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.

Highly sensitive and quick in ovo sexing of domestic chicken eggs by two-wavelength fluorescence spectroscopy.

The in ovo sexing of chicken eggs is a current task and a prerequisite to overcome the mass killing of male day-old chicks from laying lines. Although various methods have been developed and tested in recent years, practicable methods for sex determination are still missing which can be applicated in poultry hatcheries before the chicken embryo is capable of nociception and pain sensation. Optical spectroscopic methods enable an early determination of the sex. In this study, a novel method based on two-wavelength in ovo fluorescence excitation is described. More than 1600 eggs were examined. In ovo fluorescence was sequentially excited at 532 nm and 785 nm. The fluorescence intensities of the spectral regions behave inversely with respect to sex. It is shown that the observed sex-related differences in the fluorescence intensities are based on the embryonic hemoglobin synthesis. The accuracy of sex determination is 96% for both sexes. The hatching rate is not reduced compared to an equivalent reference group.

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.

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.

Brillouin and Raman imaging of domain walls in periodically-poled 5%-MgO:LiNbO3.

Recently, ferroelectric domain walls (DWs) have attracted considerable attention due to their intrinsic topological effects and their huge potential for optoelectronic applications. In contrast, many of the underlying physical properties and phenomena are not well characterized. In this regard, analyzing the vibrational properties, e.g. by Raman spectroscopy, provides direct access to the various local material properties, such as strains, defects or electric fields. While the optical phonon spectra of DWs have been widely investigated in the past, no reports on the acoustic phonon properties of DWs exist. In this work, we present a joint Raman and Brillouin visualization of ferroelectric DWs in the model ferroelectric lithium niobate. This is possible by using a combined Raman and virtually imaged phased array Brillouin setup. Here, we show that DWs can be visualized via frequency shifts observed in the acoustic phonons, as well. The observed contrast then is qualitatively explained by models adapted from Raman spectroscopy. This work, hence, provides a novel route to study ferroelectric DWs and their intrinsic mechanical properties.

Effects of Intermittent Treatment with Topical Corticosteroids and Calcineurin Inhibitors on Epidermal and Dermal Thickness Using Optical Coherence Tomography and Ultrasound.

INTRODUCTION: Proactive therapy with topical corticosteroids (TCSs) is the standard treatment for chronic inflammatory diseases such as atopic dermatitis; however, skin atrophy as TCS side effect remains a concern. METHODS: This 16-week, evaluator-blinded, within-patient placebo-controlled, randomized study enrolled volunteers with healthy skin conditions. For 12 weeks, their volar forearm and the back of their hand were applied with hydrocortisone acetate 1% cream (HC), methylprednisolone aceponate 0.1% cream (MPA), betamethasone valerate 0.1% cream (BMV), or an active agent-free base cream (Dermatop® Basiscreme) once daily twice weekly, and pimecrolimus 1% cream (PIM) twice daily twice weekly. Epidermal and dermal thickness was measured by optical coherence tomography and high-frequency ultrasound, respectively. Furthermore, skin atrophy and telangiectasia were determined by contact dermatoscopic photography (Dermaphot®). RESULTS: After 8 and 12 weeks, only BMV led to significant epidermal thinning on both sites. Four weeks after the end of treatment, epidermal thickness returned to baseline. No dermal thinning, atrophy, or telangiectasia was observed. CONCLUSIONS: MPA, HC, and PIM may be more suitable for repeated and prolonged treatment, especially in chronic diseases.

Quantifying the refractive index of ferroelectric domain walls in periodically poled LiNbO3 single crystals by polarization-sensitive optical coherence tomography.

Domain walls (DWs) in ferroelectric (FE) and multiferroic materials possess an ever-growing potential as integrated functional elements, for instance in optoelectronic nanodevices. Mandatory, however, is the profound knowledge of the local-scale electronic and optical properties, especially at DWs that are still incompletely characterized to date. Here, we quantify the refractive index of individual FE DWs in periodically-poled LiNbO3 (PPLN) single crystals. When applying polarization-sensitive optical coherence tomography (PS-OCT) at 1300 nm using circular light polarization, we are able to probe the relevant electro-optical properties close to and at the DWs, including also their ordinary and extraordinary contributions. When comparing to numerical calculations, we conclude that the DW signals recorded for ordinary and extraordinary polarization stem from an increased refractive index of at least Δn > 2·10-3 that originates from a tiny region of < 30 nm in width. PS-OCT hence provides an extremely valuable tool to decipher and quantify subtle changes of refractive index profiles for both inorganic and biomedical nanomaterial systems.

