Prediction of WHO grade and methylation class of aggressive meningiomas: Extraction of diagnostic information from infrared spectroscopic data.

Background: Infrared (IR) spectroscopy allows intraoperative, optical brain tumor diagnosis. Here, we explored it as a translational technology for the identification of aggressive meningioma types according to both, the WHO CNS grading system and the methylation classes (MC). Methods: Frozen sections of 47 meningioma were examined by IR spectroscopic imaging and different classification approaches were compared to discern samples according to WHO grade or MC. Results: IR spectroscopic differences were more pronounced between WHO grade 2 and 3 than between MC intermediate and MC malignant, although similar spectral ranges were affected. Aggressive types of meningioma exhibited reduced bands of carbohydrates (at 1024 cm-1) and nucleic acids (at 1080 cm-1), along with increased bands of phospholipids (at 1240 and 1450 cm-1). While linear discriminant analysis was able to discern spectra of WHO grade 2 and 3 meningiomas (AUC 0.89), it failed for MC (AUC 0.66). However, neural network classifiers were effective for classification according to both WHO grade (AUC 0.91) and MC (AUC 0.83), resulting in the correct classification of 20/23 meningiomas of the test set. Conclusions: IR spectroscopy proved capable of extracting information about the malignancy of meningiomas, not only according to the WHO grade, but also for a diagnostic system based on molecular tumor characteristics. In future clinical use, physicians could assess the goodness of the classification by considering classification probabilities and cross-measurement validation. This might enhance the overall accuracy and clinical utility, reinforcing the potential of IR spectroscopy in advancing precision medicine for meningioma characterization.

Label-free multiphoton microscopy enables histopathological assessment of colorectal liver metastases and supports automated classification of neoplastic tissue.

As the state of resection margins is an important prognostic factor after extirpation of colorectal liver metastases, surgeons aim to obtain negative margins, sometimes elaborated by resections of the positive resection plane after intraoperative frozen sections. However, this is time consuming and results sometimes remain unclear during surgery. Label-free multimodal multiphoton microscopy (MPM) is an optical technique that retrieves morpho-chemical information avoiding all staining and that can potentially be performed in real-time. Here, we investigated colorectal liver metastases and hepatic tissue using a combination of three endogenous nonlinear signals, namely: coherent anti-Stokes Raman scattering (CARS) to visualize lipids, two-photon excited fluorescence (TPEF) to visualize cellular patterns, and second harmonic generation (SHG) to visualize collagen fibers. We acquired and analyzed over forty thousand MPM images of metastatic and normal liver tissue of 106 patients. The morphological information with biochemical specificity produced by MPM allowed discriminating normal liver from metastatic tissue and discerning the tumor borders on cryosections as well as formalin-fixed bulk tissue. Furthermore, automated tissue type classification with a correct rate close to 95% was possible using a simple approach based on discriminant analysis of texture parameters. Therefore, MPM has the potential to increase the precision of resection margins in hepatic surgery of metastases without prolonging surgical intervention.

A new approach for clinical translation of infrared spectroscopy: exploitation of the signature of glioblastoma for general brain tumor recognition.

PURPOSE: Infrared (IR) spectroscopy has the potential for tumor delineation in neurosurgery. Previous research showed that IR spectra of brain tumors are generally characterized by reduced lipid-related and increased protein-related bands. Therefore, we propose the exploitation of these common spectral changes for brain tumor recognition. METHODS: Attenuated total reflection IR spectroscopy was performed on fresh specimens of 790 patients within minutes after resection. Using principal component analysis and linear discriminant analysis, a classification model was developed on a subset of glioblastoma (n = 135) and non-neoplastic brain (n = 27) specimens, and then applied to classify the IR spectra of several types of brain tumors. RESULTS: The model correctly classified 82% (517/628) of specimens as "tumor" or "non-tumor", respectively. While the sensitivity was limited for infiltrative glioma, this approach recognized GBM (86%), other types of primary brain tumors (92%) and brain metastases (92%) with high accuracy and all non-tumor samples were correctly identified. CONCLUSION: The concept of differentiation of brain tumors from non-tumor brain based on a common spectroscopic tumor signature will accelerate clinical translation of infrared spectroscopy and related technologies. The surgeon could use a single instrument to detect a variety of brain tumor types intraoperatively in future clinical settings. Our data suggests that this would be associated with some risk of missing infiltrative regions or tumors, but not with the risk of removing non-tumor brain.

