Springer protocols feed by Neuroscience 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Foundations of Sted Microscopy
This chapter presents the foundations of Sted microscopy with a comparison to its generalization Resolft microscopy and to stochastic imaging methods (Palm, Storm, Fpalm, and alike). (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - February 12, 2014 Category: Neuroscience Source Type: news
Super-Resolution Microscopy: Principles, Techniques, and Applications
Diffraction sets a physical limit for the theoretically achievable resolution; however, it is possible to circumvent this barrier. That’s what microscopists have been doing in recent years and in many ways at once, starting the era of super-resolution in light microscopy. High-resolution approaches come in various flavors, and each has specific advantages or disadvantages. For example, near-field techniques avoid the problems associated with the propagation of light by getting very close to the specimen. In the far-field, the strategies include increasing the light collecting capability, sharpening the point spread f...
Source: Springer protocols feed by Neuroscience - February 12, 2014 Category: Neuroscience Source Type: news
Light Microscopy and Resolution
Galileo Galilei invented the first microscope “occhiolino,” by combining a concave and a convex lens in 1600s. Robert Hooke and Anton van Leeuwenhoek later modified it to look at living things. Since then, light microscopy has gained immense popularity and has been pushing the limits of optical technology. The race to improve the power of seeing continued by introduction of new techniques, more and more powerful lenses, better optical corrections, stronger light sources, higher-sensitivity detectors, and assembly of cutting-edge systems. However, the wish for a better peek at the cellular world hit a wall in th...
Source: Springer protocols feed by Neuroscience - February 12, 2014 Category: Neuroscience Source Type: news
Probing Biological Samples in High-Resolution Microscopy: Making Sense of Spots
In recent years, microscopy techniques have reached high sensitivities and excellent resolutions, far beyond the diffraction limit. However, images of biological specimens obtained with super-resolution instruments have the tendency of being dominated by spots. The quality or faithfulness of the observed structure depends in great manner on the labeling density achieved by affinity probes. To obtain the required high labeling densities, several problems still need to be addressed. Prevalent staining methodologies are mainly based on antibodies. Due to their large size (~10–15 nm, ~150 kDa), antibodies penetrate poorl...
Source: Springer protocols feed by Neuroscience - February 12, 2014 Category: Neuroscience Source Type: news
Requirements for Samples in Super-Resolution Fluorescence Microscopy
The preparation of samples and the choice of appropriate labeling techniques have become instrumental for the development of light microscopy techniques with increasingly high resolution. Both localization microscopy and STED approaches require fluorophores with specific features, including high photostability, specific excitation–emission spectra, and selective switching of single molecules to “on” and “off” state. Additionally, at higher resolutions the limits of conventional immunostaining often become apparent, as clearly exemplified by rather fragmented stainings of continuous cellular co...
Source: Springer protocols feed by Neuroscience - February 12, 2014 Category: Neuroscience Source Type: news
Photooxidation Microscopy: Bridging the Gap Between Fluorescence and Electron Microscopy
Eighty years after its development, electron microscopy still represents the gold standard in terms of resolution. A major disadvantage is, however, the requirement for fixed specimens—especially in view of the numerous live fluorescence microscopy methods that have been developed during the last few decades. This drawback can be largely compensated by combining both microscopy techniques, live imaging and electron microscopy, by transforming a fluorescent signal into one that can be visualized in the electron microscope. This can be achieved by employing photooxidation. This procedure uses the production of reactive...
Source: Springer protocols feed by Neuroscience - February 12, 2014 Category: Neuroscience Source Type: news
Nonlinear Optics Approaches Towards Subdiffraction Resolution in CARS Imaging
In theoretical investigations, we review several nonlinear optical approaches towards subdiffraction-limited resolution in label-free imaging via coherent anti-Stokes Raman scattering (CARS). Using a density matrix model and numerical integration, we investigate various level schemes and combinations of the light fields that induce CARS along with additional control laser fields. As the key to techniques that gain far-field resolution below the diffraction limit, we identify the inhibition of the buildup of vibrational molecular coherence via saturation or depletion of population (Beeker et al. Opt Express 17:22632–2...
Source: Springer protocols feed by Neuroscience - February 12, 2014 Category: Neuroscience Source Type: news
X-Ray Microscopy for Neuroscience: Novel Opportunities by Coherent Optics
X-ray microscopy and tomography can provide the three-dimensional density distribution within cells and tissues without staining and slicing. In addition, chemical information—i.e. the elemental distribution—can be retrieved by X-ray spectro-microscopy based on contrast variation around photon absorption edges and X-ray fluorescence. For a long time, X-ray microscopy has been limited in resolution by the fabrication of zone plate lenses, in particular for the hard X-ray range, which is needed to penetrate multicellular samples. Recent progress in X-ray optics and lensless coherent imaging now pave the way for e...
