Diagnosing and Mapping Pulmonary Emphysema on X-Ray Projection Images: Incremental Value of Grating-Based X-Ray Dark-Field Imaging

Purpose

To assess whether grating-based X-ray dark-field imaging can increase the sensitivity of X-ray projection images in the diagnosis of pulmonary emphysema and allow for a more accurate assessment of emphysema distribution.

Materials and Methods

Lungs from three mice with pulmonary emphysema and three healthy mice were imaged ex vivo using a laser-driven compact synchrotron X-ray source. Median signal intensities of transmission (T), dark-field (V) and a combined parameter (normalized scatter) were compared between emphysema and control group. To determine the diagnostic value of each parameter in differentiating between healthy and emphysematous lung tissue, a receiver-operating-characteristic (ROC) curve analysis was performed both on a per-pixel and a per-individual basis. Parametric maps of emphysema distribution were generated using transmission, dark-field and normalized scatter signal and correlated with histopathology.

Results

Transmission values relative to water were higher for emphysematous lungs than for control lungs (1.11 vs. 1.06, p<0.001). There was no difference in median dark-field signal intensities between both groups (0.66 vs. 0.66). Median normalized scatter was significantly lower in the emphysematous lungs compared to controls (4.9 vs. 10.8, p<0.001), and was the best parameter for differentiation of healthy vs. emphysematous lung tissue. In a per-pixel analysis, the area under the ROC curve (AUC) for the normalized scatter value was significantly higher than for transmission (0.86 vs. 0.78, p<0.001) and dark-field value (0.86 vs. 0.52, p<0.001) alone. Normalized scatter showed very high sensitivity for a wide range of specificity values (94% sensitivity at 75% specificity). Using the normalized scatter signal to display the regional distribution of emphysema provides color-coded parametric maps, which show the best correlation with histopathology.

Conclusion

In a murine model, the complementary information provided by X-ray transmission and dark-field images adds incremental diagnostic value in detecting pulmonary emphysema and visualizing its regional distribution as compared to conventional X-ray projections.     

Dark-Field Imaging: Recent developments and potential clinical applications

This paper describes an X-ray phase contrast imaging technique using analyzer-based optics called X-ray Dark-Field Imaging that has been under development for the past 10 years. We describe the theory behind XDFI, the X-ray optics required for implementing it in practice, and algorithms used for 2D, 2.5D, and 3D image reconstruction. The XDFI optical chain consists of an asymmetrically cut, Bragg-type monochromator-collimator that provides a planar monochromatic X-ray beam, a positioning stage for the specimens, a Laue-case angle analyzer, and one or two cameras to capture the dark and bright field images. We demonstrate the soft-tissue discrimination capabilities of XDFI by reconstructing images with absorption and phase contrast. By using a variety of specimens such as breast tissue with cancer, joints with articular cartilage, ex-vivo human eye specimen, and others, we show that refraction-based contrast derived from XDFI is more effective in characterizing anatomical features, articular pathology, and neoplastic disease than conventional absorption-based images. For example, XDFI of breast tissue can discriminate between the normal and diseased terminal duct lobular unit, and between invasive and in-situ cancer. The final section of this paper is devoted to potential future developments to enable clinical and histo-pathological applications of this technique.     

Imaging Liver Lesions Using Grating-Based Phase-Contrast Computed Tomography with Bi-Lateral Filter Post-Processing

X-ray phase-contrast imaging shows improved soft-tissue contrast compared to standard absorption-based X-ray imaging. Especially the grating-based method seems to be one promising candidate for clinical implementation due to its extendibility to standard laboratory X-ray sources. Therefore the purpose of our study was to evaluate the potential of grating-based phase-contrast computed tomography in combination with a novel bi-lateral denoising method for imaging of focal liver lesions in an ex vivo feasibility study. Our study shows that grating-based phase-contrast CT (PCCT) significantly increases the soft-tissue contrast in the ex vivo liver specimens. Combining the information of both signals – absorption and phase-contrast – the bi-lateral filtering leads to an improvement of lesion detectability and higher contrast-to-noise ratios. The normal and the pathological tissue can be clearly delineated and even internal structures of the pathological tissue can be visualized, being invisible in the absorption-based CT alone. Histopathology confirmed the presence of the corresponding findings in the analyzed tissue. The results give strong evidence for a sufficiently high contrast for different liver lesions using non-contrast-enhanced PCCT. Thus, ex vivo imaging of liver lesions is possible with a polychromatic X-ray source and at a spatial resolution of ∼100 µm. The post-processing with the novel bi-lateral denoising method improves the image quality by combining the information from the absorption and the phase-contrast images.     

