Determinants of trust in times of crises: A cross-sectional study of 3,065 German-speaking adults from the D-A-CH region.

Interpersonal trust declined worldwide during the COVID-19 pandemic; strategies are needed to restore it. We surveyed 3,065 quota-sampled German-speaking adults residing in the D-A-CH region. Using multinomial logistic regression models and backward elimination for variable selection, we calculated multivariable-adjusted odds ratios (OR) and 95% confidence intervals (95% CIs) to appraise correlates of interpersonal trust using the Interpersonal Trust Short Scale (KUSIV3). Participants with high levels of interpersonal trust (top KUSIV3 tertile (T3)) tended to be older, male, residents of Switzerland, university degree holders, and workers with higher income and work satisfaction (all Pdiff<0.01) compared to those in the lowest KUSIV3 tertile (T1). Optimism was most strongly associated with high interpersonal trust (ORT3vsT1 = 5.75, 95%CI = 4.33-7.64). Also significantly associated with high interpersonal trust were: Having voted in the last national election (for the opposition, OR = 1.39, 95%CI = 1.02-1.89 or the governing party, OR = 1.61, 95%CI = 1.23-2.11) versus non-voters; perspective taking (ORT3vsT1 = 1.46, 95%CI = 1.11-1.91); being more extraverted (ORT3vsT1 = 1.99, 95%CI = 1.53-2.59) and more agreeable (ORT3vsT1 = 1.95, 95% CI = 1.46-2.61); and scoring higher on complexity thinking (ORT3vsT1 = 1.32, 95%CI = 1.01-1.72). Participants scoring significantly lower for interpersonal trust did not regularly participate in religious meetings (OR = 0.61, 95%CI = 0.44-0.84, versus participation at least monthly); were more conscientious (ORT3vsT1 = 0.68, 95%CI = 0.51-0.91) or current smokers (OR = 0.68; 95%CI = 0.53-0.87, versus never smoking); had sleep problems >5 times a week (OR = 0.48; 95%CI = 0.36-0.66, versus none); and scored high on conspiracy belief (ORT3vsT1 = 0.53; 95%CI = 0.41-0.69). Results differed minimally by gender and country. These findings may be helpful in devising targeted strategies to strengthen interpersonal trust and social engagement in European societies, especially during times of crises.

Fast and label-free intraoperative discrimination of malignant pancreatic tissue by attenuated total reflection infrared spectroscopy.

Significance: Pancreatic surgery is a highly demanding and routinely applied procedure for the treatment of several pancreatic lesions. The outcome of patients with malignant entities crucially depends on the margin resection status of the tumor. Frozen section analysis for intraoperative evaluation of tissue is still time consuming and laborious. Aim: We describe the application of fiber-based attenuated total reflection infrared (ATR IR) spectroscopy for label-free discrimination of normal pancreatic, tumorous, and pancreatitis tissue. A pilot study for the intraoperative application was performed. Approach: The method was applied for unprocessed freshly resected tissue samples of 58 patients, and a classification model for differentiating between the distinct tissue classes was established. Results: The developed three-class classification model for tissue spectra allows for the delineation of tumors from normal and pancreatitis tissues using a probability score for class assignment. Subsequently, the method was translated into intraoperative application. Fiber optic ATR IR spectra were obtained from freshly resected pancreatic tissue directly in the operating room. Conclusion: Our study shows the possibility of applying fiber-based ATR IR spectroscopy in combination with a supervised classification model for rapid pancreatic tissue identification with a high potential for transfer into intraoperative surgical diagnostics.

Topographic Mapping of the Primary Sensory Cortex Using Intraoperative Optical Imaging and Tactile Irritation.

The determination of exact tumor boundaries within eloquent brain regions is essential to maximize the extent of resection. Recent studies showed that intraoperative optical imaging (IOI) combined with median nerve stimulation is a helpful tool for visualization of the primary sensory cortex (PSC). In this technical note, we describe a novel approach of using IOI with painless tactile irritation to demonstrate the feasibility of topographic mapping of different body regions within the PSC. In addition, we compared the IOI results with preoperative functional MRI (fMRI) findings. In five patients with tumors located near the PSC who received tumor removal, IOI with tactile irritation of different body parts and fMRI was applied. We showed that tactile irritation of the hand in local and general anesthesia leads to reliable changes of cerebral blood volume during IOI. Hereby, we observed comparable IOI activation maps regarding the median nerve stimulation, fMRI and tactile irritation of the hand. The tactile irritation of different body areas revealed a plausible topographic distribution along the PSC. With this approach, IOI is also suitable for awake surgeries, since the tactile irritation is painless compared with median nerve stimulation and is congruent to fMRI findings. Further studies are ongoing to standardize this method to enable a broad application within the neurosurgical community.

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.

Label-free differentiation of human pancreatic cancer, pancreatitis, and normal pancreatic tissue by molecular spectroscopy.