Clinical Raman spectroscopy of brain tumors from an interdisciplinary perspective.

Raman spectroscopy is an optical technology that probes tissue composition and is envisioned for clinical applications in neurosurgery. Here, we provide an overview of basic and translational research addressing brain tumor delineation and diagnosis and identify potential scenarios for routine clinical use of Raman spectroscopy. Moreover, we discuss the practical technical requirements in the context of daily use as well as open questions regarding automated tissue assessment.

Learned end-to-end high-resolution lensless fiber imaging towards real-time cancer diagnosis.

Recent advances in label-free histology promise a new era for real-time diagnosis in neurosurgery. Deep learning using autofluorescence is promising for tumor classification without histochemical staining process. The high image resolution and minimally invasive diagnostics with negligible tissue damage is of great importance. The state of the art is raster scanning endoscopes, but the distal lens optics limits the size. Lensless fiber bundle endoscopy offers both small diameters of a few 100 microns and the suitability as single-use probes, which is beneficial in sterilization. The problem is the inherent honeycomb artifacts of coherent fiber bundles (CFB). For the first time, we demonstrate an end-to-end lensless fiber imaging with exploiting the near-field. The framework includes resolution enhancement and classification networks that use single-shot CFB images to provide both high-resolution imaging and tumor diagnosis. The well-trained resolution enhancement network not only recovers high-resolution features beyond the physical limitations of CFB, but also helps improving tumor recognition rate. Especially for glioblastoma, the resolution enhancement network helps increasing the classification accuracy from 90.8 to 95.6%. The novel technique enables histological real-time imaging with lensless fiber endoscopy and is promising for a quick and minimally invasive intraoperative treatment and cancer diagnosis in neurosurgery.

Correlation of biomechanics and cancer cell phenotype by combined Brillouin and Raman spectroscopy of U87-MG glioblastoma cells.

The elucidation of biomechanics furthers our understanding of brain tumour biology. Brillouin spectroscopy is a new optical method that addresses viscoelastic properties down to subcellular resolution in a contact-free manner. Moreover, it can be combined with Raman spectroscopy to obtain co-localized biochemical information. Here, we applied co-registered Brillouin and Raman spectroscopy to U87-MG human glioblastoma cells in vitro. Using two-dimensional and three-dimensional cultures, we related biomechanical properties to local biochemical composition at the subcellular level, as well as the cell phenotype. Brillouin and Raman mapping of adherent cells showed that the nucleus and nucleoli are stiffer than the perinuclear region and the cytoplasm. The biomechanics of the cell cytoplasm is affected by culturing conditions, i.e. cells grown as spheroids are stiffer than adherent cells. Inside the spheroids, the presence of lipid droplets as assessed by Raman spectroscopy revealed higher Brillouin shifts that are not related to a local increase in stiffness, but are due to a higher refractive index combined with a lower mass density. This highlights the importance of locally defined biochemical reference data for a correct interpretation of the Brillouin shift of cells and tissues in future studies investigating the biomechanics of brain tumour models by Brillouin spectroscopy.

Brillouin confocal microscopy to determine biomechanical properties of SULEEI-treated bovine pericardium for application in cardiac surgery.