Label-Free Single-Particle Imaging of the Influenza Virus by Objective-Type Total Internal Reflection Dark-Field Microscopy

Here we report label-free optical imaging of single particles of the influenza virus attached on a glass surface with a simple objective-type total internal reflection dark-field microscopy (TIRDFM). The capability of TIRDFM for the imaging of single viral particles was confirmed from fine correlation of the TIRDFM images with fluorescent immunostaining image and scanning electron microscopy image. The density of scattering spots in the TIRDFM images showed a good linearity against the virus concentration, giving the limit of detection as 1.2×10⁴ plaque-forming units per milliliter. Our label-free optical imaging method does require neither elaborated sample preparation nor complex optical systems, offering a good platform for rapid and sensitive counting of viral particles.     

Efficient Decoding of 2D Structured Illumination with Linear Phase Stepping in X-Ray Phase Contrast and Dark-Field Imaging

The ability to map the phase distribution and lateral coherence of an x-ray wavefront offers the potential for imaging the human body through phase contrast, without the need to deposit significant radiation energy. The classic means to achieve this goal is structured illumination, in which a periodic intensity modulation is introduced into the image, and changes in the phase distribution of the wavefront are detected as distortions of the modulation pattern. Two-dimensional periodic patterns are needed to fully characterize a transverse wavefront. Traditionally, the information in a 2D pattern is retrieved at high resolution by acquiring multiple images while shifting the pattern over a 2D matrix of positions. Here we describe a method to decode 2D periodic patterns with single-axis phase stepping, without either a loss of information or increasing the number of sampling steps. The method is created to reduce the instrumentation complexity of high-resolution 2D wavefront sensing in general. It is demonstrated with motionless electromagnetic phase stepping and a flexible processing algorithm in x-ray dark-field and phase contrast imaging.     

Imaging Lignin-Downregulated Alfalfa Using Coherent Anti-Stokes Raman Scattering Microscopy

Targeted lignin modification in bioenergy crops could potentially improve conversion efficiency of lignocellulosic biomass to biofuels. To better assess the impact of lignin modification on overall cell wall structure, wild-type and lignin-downregulated alfalfa lines were imaged using coherent anti-Stokes Raman scattering (CARS) microscopy. The 1,600-cm^?1 Raman mode was used in CARS imaging to specifically represent the lignin signal in the plant cell walls. The intensities of the CARS signal follow the general trend of lignin contents in cell walls from both wild-type and lignin-downregulated plants. In the downregulated lines, the overall reduction of lignin content agreed with the previously reported chemical composition. However, greater reduction of lignin content in cell corners was observed by CARS imaging, which could account for the enhanced susceptibility to chemical and enzymatic hydrolysis observed previously.     

Imaging of Lipids in Microalgae with Coherent Anti-Stokes Raman Scattering Microscopy

