"There's plenty of room at the bottom": deep brain imaging with holographic endo-microscopy.

Significance: Over more than 300 years, microscopic imaging keeps providing fundamental insights into the mechanisms of living organisms. Seeing microscopic structures beyond the reach of free-space light-based microscopy, however, requires dissection of the tissue-an intervention seriously disturbing its physiological functions. The hunt for low-invasiveness tools has led a growing community of physicists and engineers into the realm of complex media photonics. One of its activities represents exploiting multimode optical fibers (MMFs) as ultra-thin endoscopic probes. Employing wavefront shaping, these tools only recently facilitated the first peeks at cells and their sub-cellular compartments at the bottom of the mouse brain with the impact of micro-scale tissue damage. Aim: Here, we aim to highlight advances in MMF-based holographic endo-microscopy facilitating microscopic imaging throughout the whole depth of the mouse brain. Approach: We summarize the important technical and methodological prerequisites for stabile high-resolution imaging in vivo. Results: We showcase images of the microscopic building blocks of brain tissue, including neurons, neuronal processes, vessels, intracellular calcium signaling, and red blood cell velocity in individual vessels. Conclusions: This perspective article helps to understand the complexity behind the technology of holographic endo-microscopy, summarizes its recent advances and challenges, and stimulates the mind of the reader for further exploitation of this tool in the neuroscience research.

110 µm thin endo-microscope for deep-brain in vivo observations of neuronal connectivity, activity and blood flow dynamics.

Light-based in-vivo brain imaging relies on light transport over large distances of highly scattering tissues. Scattering gradually reduces imaging contrast and resolution, making it difficult to reach structures at greater depths even with the use of multiphoton techniques. To reach deeper, minimally invasive endo-microscopy techniques have been established. These most commonly exploit graded-index rod lenses and enable a variety of modalities in head-fixed and freely moving animals. A recently proposed alternative is the use of holographic control of light transport through multimode optical fibres promising much less traumatic application and superior imaging performance. We present a 110 µm thin laser-scanning endo-microscope based on this prospect, enabling in-vivo volumetric imaging throughout the whole depth of the mouse brain. The instrument is equipped with multi-wavelength detection and three-dimensional random access options, and it performs at lateral resolution below 1 µm. We showcase various modes of its application through the observations of fluorescently labelled neurones, their processes and blood vessels. Finally, we demonstrate how to exploit the instrument to monitor calcium signalling of neurones and to measure blood flow velocity in individual vessels at high speeds.

Computational image enhancement of multimode fibre-based holographic endo-microscopy: harnessing the muddy modes.

In imaging geometries, which employ wavefront-shaping to control the light transport through a multi-mode optical fibre (MMF), this terminal hair-thin optical component acts as a minimally invasive objective lens, enabling high resolution laser-scanning fluorescence microscopy inside living tissues at depths hardly accessible by any other light-based technique. Even in the most advanced systems, the diffraction-limited foci scanning the object across the focal plane are contaminated by a stray optical signal carrying typically few tens of % of the total optical power. The stray illumination takes the shape of a randomised but reproducible speckle, and is unique for each position of the focus. We experimentally demonstrate that the performance of imaging a fluorescent object can be significantly improved, when resulting images are computationally post-processed, utilising records of intensities of all speckle-contaminated foci used in the imaging procedure. We present two algorithms based on a regularised iterative inversion and regularised direct pseudo-inversion respectively which lead to enhancement of the image contrast and resolution.

A Suite of Neurophotonic Tools to Underpin the Contribution of Internal Brain States in fMRI.

Recent developments in optical microscopy, applicable for large-scale and longitudinal imaging of cortical activity in behaving animals, open unprecedented opportunities to gain a deeper understanding of neurovascular and neurometabolic coupling during different brain states. Future studies will leverage these tools to deliver foundational knowledge about brain state-dependent regulation of cerebral blood flow and metabolism as well as regulation as a function of brain maturation and aging. This knowledge is of critical importance to interpret hemodynamic signals observed with functional magnetic resonance imaging (fMRI).