BACKGROUND: Heart valves are exposed to a highly dynamic environment and underlie high tensile and shear forces during opening and closing. Therefore, analysis of mechanical performance of novel heart valve bioprostheses materials, like SULEEI-treated bovine pericardium, is essential and usually carried out by uniaxial tensile tests. Nevertheless, major drawbacks are the unidirectional strain, which does not reflect the in vivo condition and the deformation of the sample material. An alternative approach for measurement of biomechanical properties is offered by Brillouin confocal microscopy (BCM), a novel, non-invasive and three-dimensional method based on the interaction of light with acoustic waves. OBJECTIVE: BCM is a powerful tool to determine viscoelastic tissue properties and is, for the first time, applied to characterize novel biological graft materials, such as SULEEI-treated bovine pericardium. Therefore, the method has to be validated as a non-invasive alternative to conventional uniaxial tensile tests. METHODS: Vibratome sections of SULEEI-treated bovine pericardium (decellularized, riboflavin/UV-cross-linked and low-energy electron irradiated) as well as native and GA-fixed controls (n = 3) were analyzed by BCM. In addition, uniaxial tensile tests were performed on equivalent tissue samples and Young's modulus as well as length of toe region were analyzed from stress-strain diagrams. The structure of the extracellular matrix (ECM), especially collagen and elastin, was investigated by multiphoton microscopy (MPM). RESULTS: SULEEI-treated pericardium exhibited a significantly higher Brillouin shift and hence higher tissue stiffness in comparison to native and GA-fixed controls (native: 5.6±0.2 GHz; GA: 5.5±0.1 GHz; SULEEI: 6.3±0.1 GHz; n = 3, p < 0.0001). Similarly, a significantly higher Young's modulus was detected in SULEEI-treated pericardia in comparison to native tissue (native: 30.0±10.4 MPa; GA: 31.8±10.7 MPa; SULEEI: 42.1±7.0 MPa; n = 3, p = 0.027). Native pericardia showed wavy and non-directional collagen fibers as well as thin, linear elastin fibers generating a loose matrix. The fibers of GA-fixed and SULEEI-treated pericardium were aligned in one direction, whereat the SULEEI-sample exhibited a much denser matrix. CONCLUSION: BCM is an innovative and non-invasive method to analyze elastic properties of novel pericardial graft materials with special mechanical requirements, like heart valve bioprostheses.

Label-free multiphoton imaging allows brain tumor recognition based on texture analysis-a study of 382 tumor patients.

BACKGROUND: Label-free multiphoton microscopy has been suggested for intraoperative recognition and delineation of brain tumors. For any future clinical application, appropriate approaches for image acquisition and analysis have to be developed. Moreover, an evaluation of the reliability of the approach, taking into account inter- and intrapatient variability, is needed. METHODS: Coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF), and second-harmonic generation were acquired on cryosections of brain tumors of 382 patients and 28 human nontumor brain samples. Texture parameters of those images were calculated and used as input for linear discriminant analysis. RESULTS: The combined analysis of texture parameters of the CARS and TPEF signal proved to be most suited for the discrimination of nontumor brain versus brain tumors (low- and high-grade astrocytoma, oligodendroglioma, glioblastoma, recurrent glioblastoma, brain metastases of lung, colon, renal, and breast cancer and of malignant melanoma) leading to a correct rate of 96% (sensitivity: 96%, specificity: 100%). To approximate the clinical setting, the results were validated on 42 fresh, unfixed tumor biopsies. 82% of the tumors and, most important, all of the nontumor samples were correctly recognized. An image resolution of 1 µm was sufficient to distinguish brain tumors and nontumor brain. Moreover, the vast majority of single fields of view of each patient's sample were correctly classified with high probabilities, which is important for clinical translation. CONCLUSION: Label-free multiphoton imaging might allow fast and accurate intraoperative delineation of primary and secondary brain tumors in combination with endoscopic systems.

Label-free multiphoton microscopy as a tool to investigate alterations of cerebral aneurysms.

