood vessels, and mucus could be distinguished in IR images by analysis of the spectral fingerprint region (950-1480 cm(-1)). In the second step, analysis in the spectral window 1420-1480 cm(-1) enables, for the first time, IR spectroscopic distinction between the basal layer, dysplastic lesions and squamous cell carcinoma within a particular sample. The joint application of IR microspectroscopic imaging and multivariate spectral processing combines diffraction-limited lateral optical resolution on the single cell level with highly specific and sensitive spectral classification on the molecular level. Compared with previous reports our approach constitutes a significant progress in the development of optical molecular spectroscopic techniques toward an additional diagnostic tool for the early histopathological characterization of cervical cancer.
Carcinoma, Squamous Cell↗
Near infrared Raman spectroscopic mapping of native brain tissue and intracranial tumors.
This study assessed the diagnostic potential of Raman spectroscopic mapping by evaluating its ability to distinguish between normal brain tissue and the human intracranial tumors gliomas and meningeomas. Seven Raman maps of native specimens were collected ex vivo by a Raman spectrometer with 785 nm excitation coupled to a microscope with a motorized stage. Variations within each Raman map were analyzed by cluster analysis. The dependence of tissue composition on the tissue type in cluster averaged Raman spectra was shown by linear combinations of reference spectra. Normal brain tissue was found to contain higher levels of lipids, intracranial tumors have more hemoglobin and lower lipid to protein ratios, meningeomas contain more collagen with maximum collagen content in normal meninges. One sample was studied without freezing. Whereas tumor regions did not change significantly, spectral changes were observed in the hemoglobin component after snap freezing and thawing to room temperature. The results constitute a basis for subsequent Raman studies to develop classification models for diagnosis of brain tissue.
Brain Mapping↗
Identification of B and T cells in human spleen sections by infrared microspectroscopic imaging.
BACKGROUND: Infrared spectroscopy probes the chemical composition and molecular structure of complex systems such as tissue and cells. Infrared spectroscopic imaging combines this spectral information with lateral resolution near the single-cell level. We analyzed whether this method is competitive with classic immunohistochemical methods for immunologic tissue and cells. METHODS: We recorded infrared microspectroscopic mapping datasets with a 90- x 90-microm2 aperture from a 3- x 3-mm2 unstained tissue area of human spleen. A secondary follicle containing a germinal center and a T zone were studied in more detail by infrared microspectroscopic imaging with lateral resolution near 5 mum. The results were compared with consecutive sections stained by immunoglobulin D antibodies. T and B lymphocytes were extracted from human blood and served as independent test samples. RESULTS: Cluster analysis of infrared datasets produced images that distinguished anatomical features such as primary and secondary follicles, T zones, arteries, and spleen red pulp. The assignments could be confirmed in consecutive sections by immunohistochemical staining. Main spectral variances between T and B lymphocytes in high-resolution measurements were attributed to specific spectral contributions of DNA and cytosol. CONCLUSIONS: Sensitivity and specificity of the infrared based methods are comparable to those of standard staining procedures for identification of B and T cells. However, infrared spectroscopic imaging can offer advantages in velocity, data throughput, and standardization because of minimal sample preparation. The results emphasize the potential of infrared spectroscopy as an innovative tool for the distinction of cell types, in particular in immunologic tissue.
Adolescent↗
Near infrared Raman spectra of human brain lipids.
Human brain tissue, in particular white matter, contains high lipid content. These brain lipids can be divided into three principal classes: neutral lipids including the steroid cholesterol, phospholipids and sphingolipids. Major lipids in normal human brain tissue are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, sphingomyelin, galactocerebrosides, gangliosides, sulfatides and cholesterol. Minor lipids are cholesterolester and triacylglycerides. During transformation from normal brain tissue to tumors, composition and concentration of lipids change in a specific way. Therefore, analysis of lipids might be used as a diagnostic parameter to distinguish normal tissue from tumors and to determine the tumor type and tumor grade. Raman spectroscopy has been suggested as an analytical tool to detect these changes even under intra-operative conditions. We recorded Raman spectra of the 12 major and minor brain lipids with 785 nm excitation in order to identify their spectral fingerprints for qualitative and quantitative analyses.
Brain↗
Analysis of human brain tissue, brain tumors and tumor cells by infrared spectroscopic mapping.
This study uses infrared (IR) spectroscopic, point detection, mapping procedures to examine tissue samples from normal brain specimens and from astrocytic gliomas, the most frequent human brain tumors. Model systems were derived from cultured glioma cell lines. IR spectra of normal tissue sections distinguished white matter from gray matter by increased spectral contributions from lipids and cholesterol. Qualitatively the same differences were found in IR spectra of low and high grade glioma tissue sections pointing to a significant reduction of brain lipids with increasing malignancy. Whereas spectral contributions of proteins and lipids were similar in IR spectra of glioma cells and tissues, nucleic acid bands were more intense for cells suggesting higher proliferative activities. For statistical analyses of IR spectroscopic maps from 71 samples, a parameter for the lipid to protein ratio was introduced involving the CH(2) symmetric stretch band with lipids as main contributors and the amide I band of proteins. As this parameter correlated with the grade of gliomas obtained from standard histopathological examination, it was applied to classify brain tissue sections based on IR spectroscopic mapping.
Astrocytes↗
Bioanalytical applications of Raman spectroscopy.
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Animals↗
Secondary structure polymorphism in Oxytricha nova telomeric DNA.
Tandem repeats of the telomeric DNA sequence d(T4G4) of Oxytricha nova are capable of forming unusually stable secondary structures incorporating Hoogsteen hydrogen bonding interactions. The biological significance of such DNA structures is supported by evidence of specific recognition of telomere end-binding proteins in the crystal state. To further characterize structural polymorphism of Oxytricha telomeric DNAs, we have obtained and interpreted Raman, ultraviolet resonance Raman (UVRR) and circular dichroism (CD) spectra of the tandem repeats d(G4T4G4) (Oxy1.5), d(T4G4)2 (Oxy2) and dT6(T4G4)2 (T6Oxy2) and related non-telomeric isomers in aqueous salt solutions. Raman markers of Oxy1.5 identify both C2'-endo/anti and C2'-endo/syn conformations of the deoxyguanosine residues and Hoogsteen hydrogen bonded guanine quartets, consistent with the quadruplex fold determined previously by solution NMR spectroscopy. Raman, UVRR and CD signatures and Raman dynamic measurements, to monitor imino NH-->ND exchanges, show that the Oxy1.5 antiparallel quadruplex fold is distinct from the hairpin structures of Oxy2 and T6Oxy2, single-stranded structures of d(TG)8 and dT6(TG)8 and previously reported quadruplex structures of d(T4G4)4 (Oxy4) and dG12. Spectral markers of the telomeric and telomere-related DNA structures are tabulated and novel Raman and UVRR indicators of thymidine and deoxyguanosine conformations are identified. The results will be useful for probing structures of Oxytricha telomeric repeats in complexes with telomere end-binding proteins.
Animals↗