Dual Targeting of BRAF and mTOR Signaling in Melanoma Cells with Pyridinyl Imidazole Compounds.

BRAF inhibitors can delay the progression of metastatic melanoma, but resistance usually emerges, leading to relapse. Drugs simultaneously targeting two or more pathways essential for cancer growth could slow or prevent the development of resistant clones. Here, we identified pyridinyl imidazole compounds SB202190, SB203580, and SB590885 as dual inhibitors of critical proliferative pathways in human melanoma cells bearing the V600E activating mutation of BRAF kinase. We found that the drugs simultaneously disrupt the BRAF V600E-driven extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activity and the mechanistic target of rapamycin complex 1 (mTORC1) signaling in melanoma cells. Pyridinyl imidazole compounds directly inhibit BRAF V600E kinase. Moreover, they interfere with the endolysosomal compartment, promoting the accumulation of large acidic vacuole-like vesicles and dynamic changes in mTOR signaling. A transient increase in mTORC1 activity is followed by the enrichment of the Ragulator complex protein p18/LAMTOR1 at contact sites of large vesicles and delocalization of mTOR from the lysosomes. The induced disruption of the endolysosomal pathway not only disrupts mTORC1 signaling, but also renders melanoma cells sensitive to endoplasmic reticulum (ER) stress. Our findings identify new activities of pharmacologically relevant small molecule compounds and provide a biological rationale for the development of anti-melanoma therapeutics based on the pyridinyl imidazole core.

Neurovascular Network Explorer 2.0: A Simple Tool for Exploring and Sharing a Database of Optogenetically-evoked Vasomotion in Mouse Cortex In Vivo.

The importance of sharing experimental data in neuroscience grows with the amount and complexity of data acquired and various techniques used to obtain and process these data. However, the majority of experimental data, especially from individual studies of regular-sized laboratories never reach wider research community. A graphical user interface (GUI) engine called Neurovascular Network Explorer 2.0 (NNE 2.0) has been created as a tool for simple and low-cost sharing and exploring of vascular imaging data. NNE 2.0 interacts with a database containing optogenetically-evoked dilation/constriction time-courses of individual vessels measured in mice somatosensory cortex in vivo by 2-photon microscopy. NNE 2.0 enables selection and display of the time-courses based on different criteria (subject, branching order, cortical depth, vessel diameter, arteriolar tree) as well as simple mathematical manipulation (e.g. averaging, peak-normalization) and data export. It supports visualization of the vascular network in 3D and enables localization of the individual functional vessel diameter measurements within vascular trees. NNE 2.0, its source code, and the corresponding database are freely downloadable from UCSD Neurovascular Imaging Laboratory website1. The source code can be utilized by the users to explore the associated database or as a template for databasing and sharing their own experimental results provided the appropriate format.

Phasor analysis of NADH FLIM identifies pharmacological disruptions to mitochondrial metabolic processes in the rodent cerebral cortex.

Investigating cerebral metabolism in vivo at a microscopic level is essential for understanding brain function and its pathological alterations. The intricate signaling and metabolic dynamics between neurons, glia, and microvasculature requires much more detailed understanding to better comprehend the mechanisms governing brain function and its disease-related changes. We recently demonstrated that pharmacologically-induced alterations to different steps of cerebral metabolism can be distinguished utilizing 2-photon fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotide (NADH) fluorescence in vivo. Here, we evaluate the ability of the phasor analysis method to identify these pharmacological metabolic alterations and compare the method's performance with more conventional nonlinear curve-fitting analysis. Visualization of phasor data, both at the fundamental laser repetition frequency and its second harmonic, enables resolution of pharmacologically-induced alterations to mitochondrial metabolic processes from baseline cerebral metabolism. Compared to our previous classification models based on nonlinear curve-fitting, phasor-based models required fewer parameters and yielded comparable or improved classification accuracy. Fluorescence lifetime imaging of NADH and phasor analysis shows utility for detecting metabolic alterations and will lead to a deeper understanding of cerebral energetics and its pathological changes.

