8,096 research outputs found

    Image Sabilization for In Vivo Microscopic Imaging

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    Acousto-optical Scanning-Based High-Speed 3D Two-Photon Imaging In Vivo.

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    Recording of the concerted activity of neuronal assemblies and the dendritic and axonal signal integration of downstream neurons pose different challenges, preferably a single recording system should perform both operations. We present a three-dimensional (3D), high-resolution, fast, acousto-optic two-photon microscope with random-access and continuous trajectory scanning modes reaching a cubic millimeter scan range (now over 950 × 950 × 3000 μm3) which can be adapted to imaging different spatial scales. The resolution of the system allows simultaneous functional measurements in many fine neuronal processes, even in dendritic spines within a central core (>290 × 290 × 200 μm3) of the total scanned volume. Furthermore, the PSF size remained sufficiently low (PSFx < 1.9 μm, PSFz < 7.9 μm) to target individual neuronal somata in the whole scanning volume for simultaneous measurement of activity from hundreds of cells. The system contains new design concepts: it allows the acoustic frequency chirps in the deflectors to be adjusted dynamically to compensate for astigmatism and optical errors; it physically separates the z-dimension focusing and lateral scanning functions to optimize the lateral AO scanning range; it involves a custom angular compensation unit to diminish off-axis angular dispersion introduced by the AO deflectors, and it uses a high-NA, wide-field objective and high-bandwidth custom AO deflectors with large apertures. We demonstrate the use of the microscope at different spatial scales by first showing 3D optical recordings of action potential back propagation and dendritic Ca2+ spike forward propagation in long dendritic segments in vitro, at near-microsecond temporal resolution. Second, using the same microscope we show volumetric random-access Ca2+ imaging of spontaneous and visual stimulation-evoked activity from hundreds of cortical neurons in the visual cortex in vivo. The selection of active neurons in a volume that respond to a given stimulus was aided by the real-time data analysis and the 3D interactive visualization accelerated selection of regions of interest

    Adaptive Movement Compensation for In Vivo Imaging of Fast Cellular Dynamics within a Moving Tissue

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    In vivo non-linear optical microscopy has been essential to advance our knowledge of how intact biological systems work. It has been particularly enabling to decipher fast spatiotemporal cellular dynamics in neural networks. The power of the technique stems from its optical sectioning capability that in turn also limits its application to essentially immobile tissue. Only tissue not affected by movement or in which movement can be physically constrained can be imaged fast enough to conduct functional studies at high temporal resolution. Here, we show dynamic two-photon Ca2+ imaging in the spinal cord of a living rat at millisecond time scale, free of motion artifacts using an optical stabilization system. We describe a fast, non-contact adaptive movement compensation approach, applicable to rough and weakly reflective surfaces, allowing real-time functional imaging from intrinsically moving tissue in live animals. The strategy involves enslaving the position of the microscope objective to that of the tissue surface in real-time through optical monitoring and a closed feedback loop. The performance of the system allows for efficient image locking even in conditions of random or irregular movements

    INTRACELLULAR SUBSURFACE IMAGING USING A HYBRID SHEAR-FORCE FEEDBACK/ SCANNING QUANTITATIVE PHASE MICROSCOPY TECHNIQUE.

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    Quantitative phase microscopy (QPM) allows for the imaging of translucent or transparent biological specimens without the need for exogenous contrast agents. This technique is usually applied towards the investigation of simple cells such as red blood cells which are typically enucleated and can be considered to be homogenous. However, most biological cells are nucleated and contain other interesting intracellular organelles. It has been established that the physical characteristics of certain subsurface structures such as the shape and roughness of the nucleus is well correlated with onset and progress of pathological conditions such as cancer. Although the acquired quantitative phase information of biological cells contains surface information as well as coupled subsurface information, the latter has been ignored up until now. A novel scanning quantitative phase imaging system unencumbered by 2p ambiguities is hereby presented. This system is incorporated into a shear-force feedback scheme which allows for simultaneous phase and topography determination. It will be shown how subsequent image processing of these two data sets allows for the extraction of the subsurface component in the phase data and in vivo cell refractometry studies. Both fabricated samples and biological cells ranging from rat fibroblast cells to malaria infected human erythrocytes were investigated as part of this research. The results correlate quite well with that obtained via other microscopy techniques

    Structural and functional alterations of cortical neurons in Alzheimer’s disease transgenic mice assessed by two-photon in vivo imaging