A metabolic switch regulates the transition between growth and diapause in C. elegans.

BACKGROUND: Metabolic activity alternates between high and low states during different stages of an organism's life cycle. During the transition from growth to quiescence, a major metabolic shift often occurs from oxidative phosphorylation to glycolysis and gluconeogenesis. We use the entry of Caenorhabditis elegans into the dauer larval stage, a developmentally arrested stage formed in response to harsh environmental conditions, as a model to study the global metabolic changes and underlying molecular mechanisms associated with growth to quiescence transition. RESULTS: Here, we show that the metabolic switch involves the concerted activity of several regulatory pathways. Whereas the steroid hormone receptor DAF-12 controls dauer morphogenesis, the insulin pathway maintains low energy expenditure through DAF-16/FoxO, which also requires AAK-2/AMPKα. DAF-12 and AAK-2 separately promote a shift in the molar ratios between competing enzymes at two key branch points within the central carbon metabolic pathway diverting carbon atoms from the TCA cycle and directing them to gluconeogenesis. When both AAK-2 and DAF-12 are suppressed, the TCA cycle is active and the developmental arrest is bypassed. CONCLUSIONS: The metabolic status of each developmental stage is defined by stoichiometric ratios within the constellation of metabolic enzymes driving metabolic flux and controls the transition between growth and quiescence.

High-dynamic-range areal profilometry using an imaging, dispersion-encoded low-coherence interferometer.

This work presents the design and characterization of an approach for areal surface profilometry with sub-nm axial resolution. The developed approach is based on a low-coherence interferometer enhanced by an dispersive element to control the axial resolution and measurement range. Optical path differences are detected by an imaging spectrometer where equalization wavelengths are determined as a basis for fitting spectra. This enables the acquisition of surface profiles with a length of up to 1.5 mm without mechanical scanning where a minimal resolution of 0.1 nm in an axial measurement range of nearly 80 µm was achieved. The resolution calculation was based on the standard deviation of measured feature heights. In addition to the system design, its capabilities are demonstrated on samples such as height standards.

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.

Spatially Resolved Cross-Linking Characterization by Imaging Low-Coherence Interferometry.

A method to characterize cross-linking differences in polymers such as waveguide polymers has been developed. The method is based on the scan-free information acquisition utilizing a low-coherence interferometer in conjunction with an imaging spectrometer. By the introduction of a novel analyzing algorithm, the recorded spectral-phase data was interpreted as wavelength-dependent optical thickness which is matchable with the refractive index and therefore with the degree of cross-linking. In the course of this work, the method was described in its hardware and algorithmic implementation as well as in its accuracy. Comparative measurements and error estimations showed an accuracy in the range of 10-6 in terms of the refractive index. Finally, photo-lithographically produced samples with laterally defined cross-linking differences have been characterized. It could be shown, that differences in the optical thickness of ±1.5 µm are distinguishable.

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.

Advanced analysis of domain walls in Mg doped LiNbO3 crystals with high resolution OCT.

The structure of domain walls (DW) in ferroelectric media is of great interest as this material is used for frequency doublers and other applications. We show that the structure of the DWs can nicely be visualized by high resolution optical coherence tomography (OCT). While the high group refractive index of lithium niobate allows a resolution much better than 1 µm, the large dispersion can blur the image and has to be compensated. Therefore, we developed an adaptive dispersion compensation algorithm based on maximizing the intensity of the DWs. By measuring a group of DWs, the mean period of the DWs could be measured with an accuracy of less than 10 nm differentiating samples with only 30 nm distinct periods. By analyzing the peak position, amplitude and phase shift within a DW, we were able to determine steps in the DW of only 50 nm. Furthermore, the inclined course of the DWs in a fan-shaped frequency doubler could be displayed. Therefore, we conclude that OCT is able to provide valuable information about the structure of domain walls in periodically poled lithium niobate (PPLN).

Label-free multiphoton microscopy reveals altered tissue architecture in hippocampal sclerosis.