Fluorescence lifetime microscopy of NADH distinguishes alterations in cerebral metabolism in vivo.

Evaluating cerebral energy metabolism at microscopic resolution is important for comprehensively understanding healthy brain function and its pathological alterations. Here, we resolve specific alterations in cerebral metabolism in vivo in Sprague Dawley rats utilizing minimally-invasive 2-photon fluorescence lifetime imaging (2P-FLIM) measurements of reduced nicotinamide adenine dinucleotide (NADH) fluorescence. Time-resolved fluorescence lifetime measurements enable distinction of different components contributing to NADH autofluorescence. Ostensibly, these components indicate different enzyme-bound formulations of NADH. We observed distinct variations in the relative proportions of these components before and after pharmacological-induced impairments to several reactions involved in glycolytic and oxidative metabolism. Classification models were developed with the experimental data and used to predict the metabolic impairments induced during separate experiments involving bicuculline-induced seizures. The models consistently predicted that prolonged focal seizure activity results in impaired activity in the electron transport chain, likely the consequence of inadequate oxygen supply. 2P-FLIM observations of cerebral NADH will help advance our understanding of cerebral energetics at a microscopic scale. Such knowledge will aid in our evaluation of healthy and diseased cerebral physiology and guide diagnostic and therapeutic strategies that target cerebral energetics.

The roadmap for estimation of cell-type-specific neuronal activity from non-invasive measurements.

The computational properties of the human brain arise from an intricate interplay between billions of neurons connected in complex networks. However, our ability to study these networks in healthy human brain is limited by the necessity to use non-invasive technologies. This is in contrast to animal models where a rich, detailed view of cellular-level brain function with cell-type-specific molecular identity has become available due to recent advances in microscopic optical imaging and genetics. Thus, a central challenge facing neuroscience today is leveraging these mechanistic insights from animal studies to accurately draw physiological inferences from non-invasive signals in humans. On the essential path towards this goal is the development of a detailed 'bottom-up' forward model bridging neuronal activity at the level of cell-type-specific populations to non-invasive imaging signals. The general idea is that specific neuronal cell types have identifiable signatures in the way they drive changes in cerebral blood flow, cerebral metabolic rate of O2 (measurable with quantitative functional Magnetic Resonance Imaging), and electrical currents/potentials (measurable with magneto/electroencephalography). This forward model would then provide the 'ground truth' for the development of new tools for tackling the inverse problem-estimation of neuronal activity from multimodal non-invasive imaging data.This article is part of the themed issue 'Interpreting BOLD: a dialogue between cognitive and cellular neuroscience'.

Cell type specificity of neurovascular coupling in cerebral cortex.

Identification of the cellular players and molecular messengers that communicate neuronal activity to the vasculature driving cerebral hemodynamics is important for (1) the basic understanding of cerebrovascular regulation and (2) interpretation of functional Magnetic Resonance Imaging (fMRI) signals. Using a combination of optogenetic stimulation and 2-photon imaging in mice, we demonstrate that selective activation of cortical excitation and inhibition elicits distinct vascular responses and identify the vasoconstrictive mechanism as Neuropeptide Y (NPY) acting on Y1 receptors. The latter implies that task-related negative Blood Oxygenation Level Dependent (BOLD) fMRI signals in the cerebral cortex under normal physiological conditions may be mainly driven by the NPY-positive inhibitory neurons. Further, the NPY-Y1 pathway may offer a potential therapeutic target in cerebrovascular disease.

Dynamic phase differences based on quantitative phase imaging for the objective evaluation of cell behavior.

