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Voltage imaging was first conceived in the late 1960s and efforts to find better organic voltage-sensitive dyes began in the 1970s and continue until today. At the beginning it was difficult to measure an action potential signal from a squid giant axon in a single trial. Now it is possible to measure the action potential in an individual spine. Other chapters will discuss advances in voltage imaging...
The chemistry and the physics of voltage sensitive dyes (VSDs) should be understood and appreciated as a prerequisite for their optimal application to problems in neuroscience. This chapter provides a basic understanding of the properties of the large variety of available organic VSDs. The mechanisms by which the dyes respond to voltage guides the best setup of the optics for recording or imaging...
A central question in neuronal network analysis is how the interaction between individual neurons produces behavior and behavioral modifications. This task depends critically on how exactly are signals integrated by individual nerve cells functioning as complex operational units. Regional electrical properties of branching neuronal processes that determine the input–output function of any neuron are...
Voltage imaging using fluorescent voltage-sensitive dyes can be combined with other optical measurements, in particular with Ca2+ imaging, allowing for correlation of membrane potential changes with intracellular Ca2+ signals. Calibration of fluorescence voltage signals permits the comparison of membrane potential changes from different sites, allowing spatial mapping of membrane potential changes...
Optical recording with fast voltage sensitive dyes makes it possible, in suitable preparations, to simultaneously monitor the action potentials of large numbers of individual neurons. Here we describe methods for doing this, including considerations of different dyes and imaging systems, methods for correlating the optical signals with their source neurons, procedures for getting good signals, and...
Pairs of membrane-associated molecules exhibiting fluorescence resonance energy transfer (FRET) provide a sensitive technique to measure changes in a cell’s membrane potential. One of the FRET pair binds to one surface of the membrane and the other is a mobile ion that dissolves in the lipid bilayer. The voltage-related signal can be measured as a change in the fluorescence of either the donor or...
Voltage-sensitive dyes (VSDs) and optical imaging are useful tools for studying spatiotemporal patterns of population neuronal activity in cortical tissue. Using a photodiode array and absorption dyes, we are able to record simultaneously from hundreds of points with sensitivity comparable to local field potential recordings. Since absorption dyes have less phototoxicity than fluorescent dyes, the...
The functional organization of the vertebrate central nervous system (CNS) during the early phase of development has long been unclear because conventional electrophysiological means have several technical limitations. First, early embryonic neurons are small and fragile, and the application of microelectrodes is often difficult. Second, the simultaneous recording of electrical activity from multiple...
Neural computations underlying sensory perception, cognition, and motor control are performed by populations of neurons at different anatomical and temporal scales. Few techniques are currently available for exploring dynamics of local and large range populations. Voltage-sensitive dye imaging (VSDI) reveals neural population activity in areas ranging from a few tens of microns to a couple of centimeters,...
The development of functional imaging techniques applicable to neuroscience and covering a wide range of spatial and temporal scales has greatly facilitated the exploration of the relationships between cognition, behaviour and electrical brain activity. For mammals, the neocortex plays a particularly profound role in generating sensory perception, controlling voluntary movement, higher cognitive functions...
Functional imaging microscopy based on voltage-sensitive dyes (VSDs) has proven effective for revealing spatiotemporal patterns of activity in vivo and in vitro. Microscopy based on two-photon excitation (TPE) of fluorescent VSDs offers the possibility of three-dimensional recording of membrane potential changes on subcellular length scales hundreds of microns below the brain’s surface. Here we describe...
Studies in several important areas of neuroscience, including analysis of single neurons as well as neural networks, continue to be limited by currently available experimental tools. By combining molecular probes of cellular function, such as voltage-sensitive or calcium-sensitive dyes, with advanced microscopy techniques such as multiphoton microscopy, modern experimental neurophysiologists have...
The non-linear optical effect known as second harmonic generation (SHG) has been recognized since the earliest days of the laser. But it has only been in the last few years that it has begun to emerge as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function. This is because only small modifications are required to equip a standard laser scanning...
Organic voltage-sensitive dyes offer very high spatial and temporal resolution for imaging neuronal function. Further progress in imaging activity is expected from the emergent development of genetically encoded fluorescent sensors of membrane potential. These fluorescent protein (FP) voltage sensors overcome some drawbacks of organic voltage sensitive dyes such as non-specificity of cell staining...
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