Source: Springer protocols feed by Neuroscience - February 12, 2014 Category: Neuroscience Source Type: news
Atomic Force Microscopy of Living Cells
Atomic force microscopy (AFM) is a powerful technique for analyzing the structure, properties, and interactions of living cells down to molecular resolution. Rather than using an incident beam as in optical and electron microscopies, AFM measures the tiny forces acting between a sharp tip and the sample surface. While AFM imaging provides information about the nanoscale surface architecture of living cells in real time, single-molecule force spectroscopy analyzes the localization, mechanics, and interactions of the individual cell surface constituents, thereby contributing to elucidate the molecular bases of cellular event...
Source: Springer protocols feed by Neuroscience - February 12, 2014 Category: Neuroscience Source Type: news
Two-Photon Microscopy to Measure Blood Flow and Concurrent Brain Cell Activity
The cerebral vascular system services the constant demand for energy during neuronal activity in the brain. Attempts to delineate the logic of neurovascular coupling have been greatly aided by the advent of two-photon laser scanning microscopy to concurrently image blood flow and the activity of individual neurons and astrocytes involved in the control of the flow. Here we review the procedures to generate optical access to the cortex for both rats and mice, determine the receptive fields of the exposed cortical areas, and use two-photon microscopy to accurately measure blood flow in individual cortical vessels concurrent ...
Source: Springer protocols feed by Neuroscience - January 27, 2014 Category: Neuroscience Source Type: news
Laser Speckle Imaging of Cerebral Blood Flow
Optical imaging of cerebral hemodynamics has been used extensively to investigate the complex interplay between neurons and the blood-borne supply of oxygen and energy substrates. Among several techniques, the so-called laser speckle imaging (LSI) has found widespread applications for studying (patho-)physiological aspects of neurovascular coupling. LSI is a rather simple and cost-effective method to acquire two-dimensional maps of cerebral blood flow from the cortex of experimental animals. In this chapter, we introduce the physical underpinnings as well as the most important technical and practical aspects of its impleme...
Source: Springer protocols feed by Neuroscience - January 27, 2014 Category: Neuroscience Source Type: news
Functional Imaging of Cerebral Oxygenation with Intrinsic Optical Contrast and Phosphorescent Probes
Microscopic in vivo measurements of cerebral oxygenation are of key importance for understanding normal cerebral energy metabolism and its dysregulation in a wide range of clinical conditions. Relevant cerebral pathologies include compromised blood perfusion following stroke and a decrease in efficiency of single-cell respiratory processes that occurs in neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. In this chapter we review a number of quantitative optical approaches to measuring oxygenation of blood and cerebral tissue. These methods can be applied to map the hemodynamic response and...
Source: Springer protocols feed by Neuroscience - January 27, 2014 Category: Neuroscience Source Type: news
Imaging Morphology and Function of Cortical Microglia
Microglial cells are the innate immune cells of the central nervous system. In the healthy adult brain “resting” ramified microglia continuously palpate their environment to monitor the integrity of and to react to any disturbance of brain homeostasis. During injury, inflammation, and in the course of neurodegenerative diseases microglia become activated, proliferate, and release a plethora of cytokines as well as reactive oxygen species. In addition to their well known role in disease, it has become increasingly clear that “resting” microglia also contribute to normal brain physiology, both during ...
Source: Springer protocols feed by Neuroscience - January 27, 2014 Category: Neuroscience Source Type: news
Imaging of Astrocytic Activity in Living Rodents
Mounting evidence from in vitro experiments supports bidirectional interactions between astrocytes and neurons, suggesting glial involvement in neuronal information processing in the brain. Elevation of the cytosolic calcium ion (Ca2+) concentration has been suggested to be important for gliotransmission; however, the study of Ca2+ dynamics in cerebral cortical astrocytes in vivo became possible only recently. Here, we describe a set of procedures to monitor Ca2+ concentration fluctuations in a population of astrocytes or in astrocytic processes using in vivo two-photon microscopy. Simultaneous recording of neuronal activi...
Source: Springer protocols feed by Neuroscience - January 27, 2014 Category: Neuroscience Source Type: news
Two-Photon Imaging of Neuronal Network Dynamics in Neocortex
Optical investigation of fast neuronal network dynamics in the intact neocortex—using appropriate activity-dependent indicators—requires single-cell resolution at large imaging depths and sufficient acquisition speed. These requirements are met by two-photon laser scanning microscopy, which has become one of the key methods for functional measurements of neuronal population activity in vivo, primarily in combination with calcium indicators. In this chapter we focus on various advanced two-photon imaging techniques that were recently developed to improve scanning speed, to enable 3D sampling from large numbers o...
Source: Springer protocols feed by Neuroscience - January 27, 2014 Category: Neuroscience Source Type: news