Quantitative phase imaging (QPI) brought innovation to noninvasive observation of live cell dynamics seen as cell behavior. Unlike the Zernike phase contrast or differential interference contrast, QPI provides quantitative information about cell dry mass distribution. We used such data for objective evaluation of live cell behavioral dynamics by the advanced method of dynamic phase differences (DPDs). The DPDs method is considered a rational instrument offered by QPI. By subtracting the antecedent from the subsequent image in a time-lapse series, only the changes in mass distribution in the cell are detected. The result is either visualized as a two dimensional color-coded projection of these two states of the cell or as a time dependence of changes quantified in picograms. Then in a series of time-lapse recordings, the chain of cell mass distribution changes that would otherwise escape attention is revealed. Consequently, new salient features of live cell behavior should emerge. Construction of the DPDs method and results exhibiting the approach are presented. Advantage of the DPDs application is demonstrated on cells exposed to an osmotic challenge. For time-lapse acquisition of quantitative phase images, the recently developed coherence-controlled holographic microscope was employed.

In vivo stimulus-induced vasodilation occurs without IP3 receptor activation and may precede astrocytic calcium increase.

Calcium-dependent release of vasoactive gliotransmitters is widely assumed to trigger vasodilation associated with rapid increases in neuronal activity. Inconsistent with this hypothesis, intact stimulus-induced vasodilation was observed in inositol 1,4,5-triphosphate (IP3) type-2 receptor (R2) knock-out (KO) mice, in which the primary mechanism of astrocytic calcium increase-the release of calcium from intracellular stores following activation of an IP3-dependent pathway-is lacking. Further, our results in wild-type (WT) mice indicate that in vivo onset of astrocytic calcium increase in response to sensory stimulus could be considerably delayed relative to the simultaneously measured onset of arteriolar dilation. Delayed calcium increases in WT mice were observed in both astrocytic cell bodies and perivascular endfeet. Thus, astrocytes may not play a role in the initiation of blood flow response, at least not via calcium-dependent mechanisms. Moreover, an increase in astrocytic intracellular calcium was not required for normal vasodilation in the IP3R2-KO animals.

Monitoring of ozone effects on the vitality and increment of Norway spruce and European beech in the Central European forests.

The ozone effect on Norway spruce (Picea abies (L) Karst.) and European beech (Fagus sylvatica L.) was studied on 48 monitoring plots in 2005-2008. These plots represent two major forest tree species stands of different ages in eight regions of the Czech Republic. The forest conditions were represented by defoliation and the annual radial increment of individual trees. The ozone exposure was assessed by using modeled values of mean annual O(3) concentration and the AOT40 index. The malondialdehyde (MDA) content of the foliage was analysed and used as an indicator of oxidative stress. The correlation analysis showed a significant relation of Norway spruce defoliation to the AOT40 exposure index, and European beech defoliation to the MDA level. The radial increment response to ozone was significant only for the European beech: (a) the correlation analysis showed its decrease with increasing AOT40; (b) the regression model showed its decrease with increasing mean annual ozone concentration only at lower altitudes (<700 m a.s.l.).

The impact of ambient ozone on mountain spruce forests in the Czech Republic as indicated by malondialdehyde.

Malondialdehyde (MDA), a product of lipid peroxidation and biomarker of oxidative stress, is measured over the long term in spruce Picea abies needles under real conditions in three Czech mountain border areas. The trends presented collate the MDA content in spruce needles with ambient ozone, temperature and precipitation as casual, and defoliation as a subsequent factor for the period 1994-2006. We have found the overall decreasing trends in MDA and defoliation. The highest MDA and defoliation are recorded in the Jizerske, the lowest in the Krusne hory Mts. Out of the examined variables the MDA is predicted best by mean temperature in vegetation season, median of O(3) concentrations and AOT40; these three variables account for 34% of MDA1 and 36% of MDA2 variability. Our hypothesis that higher ambient O(3) exposure results in higher MDA contents in P. abies needles under real conditions has not been approved.

Monitoring of ozone risk for forests in the Czech Republic: preliminary results.