Cerebral aneurysms are abnormal focal dilatations of arterial vessel walls with pathological vessel structure alterations. Sudden rupture can lead to a subarachnoid hemorrhage, which is associated with a high mortality. Therefore, the origin of cerebral aneurysms as well as the progression to the point of rupture needs to be further investigated. Label-free multimodal multiphoton microscopy (MPM) was performed on resected human aneurysm domes and integrated three modalities: coherent anti-Stokes Raman scattering, endogenous two-photon fluorescence and second harmonic generation. We showed that MPM is a completely label-free and real-time powerful tool to detect pathognomonic histopathological changes in aneurysms, e.g. thickening and thinning of vessel walls, intimal hyperplasia, intra-wall haemorrhage, calcification as well as atherosclerotic changes. In particular, the loss or fragmentation of elastin as well as fibromatous wall remodelling appeared very distinct. Remarkably, cholesterol and lipid deposits were clearly visible in the multiphoton images. MPM provides morphological and biochemical information that are crucial for understanding the mechanisms of aneurysm formation and progression.

Rapid Label-Free Analysis of Brain Tumor Biopsies by Near Infrared Raman and Fluorescence Spectroscopy-A Study of 209 Patients.

In brain surgery, novel technologies are continuously developed to achieve better tumor delineation and maximize the extent of resection. Raman spectroscopy is an optical method that enables to retrieve a molecular signature of tissue biochemical composition in order to identify tumor and normal tissue. Here, the translation of Raman spectroscopy to the surgical practice for discerning a variety of different tumor entities from non-neoplastic brain parenchyma was investigated. Fresh unprocessed biopsies obtained from brain tumor surgery were analyzed over 1.5 years including all patients that gave consent. Measurements were performed with a Raman microscope by medical personnel as routine activity. The Raman and fluorescence signals of the acquired spectra were analyzed by principal component analysis, followed by supervised classification to discriminate non-tumor tissue vs. tumor and distinguish tumor entities. Histopathology of the measured biopsies was performed as reference. Classification led to the correct recognition of all non-neoplastic biopsies (7/7) and of 97% of the investigated tumor biopsies (195/202). For instance, GBM was recognized as tumor with a correct rate of 94% if primary, and of 100% if recurrent. Astrocytoma and oligodendroglioma were recognized as tumor with correct rates of 86 and 90%, respectively. All brain metastases, meningioma and schwannoma were correctly recognized as tumor and distinguished from non-neoplastic brain tissue. Furthermore, metastases were discerned from glioma with correct rate of 90%. Oligodendroglioma and astrocytoma IDH1-mutant, which differ in the presence of 1p/19q codeletion, were discerned with a correct rate of 81%. These results demonstrate the feasibility of rapid brain tumors recognition and extraction of diagnostic information by Raman spectroscopy, using a protocol that can be easily included in the routine surgical workflow.

Identification of distinctive features in human intracranial tumors by label-free nonlinear multimodal microscopy.

Nonlinear multimodal microscopy offers a series of label-free techniques with potential for intraoperative identification of tumor borders in situ using novel endoscopic devices. Here, we combined coherent anti-Stokes Raman scattering, two-photon excited fluorescence (TPEF) and second harmonic generation imaging to analyze biopsies of different human brain tumors, with the aim to understand whether the morphological information carried by single field of view images, similar to what delivered by present endoscopic systems, is sufficient for tumor recognition. We imaged 40 human biopsies of high and low grade glioma, meningioma, as well as brain metastases of melanoma, breast, lung and renal carcinoma, in comparison with normal brain parenchyma. Furthermore, five biopsies of schwannoma were analyzed and compared with nonpathological nerve tissue. Besides the high cellularity, the typical features of tumor, which were identified and quantified, are intracellular and extracellular lipid droplets, aberrant vessels, extracellular matrix collagen and diffuse TPEF. Each tumor type displayed a particular morphochemistry characterized by specific patterns of the above-mentioned features. Nonlinear multimodal microscopy performed on fresh unprocessed biopsies confirmed that the technique has the ability to visualize tumor structures and discern normal from neoplastic tissue likewise in conditions close to in situ.

Subclinical Endocarditis Might be a Hidden Trigger of Early Prosthetic Valve Calcification: A Histological Study.