Microalgae have great prospects as a sustainable resource of lipids for refinement into nutraceuticals and biodiesel, which increases the need for detailed insights into their intracellular lipid synthesis/storage mechanisms. As an alternative strategy to solvent- and label-based lipid quantification techniques, we introduce time-gated coherent anti-Stokes Raman scattering (CARS) microscopy for monitoring lipid contents in living algae, despite strong autofluorescence from the chloroplasts, at approximately picogram and subcellular levels by probing inherent molecular vibrations. Intracellular lipid droplet synthesis was followed in Phaeodactylum tricornutum algae grown under (1) light/nutrient-replete (control [Ctrl]), (2) light-limited (LL), and (3) nitrogen-starved (NS) conditions. Good correlation (r² = 0.924) was found between lipid volume data yielded by CARS microscopy and total fatty acid content obtained from gas chromatography-mass spectrometry analysis. In Ctrl and LL cells, micron-sized lipid droplets were found to increase in number throughout the growth phases, particularly in the stationary phase. During more excessive lipid accumulation, as observed in NS cells, promising commercial harvest as biofuels and nutritional lipids, several micron-sized droplets were present already initially during cultivation, which then fused into a single giant droplet toward stationary phase alongside with new droplets emerging. CARS microspectroscopy further indicated lower lipid fluidity in NS cells than in Ctrl and LL cells, potentially due to higher fatty acid saturation. This agreed with the fatty acid profiles gathered by gas chromatography-mass spectrometry. CARS microscopy could thus provide quantitative and semiqualitative data at the single-cell level along with important insights into lipid-accumulating mechanisms, here revealing two different modes for normal and excessive lipid accumulation.The accumulation of lipids in microalgae is currently a field of intense research: with their high photosynthetic efficiency and rapid growth rates, these organisms hold great potential both for sustainable production of biofuels (Chisti, 2007) and as a nutrition source (de Jesus Raposo et al., 2013). As in all living cells, lipids in microalgae are present in membranes, such as the plasma and organelle membranes. Some microalgae also accumulate lipids, mainly triacylglycerols, in intracellular droplets (De Martino et al., 2011; White et al., 2012). One such microalga is Phaeodactylum tricornutum, a unicellular photoautotrophic diatom and a well-studied model organism. It has a sequenced genome and is known to produce long-chain n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA), and small amounts of docosahexaenoic acid (DHA; Alonso et al., 1998). The long-chain n-3 PUFA-producing properties has made it particularly interesting within the areas of functional food and nutraceuticals. Under conditions of nitrogen starvation, P. tricornutum accumulates larger amounts of fatty acids, albeit of the more saturated nature (Yongmanitchai and Ward, 1991). However, in response to low irradiance, P. tricornutum has been reported to increase its content of PUFAs, especially EPA (Thompson et al., 1990). In order to optimize strain selection and algal cultivation conditions in relation to lipid accumulation/lipid profile, accurate tools to quantify total lipids as well as the degree of unsaturation are required.Solvent extractions followed by methylation, gas chromatography coupled to flame ionization detection, and gas chromatography-mass spectrometry (GC-MS) detection belong to the standard techniques used for algal lipid analysis. However, these are cumbersome and require relatively large amounts of solvents and sample. The resulting quantitative information on lipid amounts is related to total cell mass, which may introduce artifacts, as the cell mass is also influenced by other metabolic parameters. Furthermore, these bioanalytical techniques provide rough population averages without information on the intracellular location or distribution. As an alternative, microscopy techniques are increasingly being used, with the benefit that lipid droplets can be evaluated directly in single cells with high precision. Fluorescence microscopy is the most widespread technique, relying on lipid-specific fluorescent markers: Nile Red is commonly used, but its poor permeability through the cell walls causes difficulties, as does its nonspecific binding (Chen et al., 2009). Other fluorophores like BODIPY 505/515 also have been suggested for live-cell studies (Cooper et al., 2010; Wong and Franz, 2013). Still, invasive techniques are needed, requiring solvents to facilitate the transport of the labeling molecules into the cell, potentially inducing stress responses that may affect cell metabolism. Furthermore, there is little knowledge available on how the accumulation of lipophilic dyes in lipid droplets and the fluorescence emission are influenced by environmental conditions such as temperature, pH, and deposited light doses. It has also been shown that the fluorescence intensity emitted from the dyes cannot be related directly to the local lipid concentration, excluding quantitative measurements (De la Hoz Siegler et al., 2012). In algae/plant cell biology, the applicability of fluorescence microscopy is also limited due to the fact that algae/plant cells generate strong autofluorescence, potentially interfering with the lipid/fatty acid signals. In order to take algal lipid quantification one step further, microscopy techniques, not being dependent on exogenous fluorophores and allowing efficient separation of the lipid/fatty acid signal from the autofluorescence, are desirable.In label-free coherent anti-Stokes Raman scattering (CARS) microscopy, images are formed by probing intrinsic molecular vibrations through a nonlinear four-wave-mixing process (Cheng and Xie, 2004). Briefly, the frequency difference of two coherent near-infrared excitation beams (the pump beam at shorter wavelengths and the Stokes beam at longer wavelengths) are tuned to match the frequency of the target molecular vibration_. As a result, resonant oscillators are coherently driven in the sample focal volume and an enhanced blue-shifted CARS signal is generated. Due to the nonlinear nature of the CARS process, emission is generated only in the high-intensity region of focused laser beams, allowing optical sectioning of the specimen. By tuning the frequency difference of the excitation beams to match the resonance frequency of carbon-hydrogen (C-H) vibrations, especially abundant in lipids, three-dimensional images of lipids can be recorded without any staining (Enejder et al., 2010). As the CARS emission scales with the square of the concentration of C-H bonds, quantitative data on intracellular amounts of lipids can be extracted (Cheng and Xie, 2004). However, cells with chromophores, such as algae and plant cells, generate exceptionally strong two-photon fluorescence, the spectral tails of which tend to bleed through the most efficient optical filters typically used for separation of the CARS signal. As an alternative approach, we have incorporated a time-correlated single-photon counting system (Enejder et al., 2010), allowing us to distinguish the long-decay fluorescence component from the instant CARS signal by time gating.The capability of conventional CARS imaging for microalgae was recently demonstrated with proof-of-principle data, showing that individual, larger lipid droplets can be resolved visually, in contrast to conventional Raman microscopy (He et al., 2012). In this study, we introduce CARS microscopy with time-gated detection, also enabling the identification of subpicogram lipid-rich regions in the vicinity of strongly autofluorescent chloroplasts. This is particularly important because cellular storage lipids in algae are primarily synthesized in the chloroplasts and then budded off from the envelope membranes as nascent lipid droplets (Fan et al., 2011). Hence, time-gated CARS microscopy paves the way for high-precision quantification of the complete intracellular distribution of lipid stores, including the emerging droplets within and adjacent to chloroplasts. We show the feasibility of the approach for quantitative lipid analysis of large populations of living P. tricornutum cultivated under three different growth conditions: a control (Ctrl) group, a light-limited (LL) group to increase the EPA level, and a nitrogen-starved (NS) group to increase total lipid accumulation. We studied the lipid metabolism in approximately 100 cells per category over time and report detailed visual information on the dynamics of lipid droplet formation throughout a life cycle from budding droplets to, in some cases, giant lipid stores. We show that biologically relevant quantitative and qualitative data on intracellular lipid stores can be extracted at a precision of less than 1 pg cell⁻¹ despite adjacent chromophores. Data extracted from the CARS images are further compared with solvent extraction and quantification of fatty acids using GC-MS. We further illustrate the potential to assess whether the different growth conditions promote the synthesis of more PUFAs by detecting shifts in lipid fluidity and saturation per individual lipid droplet from CARS C-H vibration ratio images.     