Significance: Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer deaths with a best median survival of only 40 to 50 months for localized disease despite multimodal treatment. The standard tissue differentiation method continues to be pathology with histological staining analysis. Microscopic discrimination between inflammatory pancreatitis and malignancies is demanding. Aim: We aim to accurately distinguish native pancreatic tissue using infrared (IR) spectroscopy in a fast and label-free manner. Approach: Twenty cryopreserved human pancreatic tissue samples were collected from surgical resections. In total, more than 980,000 IR spectra were collected and analyzed using aMATLAB package. For differentiation of PDAC, pancreatitis, and normal tissue, a three-class training set for supervised classification was created with 25,000 spectra and the principal component analysis (PCA) score values for each cohort. Cross-validation was performed using the leaveone- out method. Validation of the algorithm was accomplished with 13 independent test samples. Results: Reclassification of the training set and the independent test samples revealed an overall accuracy of more than 90% using a discrimination algorithm. Conclusion: IR spectroscopy in combination with PCA and supervised classification is an efficient analytical method to reliably distinguish between benign and malignant pancreatic tissues. It opens up a wide research field for oncological and surgical applications.

Characterization of cortical hemodynamic changes following sensory, visual, and speech activation by intraoperative optical imaging utilizing phase-based evaluation methods.

Alterations within cerebral hemodynamics are the intrinsic signal source for a wide variety of neuroimaging techniques. Stimulation of specific functions leads due to neurovascular coupling, to changes in regional cerebral blood flow, oxygenation and volume. In this study, we investigated the temporal characteristics of cortical hemodynamic responses following electrical, tactile, visual, and speech activation for different stimulation paradigms using Intraoperative Optical Imaging (IOI). Image datasets from a total of 22 patients that underwent surgical resection of brain tumors were evaluated. The measured reflectance changes at different light wavelength bands, representing alterations in regional cortical blood volume (CBV), and deoxyhemoglobin (HbR) concentration, were assessed by using Fourier-based evaluation methods. We found a decrease of CBV connected to an increase of HbR within the contralateral primary sensory cortex (SI) in patients that were prolonged (30 s/15 s) electrically stimulated. Additionally, we found differences in amplitude as well as localization of activated areas for different stimulation patterns. Contrary to electrical stimulation, prolonged tactile as well as prolonged visual stimulation are provoking increases in CBV within the corresponding activated areas (SI, visual cortex). The processing of the acquired data from awake patients performing speech tasks reveals areas with increased, as well as areas with decreased CBV. The results lead us to the conclusion, that the CBV decreases in connection with HbR increases in SI are associated to processing of nociceptive stimuli and that stimulation type, as well as paradigm have a nonnegligible impact on the temporal characteristics of the following hemodynamic response.

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.

Fiber attenuated total reflection infrared spectroscopy of kidney tissue during live surgery.

More than 90% of solid kidney tumors are cancerous and have to be treated by surgical resection where surgical outcomes and patient prognosis are dependent on the tumor discrimination. The development of alternative approaches based on a new generation of fiber attenuated total reflection (ATR) probes could aid tumor identification even under intrasurgical conditions. Herein, fiber ATR IR spectroscopy is employed to distinguish normal and cancerous kidney tissues. Freshly resected tissue samples from 34 patients are investigated under nearly native conditions. Spectral marker bands that allow a reliable discrimination between tumor and normal tissue are identified by a supervised classification algorithm. The absorbance values of the bands at 1025, 1155 and 1240 cm-1 assigned to glycogen and fructose 1,6-bisphosphatase are used as the clearest markers for the tissue discrimination. Absorbance threshold values for tumor and normal tissue are determined by discriminant analysis. This new approach allows the surgeon to make a clinical diagnosis.

Mapping of language and motor function during awake neurosurgery with intraoperative optical imaging.

Intraoperative optical imaging (IOI) is a marker-free, contactless, and noninvasive imaging technique that is able to visualize metabolic changes of the brain surface following neuronal activation. Although it has been used in the past mainly for the identification of functional brain areas under general anesthesia, the authors investigated the potential of the method during awake surgery. Measurements were performed in 10 patients who underwent resection of lesions within or adjacent to cortical language or motor sites. IOI was applied in 3 different scenarios: identification of motor areas by using finger-tapping tasks, identification of language areas by using speech tasks (overt and silent speech), and a novel approach-the application of IOI as a feedback tool during direct electrical stimulation (DES) mapping of language. The functional maps, which were calculated from the IOI data (activity maps), were qualitatively compared with the functional MRI (fMRI) and the electrophysiological testing results during the surgical procedure to assess their potential benefit for surgical decision-making.The results reveal that the intraoperative identification of motor sites with IOI in good agreement with the preoperatively acquired fMRI and the intraoperative electrophysiological measurements is possible. Because IOI provides spatially highly resolved maps with minimal additional hardware effort, the application of the technique for motor site identification seems to be beneficial in awake procedures. The identification of language processing sites with IOI was also possible, but in the majority of cases significant differences between fMRI, IOI, and DES were visible, and therefore according to the authors' findings the IOI results are too unspecific to be useful for intraoperative decision-making with respect to exact language localization. For this purpose, DES mapping will remain the method of choice.Nevertheless, the IOI technique can provide additional value during the language mapping procedure with DES. Using a simple difference imaging approach, the authors were able to visualize and calculate the spatial extent of activation for each stimulation. This might enable surgeons in the future to optimize the mapping process. Additionally, differences between tumor and nontumor stimulation sites were observed with respect to the spatial extent of the changes in cortical optical properties. These findings provide further evidence that the method allows the assessment of the functional state of neurovascular coupling and is therefore suited for the delineation of pathologically altered tissue.