OBJECTIVE: Despite various improvements in valve prosthetics, early valve deterioration still occurs, leading to prosthetic failure. Studying the early phase of this deterioration is quite difficult, as the prosthesis to be examined is almost always explanted only after extensive deterioration. The objective of this research is to study the pathology of early valve deterioration in an early stage in order to reveal the possible trigger of the process. METHODS: Three cusps of the same type of bovine pericardium valve prosthesis underwent comparative examination. Two cusps (cusps 1 and 2) were retrieved from a valve prosthesis explanted three months post-implantation, and the third cusp was from a non-implanted valve prosthesis and used as a reference cusp (ref. cusp). The examination included macroscopic examination, Non-linear Optical Microscopy using a multiphoton microscope, and histological examination with staining, using Hematoxylin and Eosin, Movat Pentachrome stain, Von-Kossa stain, and Alizirin-Red stain. Parallel sections were decalcified using Osteosoft® solution prior to Von-Kossa and Alizirin-Red staining to exclude false positive results. RESULTS: Macroscopically, cusp 1 showed early deterioration, and cusp 2 showed endocarditic vegetations. Histologically, cusp 1 showed calcifications in acellular deposits on the surface of the cusp, with pathological signs of subacute/healed endocarditis and intact cusp tissue. The examination did not show calcifications of the cellular remnants within the valve tissue. Cusp 2 showed florid endocarditis, with microscopic destruction of the valve tissue. CONCLUSION: Early prosthetic valve deterioration can exist as early as three months post-implantation. Subacute or subclinical endocarditis can be the cause for early valve calcification and deterioration.

IDH1 mutation in human glioma induces chemical alterations that are amenable to optical Raman spectroscopy.

INTRODUCTION: Mutations in the isocytrate dehydrogenase 1 (IDH1) gene are early genetic events in glioma pathogenesis and cause profound metabolic changes. Because this genotype is found in virtually every tumor cell, therapies targeting mutant IDH1 protein are being developed. The intraoperative administration of those therapies would require fast technologies for the determination of IDH1 genotype. As of today, there is no such diagnostic test available. Recently, infrared spectroscopy was shown to bridge this gap. Here, we tested Raman spectroscopy for analysis of IDH1 genotype in glioma, which constitutes an alternative contact-free technique with the potential of being applicable in situ. METHODS: Human glioma samples (n = 36) were obtained during surgery and cryosections were prepared. IDH1 mutations were assessed using DNA sequencing and 100 Raman spectra were obtained for each sample. RESULTS: Analysis of Raman spectra revealed increased intensities in spectral bands related to DNA in IDH1 mutant glioma while bands assigned to molecular vibrations of lipids were significantly decreased. Moreover, intensities of Raman bands assigned to proteins differed in IDH1 mutant and IDH1 wild-type glioma, suggesting alterations in the protein profile. The selection of five bands (498, 826, 1003, 1174 and 1337 cm-1) allowed the classification of Raman spectra according to IDH1 genotype with a correct rate of 89%. CONCLUSION: Raman spectroscopy constitutes a simple, rapid and safe procedure for determination of the IDH1 mutation that shows great promise for clinically relevant in situ diagnostics.

Optical molecular imaging of corpora amylacea in human brain tissue.

Label-free multiphoton imaging constitutes a promising technique for clinical diagnosis and therapeutic monitoring. Corpora amylacea (CoA) are starch-like structures often found in the diseased brain, whose origin and role in nervous pathologies are still a matter of debate. Recently, CoA in the diseased human hippocampus were found to be second harmonic generation (SHG) active. Here, we show that CoA formed in other parts of the diseased brain and in brain neoplasms display a similar SHG activity. The SHG pattern of CoA depended on laser polarization, indicating that a radial structure is responsible for their nonlinear activity. Vibrational spectroscopy was used to study the biochemistry underlying the SHG activity. Infrared (IR) and Raman spectroscopy showed that CoA contain polyglucosans that are biochemically similar to glycogen, but with an unusual structure that is similar to amylopectin, which justifies the nonlinear activity of CoA. Our findings explain the SHG activity of CoA and demonstrate that CoA in the pathological brain are amenable to label-free multiphoton imaging. Further research will clarify whether intraoperative assessment of CoA can be diagnostically exploited.