Pluronic Triblock Copolymer Encapsulated Gold Nanorods as Biocompatible Localized Plasmon Resonance-Enhanced Scattering Probes for Dark-Field Imaging of Cancer Cells

Gold nanorods (GNR) are synthesized using cetylmethylammonium bromide (CTAB) surfactants which function as structure-directing agents. However, CTAB forms a tightly bound cationic bilayer on GNR surface with the cationic trimethylammonium head group exposed to the aqueous media, which is known to be highly toxic in vitro and in vivo. Pluronic is a non-ionic triblock polymer, which can associate with CTAB and form stable CTAB?Cpolymer complexes due to hydrophobic interactions. In this work, two types of Pluronic triblock copolymers were used to encapsulate GNR to reduce their cytotoxicity and improve colloidal and optical stability for biological applications. These formulations were characterized by UV?Cvis absorption spectra analysis, transmission electron microscopy, cell viability studies, differential interference contrast microscopy and dark-field imaging.     

Coherent X-Ray Imaging of Collagen Fibril Distributions within Intact Tendons

The characterization of the structure of highly hierarchical biosamples such as collagen-based tissues at the scale of tens of nanometers is essential to correlate the tissue structure with its growth processes. Coherent x-ray Bragg ptychography is an innovative imaging technique that gives high resolution images of the ordered parts of such samples. Herein, we report how we used this method to image the collagen fibrillar ultrastructure of intact rat tail tendons. The images show ordered fibrils extending over 10–20 μm in length, with a quantifiable D-banding spacing variation of 0.2%. Occasional defects in the fibrils distribution have also been observed, likely indicating fibrillar fusion events.     