Ozone (O3) is supposed to represent a significant risk for the health of forest ecosystems in Central Europe. So far, however, its impact on stands growing under natural conditions has not been clearly proved. A new project of the National Agency for the Research in Agriculture is focused on the O3 effect on selected parameters of forest health. This paper presents the results of the first year of monitoring, 2005. In 2005, high O3 concentrations were measured, mainly in the spring. In the summer, due to wet and cold weather, the O3 load was comparatively low. In the plots investigated, the concentrations of O3 were higher with the altitude. The amount of epicuticular waxes on 1-year-old Norway spruce needles was the only factor showing significant correlation to O3 concentration. Defoliation of the stands depended only on the stand age. The amount of malondialdehyde (MDA), an oxidative stress marker, was related to the altitude, and only for European beech. The results are preliminary, as the summer O3 development was not typical in 2005, and the results may change over the next monitoring periods.

Determination of trichloroacetic acid in environmental studies using carbon 14 and chlorine 36.

Radioisotopes carbon 14 and chlorine 36 were used to elucidate the environmental role of trichloroacetic acid (TCA) formerly taken to be a herbicide and a secondary air pollutant with phytotoxic effects. However, use of 14C-labeling posed again known analytical problems, especially in TCA extraction from the sample matrix. Therefore--after evaluation of available methods--a new procedure using decarboxylation of [1,2-14C]TCA combined with extraction of the resultant 14C-chloroform with a non-polar solvent and its subsequent radiometric measurement was developed. The method solves previous difficulties and permits an easy determination of amounts between 0.4 and 20 kBq (10 - 500 ng g(-1)) of carrier-less [1,2-14C]TCA in samples from environmental investigations. The procedure is, however, not suitable for direct [36Cl]TCA determination in chlorination studies with 36Cl. Because TCA might be microbially degraded in soil during extraction and sample storage and its extraction from soil or needles is never complete, the decarboxylation method--i.e. 2 h TCA decomposition to chloroform and CO2 in aqueous solution or suspension in closed vial at 90 degrees C and pH 4.6 with subsequent CHCl3 extraction-is recommended here, estimated V < 7%. Moreover, the influence of pH and temperature on the decarboxylation of TCA in aqueous solution was studied in a broad range and its environmental relevance is shown in the case of TCA decarboxylation in spruce needles which takes place also at ambient temperatures and might amount more than 10-20% after a growing season. A study of TCA distribution in spruce needles after below-ground uptake shows the highest uptake rate into current needles which have, however, a lower TCA content than older needle-year classes, TCA biodegradation in forest soil leads predominatingly to CO2.

Forest health status in the Carpathian Mountains over the period 1997-2001.

The results of forest health status assessments in the Carpathian Mountains from the monitoring networks developed by the European Union Scheme on the Protection of Forest Against Atmospheric Pollution (EU Scheme) and International Co-operative Programme on Assessment and Monitoring of Air Pollution Effects on Forests (ICP-Forests), have led to a better understanding of the impact of air pollution and other stressors on forests at the regional scale. During the period 1997-2001, forests in the Carpathian Mountains were severely affected by air pollution and natural stresses with 29.7-34.9% of the trees included in defoliation classes 2-4. The broadleaves were slightly healthier than the conifers, and European beech (Fagus sylvatica) was the least affected species. Norway spruce (Picea abies) has poor health status, with 42.9-46.6% of the trees damaged (2-4% defoliation classes). Silver fir (Abies alba) damage was also high, with 46.0-50.9% in defoliation classes 2-4. Pines (primarily Pinus sylvestris) were the least affected of the conifers, with 24.9-33.8% in defoliation classes 2-4. The results from the transnational networks (16 x 16 km) show that the Carpathian forests are slightly more damaged than the average for the entire Europe. The correlative studies performed in individual European countries show the relationships between air pollution stressors with trends in defoliation and a possible effect of natural stresses at each site. More specific, effects of tree age, drought, ozone and acid deposition critical level exceedances were demonstrated to affect crown condition.