Optical Analysis of Glioma: Fourier-Transform Infrared Spectroscopy Reveals the IDH1 Mutation Status.

Purpose: Somatic mutations in the human cytosolic isocitrate dehydrogenase 1 (IDH1) gene cause profound changes in cell metabolism and are a common feature of gliomas with unprecedented predictive and prognostic impact. Fourier-transform infrared (FT-IR) spectroscopy addresses the molecular composition of cells and tissue and was investigated to deduct the IDH1 mutation status.Experimental Design: We tested the technique on human cell lines that were transduced with wild-type IDH1 or mutated IDH1 and on 34 human glioma samples. IR spectra were acquired at 256 positions from cell pellets or tissue cryosections. Moreover, IR spectra were obtained from fresh, unprocessed biopsies of 64 patients with glioma.Results:IDH1 mutation was linked to changes in spectral bands assigned to molecular groups of lipids and proteins in cell lines and human glioma. The spectra of cryosections of brain tumor samples showed high interpatient variability, for example, bands related to calcifications at 1113 cm-1 However, supervised classification recognized relevant spectral regions at 1103, 1362, 1441, 1485, and 1553 cm-1 and assigned 88% of the tumor samples to the correct group. Similar spectral positions allowed the classification of spectra of fresh biopsies with an accuracy of 86%.Conclusions: Here, we show that vibrational spectroscopy reveals the IDH1 genotype of glioma. Because it can provide information in seconds, an implementation into the intraoperative workflow might allow simple and rapid online diagnosis of the IDH1 genotype. The intraoperative confirmation of IDH1 mutation status might guide the decision to pursue definitive neurosurgical resection and guide future in situ therapies of infiltrative gliomas. Clin Cancer Res; 24(11); 2530-8. ©2017 AACRSee related commentary by Hollon and Orringer, p. 2467.

Assessing the efficacy of coherent anti-Stokes Raman scattering microscopy for the detection of infiltrating glioblastoma in fresh brain samples.

Coherent anti-Stokes Raman scattering (CARS) microscopy is an emerging technique for identification of brain tumors. However, tumor identification by CARS microscopy on bulk samples and in vivo has been so far verified retrospectively on histological sections, which only provide a gross reference for the interpretation of CARS images without matching at cellular level. Therefore, fluorescent labels were exploited for direct assessment of the interpretation of CARS images of solid and infiltrative tumors. Glioblastoma cells expressing green fluorescent protein (GFP) were used for induction of tumors in mice (n = 7). The neoplastic nature of cells imaged by CARS microscopy was unequivocally verified by addressing two-photon fluorescence of GFP on fresh brain slices and in vivo. In fresh unfixed biopsies of human glioblastoma (n = 10), the fluorescence of 5-aminolevulinic acid-induced protoporphyrin IX was used for identification of tumorous tissue. Distinctive morphological features of glioblastoma cells, i.e. larger nuclei, evident nuclear membrane and nucleolus, were identified in the CARS images of both mouse and human brain tumors. This approach demonstrates that the chemical contrast provided by CARS allows the localization of infiltrating tumor cells in fresh tissue and that the cell morphology in CARS images is useful for tumor recognition. Experimental glioblastoma expressing green fluorescent protein.

Heart valve stenosis in laser spotlights: insights into a complex disease.

Degenerative heart valve disease is a life-threatening disease affecting about 3% of the population over 65 years. Up to date, cardiac surgery with heart valve replacement is the only available therapy. The disease is characterized by degenerative disorganization of the heart valve structure and alterations in the residing cell populations. Causes and mechanisms of disease genesis are still not fully understood and until now pharmacological therapies are not available. Thus there is enormous interest in new technologies that enable a better characterization of structure and composition of diseased valves. Currently most research techniques demand for extensive processing of extracted valve material. We present a novel approach combining coherent anti-Stokes Raman scattering, endogenous two-photon excited fluorescence and second harmonic generation. Cusp constituents can be examined simultaneously, three-dimensionally and without extensive manipulation of the sample enabling impressive insights into a complex disease.