基于单分子量子相干调制的细胞温度成像技术

目的 细胞温度成像可以帮助科学家研究和理解细胞内部的温度分布,揭示细胞代谢和生物化学过程的关键信息。目前,基于荧光温度探针的细胞温度成像技术存在低温度分辨率和有限测量范围等限制。本文旨在利用单分子量子相干过程依赖温度的特性,开发一种单细胞温度成像和实时检测技术。方法 基于飞秒脉冲激光制备延时和相位可调的飞秒脉冲对,调制的脉冲对通过显微系统激发细胞内标记的荧光单分子,之后收集并记录每个荧光光子的到达时间。利用单分子相干过程与周围环境温度的关系,定义单分子量子相干可视度（V）,建立V与环境温度的对应关系。通过调制解调荧光光子的到达时间,获取单分子周围环境温度,结合扫描成像,实现细胞的温度成像和实时检测。结果 该方法可以实现高精度（温度分辨率<0.1℃）和大范围温度（10～50℃）的温度成像和测量,并观测到了单个细胞代谢相关的温度变化。结论 该研究有助于深入了解细胞代谢、蛋白质功能和疾病机制,为生物医学研究提供重要工具。     

Tomography of a Cryo-immobilized Yeast Cell Using Ptychographic Coherent X-Ray Diffractive Imaging

The structural investigation of noncrystalline, soft biological matter using x-rays is of rapidly increasing interest. Large-scale x-ray sources, such as synchrotrons and x-ray free electron lasers, are becoming ever brighter and make the study of such weakly scattering materials more feasible. Variants of coherent diffractive imaging (CDI) are particularly attractive, as the absence of an objective lens between sample and detector ensures that no x-ray photons scattered by a sample are lost in a limited-efficiency imaging system. Furthermore, the reconstructed complex image contains quantitative density information, most directly accessible through its phase, which is proportional to the projected electron density of the sample. If applied in three dimensions, CDI can thus recover the sample''s electron density distribution. As the extension to three dimensions is accompanied by a considerable dose applied to the sample, cryogenic cooling is necessary to optimize the structural preservation of a unique sample in the beam. This, however, imposes considerable technical challenges on the experimental realization. Here, we show a route toward the solution of these challenges using ptychographic CDI (PCDI), a scanning variant of coherent imaging. We present an experimental demonstration of the combination of three-dimensional structure determination through PCDI with a cryogenically cooled biological sample—a budding yeast cell (Saccharomyces cerevisiae)—using hard (7.9 keV) synchrotron x-rays. This proof-of-principle demonstration in particular illustrates the potential of PCDI for highly sensitive, quantitative three-dimensional density determination of cryogenically cooled, hydrated, and unstained biological matter and paves the way to future studies of unique, nonreproducible biological cells at higher resolution.     

萤火虫复眼光学模型重叠成象的研究与模拟

以北京萤火虫复眼结构与生理参数为依据,基于已知的梯度折射率透镜特征,对复眼的整个光学系统进行模型化.模型的尺寸与折射率分布依据实际晶锥的测量值.通过对模型复眼的光线追迹,证明北京萤火虫复眼是一个折射重叠型复眼.重叠孔径以内入射的平行光线在视网膜水平形成一半宽度为一定值的模糊圈.利用计算机光线追迹模拟了复眼的重叠成象过程,随着象平面与晶锥近端距离的增加,信号光线的数目也逐渐增加,信噪比逐渐提高,在视网膜水平达到最大值.     

The Use of Dark-Field Microscopy for the Visualization of Acetylcholinesterase in Cholinergic Neurons

Acetylcholinesterase (ACEase) was investigated histochemically in hearts, kidneys and urinary bladders of the following animals: man, dog, cat, hamster, rat, tree shrew (Tupaia glis) and lesser bush baby (Galago senegalensis). Slides examined with both bright-field and dark-field illumination showed that use of dark-field microscopy greatly enhanced the visibility of sites of ACEase activity. ACEase-positive nerve fibers which had low enzyme activity were difficult to identify by brght-field, due to their pale staining, but these fibers were easily identified as iridescent structures in the dark-field.     

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity ¹. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations ^1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted ³. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals ⁴. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm^-1 CH stretch vibration in lipids and the 2120 cm^-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule''s vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.     

Analytic 3D Imaging of Mammalian Nucleus at Nanoscale Using Coherent X-Rays and Optical Fluorescence Microscopy

Despite the notable progress that has been made with nano-bio imaging probes, quantitative nanoscale imaging of multistructured specimens such as mammalian cells remains challenging due to their inherent structural complexity. Here, we successfully performed three-dimensional (3D) imaging of mammalian nuclei by combining coherent x-ray diffraction microscopy, explicitly visualizing nuclear substructures at several tens of nanometer resolution, and optical fluorescence microscopy, cross confirming the substructures with immunostaining. This demonstrates the successful application of coherent x-rays to obtain the 3D ultrastructure of mammalian nuclei and establishes a solid route to nanoscale imaging of complex specimens.     