The properties and structure of tissue can be visualized without labeling or preparation by multiphoton microscopy combining coherent anti-Stokes Raman scattering (CARS), addressing lipid content, second harmonic generation (SHG) showing collagen, and two-photon excited fluorescence (TPEF) of endogenous fluorophores. We compared samples of sclerotic and nonsclerotic human hippocampus to detect pathologic changes in the brain of patients with pharmacoresistant temporomesial epilepsy (n = 15). Multiphoton microscopy of cryosections and bulk tissue revealed hippocampal layering and micromorphologic details in accordance with reference histology: CARS displayed white and gray matter layering and allowed the assessment of axonal myelin. SHG visualized blood vessels based on adventitial collagen. In addition, corpora amylacea (CoA) were found to be SHG-active. Pyramidal cell bodies were characterized by intense cytoplasmic endogenous TPEF. Furthermore, diffuse TPEF around blood vessels was observed that co-localized with positive albumin immunohistochemistry and might indicate degeneration-associated vascular leakage. We present a label-free and fast optical approach that analyzes pathologic aspects of HS. Hippocampal layering, loss of pyramidal cells, and presence of CoA indicative of sclerosis are visualized. Label-free multiphoton microscopy has the potential to extend the histopathologic armamentarium for ex vivo assessment of changes of the hippocampal formation on fresh tissue and prospectively in vivo.

In ovo sexing of chicken eggs by fluorescence spectroscopy.

Culling of day-old male chicks in production of laying hen strains involves several millions of animals every year worldwide and is ethically controversial. In an attempt to provide an alternative, optical spectroscopy was investigated to determine nondestructively in ovo the sex of early embryos of the domestic chicken. The extraembryonic blood circulation system was accessed by producing a window in the egg shell and the flowing blood was illuminated with a near-infrared laser. The strong fluorescence and the weak Raman signals were acquired and spectroscopically analyzed between 800 and 1000 nm. The increase of fluorescence intensity between 3.5 and 11.5 days of incubation was found to be in agreement with the erythropoietic stages, thus enabling to identify hemoglobin as fluorescence source. Sex-related differences in the fluorescence spectrum were found at day 3.5, and principal component (PC) analysis showed that the blood of males was characterized by a specific fluorescence band located at ∼910 nm. Supervised classification of the PC scores enabled the determination of the sex of 380 eggs at day 3.5 of incubation with a correct rate up to 93% by combining the information derived from both fluorescence and Raman scattering. Graphical abstract The fluorescence of blood obtained in ovo by illumination of embryonic vessels with a IR laser displays spectral differences that can be employed for sexing of eggs in early stage of incubation, before onset of embryo sensitivity and without hindering its development into a healthy chick.

Impact of a detector dead time in phase-resolved Doppler analysis using spectral domain optical coherence tomography.

For any oblique sample movement containing a transverse velocity component, the commonly used linear relationship between the phase shift and the axial velocity component is erroneous for spectrometer-based optical coherence tomography (spectral domain OCT, SD-OCT). We recently proposed a new Doppler model that assumes a continuous integration of the photocurrent. In this research, we extend the model for detectors with a shutter control by taking detector dead time into account. We present the new relation between phase shift and oblique sample displacement as well as the correlation of the phases of consecutive depth scans, in dependency on the detector dead times ranging from 5% to 90%, as numerically calculated universal contour plots, which are valid for any center wavelength and sample beam size. We found that detector dead time is recommended, especially for oblique sample motion. The reason for this recommendation is the achieved linear relation between the phase shift and the axial velocity component in the velocity range relevant for in vivo measurements, despite the signal damping due to shorter exposure time of the line detector. The theoretical Doppler model is verified using a 1% Intralipid flow phantom model. Because of the results of this research, we believe future measurements in Doppler SD-OCT can be more simple and more accurate by setting a shutter control for the line detector used.

Acute Lung Injury Causes Asynchronous Alveolar Ventilation That Can Be Corrected by Individual Sighs.

RATIONALE: Improved ventilation strategies have been the mainstay for reducing mortality in acute respiratory distress syndrome. Their unique clinical effectiveness is, however, unmatched by our understanding of the underlying mechanobiology, and their impact on alveolar dynamics and gas exchange remains largely speculative. OBJECTIVES: To assess changes in alveolar dynamics and associated effects on local gas exchange in experimental models of acute lung injury (ALI) and their responsiveness to sighs. METHODS: Alveolar dynamics and local gas exchange were studied in vivo by darkfield microscopy and multispectral oximetry in experimental murine models of ALI induced by hydrochloric acid, Tween instillation, or in antibody-mediated transfusion-related ALI. MEASUREMENTS AND MAIN RESULTS: Independent of injury mode, ALI resulted in asynchronous alveolar ventilation characteristic of alveolar pendelluft, which either spontaneously resolved or progressed to a complete cessation or even inversion of alveolar ventilation. The functional relevance of the latter phenomena was evident as impaired blood oxygenation in juxtaposed lung capillaries. Individual sighs (2 × 10 s at inspiratory plateau pressure of 30 cm H2O) largely restored normal alveolar dynamics and gas exchange in acid-induced ALI, yet not in Tween-induced surfactant depletion. CONCLUSIONS: We describe for the first time in detail the different forms and temporal sequence of impaired alveolar dynamics in the acutely injured lung and report the first direct visualization of alveolar pendelluft. Moreover, we identify individual sighs as an effective strategy to restore intact alveolar ventilation by a mechanism independent of alveolar collapse and reopening.