Intrinsic indicator of photodamage during label-free multiphoton microscopy of cells and tissues.

Multiphoton imaging has evolved as an indispensable tool in cell biology and holds prospects for clinical applications. When addressing endogenous signals such as coherent anti-Stokes Raman scattering (CARS) or second harmonic generation, it requires intense laser irradiation that may cause photodamage. We report that increasing endogenous fluorescence signal upon multiphoton imaging constitutes a marker of photodamage. The effect was studied on mouse brain in vivo and ex vivo, on ex vivo human brain tissue samples, as well as on glioblastoma cells in vitro, demonstrating that this phenomenon is common to a variety of different systems, both ex vivo and in vivo. CARS microscopy and vibrational spectroscopy were used to analyze the photodamage. The development of a standard easy-to-use model that employs rehydrated cryosections allowed the characterization of the irradiation-induced fluorescence and related it to nonlinear photodamage. In conclusion, the monitoring of endogenous two-photon excited fluorescence during label-free multiphoton microscopy enables to estimate damage thresholds ex vivo as well as detect photodamage during in vivo experiments.

Label-free delineation of brain tumors by coherent anti-Stokes Raman scattering microscopy in an orthotopic mouse model and human glioblastoma.

BACKGROUND: Coherent anti-Stokes Raman scattering (CARS) microscopy provides fine resolution imaging and displays morphochemical properties of unstained tissue. Here, we evaluated this technique to delineate and identify brain tumors. METHODS: Different human tumors (glioblastoma, brain metastases of melanoma and breast cancer) were induced in an orthotopic mouse model. Cryosections were investigated by CARS imaging tuned to probe C-H molecular vibrations, thereby addressing the lipid content of the sample. Raman microspectroscopy was used as reference. Histopathology provided information about the tumor's localization, cell proliferation and vascularization. RESULTS: The morphochemical contrast of CARS images enabled identifying brain tumors irrespective of the tumor type and properties: All tumors were characterized by a lower CARS signal intensity than the normal parenchyma. On this basis, tumor borders and infiltrations could be identified with cellular resolution. Quantitative analysis revealed that the tumor-related reduction of CARS signal intensity was more pronounced in glioblastoma than in metastases. Raman spectroscopy enabled relating the CARS intensity variation to the decline of total lipid content in the tumors. The analysis of the immunohistochemical stainings revealed no correlation between tumor-induced cytological changes and the extent of CARS signal intensity reductions. The results were confirmed on samples of human glioblastoma. CONCLUSIONS: CARS imaging enables label-free, rapid and objective identification of primary and secondary brain tumors. Therefore, it is a potential tool for diagnostic neuropathology as well as for intraoperative tumor delineation.

Label-free identification of the glioma stem-like cell fraction using Fourier-transform infrared spectroscopy.

PURPOSE: Vibrational spectroscopy enables the label-free characterization of cells and tissue by probing the biochemical composition. Here, we evaluated these techniques to identify glioblastoma stem cells. MATERIALS AND METHODS: The biochemical fingerprints of glioblastoma cells were established in human cell lines with high and low content of CD133 (cluster of differentiation 133)-positive cells using attenuated total reflection Fourier-transform infrared (ATR FT-IR) on vital cells and FT-IR mapping, which delivers spatially resolved spectroscopic datasets. After data preprocessing, unsupervised cluster analysis was applied. CD133 was addressed with flow cytometry and immunohistochemistry and used as a stemness marker. RESULTS: In all preparations, the algorithm was able to correctly classify the spectra, differentiating CD133-rich and -poor populations. The main spectral differences were found in the region of 1000 cm(- 1) to 1150 cm(- 1) that can be assigned to vibrations of chemical bonds of DNA, RNA, carbohydrates and phospholipids. Interestingly, this spectral region is a key feature to discern glioblastoma from normal brain parenchyma, as FT-IR spectroscopic mapping of experimental brain tumors demonstrated. CONCLUSIONS: We were able to show biochemical differences between glioblastoma cell populations with high and low content of cancer stem cells that are presumably related to changes in the RNA/DNA content.