Analytic 3D Imaging of Mammalian Nucleus at Nanoscale Using Coherent X-Rays and Optical Fluorescence Microscopy

Despite the notable progress that has been made with nano-bio imaging probes, quantitative nanoscale imaging of multistructured specimens such as mammalian cells remains challenging due to their inherent structural complexity. Here, we successfully performed three-dimensional (3D) imaging of mammalian nuclei by combining coherent x-ray diffraction microscopy, explicitly visualizing nuclear substructures at several tens of nanometer resolution, and optical fluorescence microscopy, cross confirming the substructures with immunostaining. This demonstrates the successful application of coherent x-rays to obtain the 3D ultrastructure of mammalian nuclei and establishes a solid route to nanoscale imaging of complex specimens.     

Dynamic Imaging of Coherent Sources Reveals Different Network Connectivity Underlying the Generation and Perpetuation of Epileptic Seizures

The concept of focal epilepsies includes a seizure origin in brain regions with hyper synchronous activity (epileptogenic zone and seizure onset zone) and a complex epileptic network of different brain areas involved in the generation, propagation, and modulation of seizures. The purpose of this work was to study functional and effective connectivity between regions involved in networks of epileptic seizures. The beginning and middle part of focal seizures from ictal surface EEG data were analyzed using dynamic imaging of coherent sources (DICS), an inverse solution in the frequency domain which describes neuronal networks and coherences of oscillatory brain activities. The information flow (effective connectivity) between coherent sources was investigated using the renormalized partial directed coherence (RPDC) method. In 8/11 patients, the first and second source of epileptic activity as found by DICS were concordant with the operative resection site; these patients became seizure free after epilepsy surgery. In the remaining 3 patients, the results of DICS / RPDC calculations and the resection site were discordant; these patients had a poorer post-operative outcome. The first sources as found by DICS were located predominantly in cortical structures; subsequent sources included some subcortical structures: thalamus, Nucl. Subthalamicus and cerebellum. DICS seems to be a powerful tool to define the seizure onset zone and the epileptic networks involved. Seizure generation seems to be related to the propagation of epileptic activity from the primary source in the seizure onset zone, and maintenance of seizures is attributed to the perpetuation of epileptic activity between nodes in the epileptic network. Despite of these promising results, this proof of principle study needs further confirmation prior to the use of the described methods in the clinical praxis.     

Directional cell migration in vivo

Directional cell migration is essential for almost all organisms during embryonic development, in adult life and contributes to pathological conditions. This is particularly critical during embryogenesis where it is essential that cells end up in their correct, precise locations in order to build a normal embryo. Many cells have solved this problem by following a gradient of a chemoattractant usually secreted by their target tissues. Our recent research has found an alternative, complimentary, mechanism where intracellular signals are able to generate cell polarity and directional migration in absence of any external chemoattactant. We used neural crest cells to study cell migration in vivo, by performing live imagining of the neural crest cell migrating during embryo development. We show that the Planar Cell Polarity (PCP) or non-canonical Wnt signaling pathway interacts with the proteoglycan syndecan-4 to control the direction in which cell protrusions are generated, and in consequence, the direction of migration. By analyzing the activity of the small GTPases using in vivo FRET imaging we showed that PCP signaling activates RhoA, while syndecan-4 inhibits Rac, both at the back of the neural crest cell. Here we discuss a model where these signals are integrated to generate directional migration in vivo.     

Coherent scattering in multi-harmonic light microscopy

By focusing a pulsed laser beam into a sample, harmonic up-conversion can be generated as well as multi-photon excited fluorescence. Whereas multi-photon excited fluorescence microscopy is well established, the use of multi-harmonic generation for three-dimensional image contrast is very recent. Both techniques can provide similar resolution and, for adequate radiating source density, comparable signal levels, allowing them to be combined in a single versatile instrument. However, harmonic generation differs fundamentally from fluorescence generation in that it is coherent and produces radiation patterns that are highly sensitive to phase. As such, multi-harmonic generation microscopy provides a unique window into molecular spatial organization that is inaccessible to fluorescence.