NADPH oxidase 4 protects against development of endothelial dysfunction and atherosclerosis in LDL receptor deficient mice.

AIMS: Endothelial dysfunction is an early step in the development of atherosclerosis. Increased formation of superoxide anions by NADPH oxidase Nox1, 2, and 5 reduces nitric oxide availability and can promote endothelial dysfunction. In contrast, recent evidence supports a vasoprotective role of H2O2 produced by main endothelial isoform Nox4. Therefore, we analysed the impact of genetic deletion of Nox4 on endothelial dysfunction and atherosclerosis in the low-density lipoprotein receptor (Ldlr) knockout model. METHODS AND RESULTS: Ex vivo analysis of endothelial function by Mulvany myograph showed impaired endothelial function in thoracic aorta of Nox4(-/-)/Ldlr(-/-) mice. Further progression of endothelial dysfunction due to high-fat diet increased atherosclerotic plaque burden and galectin-3 staining in Nox4(-/-)/Ldlr(-/-) mice compared with Ldlr(-/-) mice. Under physiological conditions, loss of Nox4 does not influence aortic vascular function. In this setting, loss of Nox4-derived H2O2 production could be partially compensated for by nNOS upregulation. Using an innovative optical coherence tomography approach, we were able to analyse endothelial function by flow-mediated vasodilation in the murine saphenous artery in vivo. This new approach revealed an altered flow-mediated dilation in Nox4(-/-) mice, indicating a role for Nox4 under physiological conditions in peripheral arteries in vivo. CONCLUSIONS: Nox4 plays an important role in maintaining endothelial function under physiological and pathological conditions. Loss of Nox4-derived H2O2 could be partially compensated for by nNOS upregulation, but severe endothelial dysfunction is not reversible. This leads to increased atherosclerosis under atherosclerotic prone conditions.

In Ovo Sexing of Domestic Chicken Eggs by Raman Spectroscopy.

Male birds of egg-laying hen strains have no commercial value and are culled immediately after hatching, raising concerns for animal welfare. Existing experimental methods for in ovo sexing require taking samples and are applicable after embryos' sexual differentiation. We demonstrate that Raman spectroscopy enables contactless in ovo sex determination of the domestic chicken (Gallus gallus f. dom.) already at day 3.5 of egg incubation. A sexing accuracy of 90% was obtained by analyzing the spectra of blood circulating in the extraembryonic vessels. The measurement is damage-free and barely affects the hatching rate. Sex recognition is achieved before the onset of sensitivity. Therefore, Raman spectroscopy provides an alternative to the culling of 1-day-old male chicks in laying hen production.

Relation of joint spectral and time domain optical coherence tomography (jSTdOCT) and phase-resolved Doppler OCT.

A variety of promising approaches for quantitative flow velocity measurement in OCT have been proposed in recent years. The question is: Which method gets the most precise flow velocity out of the interference signals detected. We have compared the promising joint spectral and time domain optical coherence tomography (jSTdOCT) and the commonly used phase-resolved Doppler OCT (DOCT) and describe the link between these two proven methods for OCT in the Fourier domain (FD OCT). First, we show that jSTdOCT can be significantly improved by calculating the center of gravity via an unbiased complex algorithm instead of detecting the maximum intensity signal of the broadened Doppler frequency spectrum. Secondly, we introduce a unified mathematical description for DOCT and jSTdOCT that differs only in one exponent and call it enhjSTdOCT. Third, we present that enhjSTdOCT has the potential to significantly reduce the noise of the velocity measurement by choosing an exponent depending on the transverse sample velocity component and the signal-to-noise ratio. EnhjSTdOCT is verified numerically and experimentally to find the optimal parameters for maximal velocity noise reduction.