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.

Treatment with XAV-939 prevents in vitro calcification of human valvular interstitial cells.

The development of a substance or inhibitor-based treatment strategy for the prevention of aortic valve stenosis is a challenge and a main focus of medical research in this area. One strategy may be to use the tankyrase inhibitor XAV-939, which leads to Axin stabilisation and subsequent destruction of the ß-catenin complex and dephosphorylation of ß-catenin. The dephosphorylated active form of ß-catenin (non-phospho-ß-catenin) then promotes nuclear transcription that leads to osteogenesis. The aims of the present study were to develop an experimental system for inducing in vitro calcification of human aortic valvular interstitial cells (VICs) to investigate the potential anti-calcific effect of XAV-939 and to analyse expression of the Wnt signalling proteins and Sox9, a chondrogenesis regulator, in this model. Calcification of human VIC cultures was induced by cultivation in an osteogenic medium and the effect of co-incubation with 1µM XAV-939 was monitored. Calcification was quantified when mineral deposits were visible in culture and was histologically verified by von Kossa or Alizarin red staining and by IR-spectroscopy. Protein expression of alkaline phosphatase, Axin, ß-catenin and Sox9 were quantified by western blotting. In 58% of the VIC preparations, calcification was induced in an osteogenic culture medium and was accompanied by upregulation of alkaline phosphatase. The calcification induction was prevented by the XAV-939 co-treatment and the alkaline phosphatase upregulation was suppressed. As expected, Axin was upregulated, but the levels of active non-phospho-ß-catenin were also enhanced. Sox9 was induced during XAV-939 treatment but apparently not as a result of downregulation of ß-catenin signalling. XAV-939 was therefore able to prevent calcification of human VIC cultures, and XAV-939 treatment was accompanied by upregulation of active non-phospho-ß-catenin. Although XAV-939 does not downregulate active ß-catenin, treatment with XAV-939 results in Sox9 upregulation that may prevent the calcification process.

Intraoperative mapping of the sensory cortex by time-resolved thermal imaging.

The resection of brain tumor requires a precise distinction between eloquent areas of the brain and pathological tumor tissue in order to improve the extent of resection as well as the patient's progression free survival time. In this study, we discuss mathematical tools necessary to recognize neural activity using thermal imaging cameras. The main contribution to thermal radiation of the exposed human cortex is regional cerebral blood flow (CBF). In fact, neurovascular coupling links neural activity to changes in regional CBF which in turn affects the cortical temperature. We propose a statistically sound framework to visualize neural activity of the primary somatosensory cortex. The framework incorporates a priori known experimental conditions such as the thermal response to neural activity as well as unrelated effects induced by random neural activity and autoregulation. These experimental conditions can be adopted to certain electrical stimulation protocols so that the framework allows to unveil arbitrary evoked neural activity. The method was applied to semisynthetic as well as two intraoperative cases with promising results as we were able to map the eloquent sensory cortex with high sensitivity. Furthermore, the results were validated by anatomical localization and electrophysiological measurements.

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.

Rapid intra-operative diagnosis of kidney cancer by attenuated total reflection infrared spectroscopy of tissue smears.

Herein, a technique to analyze air-dried kidney tissue impression smears by means of attenuated total reflection infrared (ATR-IR) spectroscopy is presented. Spectral tumor markers-absorption bands of glycogen-are identified in the ATR-IR spectra of the kidney tissue smear samples. Thin kidney tissue cryo-sections currently used for IR spectroscopic analysis lack such spectral markers as the sample preparation causes irreversible molecular changes in the tissue. In particular, freeze-thaw cycle results in degradation of the glycogen and reduction or complete dissolution of its content. Supervised spectral classification was applied to the recorded spectra of the smears and the test spectra were classified with a high accuracy of 92% for normal tissue and 94% for tumor tissue, respectively. For further development, we propose that combination of the method with optical fiber ATR probes could potentially be used for rapid real-time intra-operative tissue analysis without interfering with either the established protocols of pathological examination or the ordinary workflow of operating surgeon. Such approach could ensure easier transition of the method to clinical applications where it may complement the results of gold standard histopathology examination and aid in more precise resection of kidney tumors.

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.

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.