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    Alzheimer’s disease (AD), the most common form of dementia, has been proposed to result from the degeneration of synapses, putatively caused by assemblies of the amyloid-β peptide (Aβ). The spatiotemporal dynamics of this synaptopathy, its potential reversibility as well as its consequences on the function of single neurons and neuronal circuits, however, are not fully understood to date. In order to address these questions, I assessed structural and functional alterations of neurons in the neocortex in a transgenic mouse model of Alzheimer’s disease, namely APP/PS1 (APPswe, PS1L166P) mice, using in vivo two-photon imaging. Chronic imaging of dendrites and axons over the course of four weeks revealed not only a reduction in dendritic spine density close to amyloid plaques (proteinaceous extracellular deposits typical of AD), but I also identified synaptic instability as a main aspect contributing to AD pathology. Importantly, while synapse loss was confined to the immediate plaque vicinity (up to 15µm from the histological plaque border), synaptic instability was evident in a much larger region surrounding plaques (50 µm) and affected both, pre- and postsynaptic compartments. As the prevailing hypothesis in AD holds that Aβ conveys these detrimental effects on synapses one therapeutic approach is based on the pharmacological inhibition of Aβ generation. I thus assessed the impact of a novel selective γ-secretase inhibitor (GSI), a compound that prevents the last cleavage step necessary for the release of Aβ from the longer transmembrane amyloid precursor protein (APP). Notably, the GSI used here primarily interferes with the processing of APP and still allows for processing of other γ-secretase substrates, and hence should largely reduce side effects seen with earlier generations of GSIs before. Daily treatment with the GSI reduced the deposition of Aβ as evidenced by the initial reduction in the number of new plaques and a sustained decrease in the growth of these newly deposited plaques. Importantly, it also ameliorated the plaque-associated synaptic instability, without displaying overt adverse effects on dendritic spines in WT mice. These data represent the first in vivo evidence that selective pharmacological inhibition of the γ-secretase mediated APP cleavage can have beneficial effects on synaptic pathology in AD. Given the widespread impact of Aβ assemblies on neuronal structures, I then asked to which extent these structural alterations affect the function of neurons. To address this question, I recorded neuronal response properties in the primary visual cortex of behaving APP/PS1 mice, employing in vivo two-photon calcium imaging using the genetically encoded calcium indicator GCaMP6m. In order to probe the impact of AD related pathology on specific aspects of information processing, which rely on multiple neuronal circuits, I characterized visually driven and motor-related activity, as well as signals based on mismatches between actual and expected visual input. My data reveal a massive reduction in responsiveness under almost all conditions tested, which is line with the profound impact on neuronal structure. Stimulus selectivity, like orientation or direction tuning, were not altered in APP/PS1 mice, indicating that the main effect is caused by a change in response gain. Along with the massive decrease in feedforward signals, I observed an increase in spontaneous, hence uncorrelated neuronal activity in AD transgenic mice. Both features jointly affected the coding accuracy of the network, and I propose that this combination may represent a common characteristic leading to impaired information processing in AD. Surprisingly, I found that responses elicited after a discordance of actual and expected visual flow during running, i.e. a visuomotor mismatch, were selectively spared in APP/PS1 mice, suggesting a particular resilience of this very signal. Together, both studies demonstrate that global widespread structural changes of neurons in the AD brain are accompanied by a severe impact on information processing, most prominently seen in a strong reduction of feedforward signals. My data, thus, provide a correlate of impaired cognition in AD at the level of single neurons and neural circuits

    Aerospace medicine and biology: A continuing bibliography with indexes, supplement 204

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    This bibliography lists 140 reports, articles, and other documents introduced into the NASA scientific and technical information system in February 1980

    High Resolution Intravital Imaging of Subcellular Structures of Mouse Abdominal Organs Using a Microstage Device

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    Intravital imaging of brain and bone marrow cells in the skull with subcellular resolution has revolutionized neurobiology, immunology and hematology. However, the application of this powerful technology in studies of abdominal organs has long been impeded by organ motion caused by breathing and heartbeat. Here we describe for the first time a simple device designated ‘microstage’ that effectively reduces organ motions without causing tissue lesions. Combining this microstage device with an upright intravital laser scanning microscope equipped with a unique stick-type objective lens, the system enables subcellular-level imaging of abdominal organs in live mice. We demonstrate that this technique allows for the quantitative analysis of subcellular structures and gene expressions in cells, the tracking of intracellular processes in real-time as well as three-dimensional image construction in the pancreas and liver of the live mouse. As the aforementioned analyses based on subcellular imaging could be extended to other intraperitoneal organs, the technique should offer great potential for investigation of physiological and disease-specific events of abdominal organs. The microstage approach adds an exciting new technique to the in vivo imaging toolbox

    Aerospace Medicine and Biology: A continuing bibliography with indexes, supplement 140

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    This bibliography lists 306 reports, articles, and other documents introduced into the NASA scientific and technical information system in March 1975

    Investigation of the Drosophila motion vision circuit using remote-focusing two-photon microscopy

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