ABSTRACT
To study the complex synaptic interactions underpinning dendritic information processing in single neurons, experimenters require methods to mimic presynaptic neurotransmitter release at multiple sites with no physiological damage. We show that laser scanning systems built around large-aperture acousto-optic deflectors and high numerical aperture objective lenses provide the sub-millisecond, sub-micron precision necessary to achieve physiological, exogenous synaptic stimulation. Our laser scanning systems can produce the sophisticated spatio-temporal patterns of synaptic input that are necessary to investigate single-neuron dendritic physiology.
Subject(s)
Acoustic Stimulation/instrumentation , Acoustics/instrumentation , Action Potentials/physiology , Microscopy, Confocal/instrumentation , Neurons/cytology , Neurons/physiology , Photic Stimulation/instrumentation , Animals , Cells, Cultured , Equipment Design , Equipment Failure Analysis , Hippocampus/cytology , Hippocampus/physiology , Hippocampus/radiation effects , Light , Neurons/radiation effects , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
We developed a two-photon microscope optimized for physiologically manipulating single neurons through their postsynaptic receptors. The optical layout fulfills the stringent design criteria required for high-speed, high-resolution imaging in scattering brain tissue with minimal photodamage. We detail the practical compensation of spectral and temporal dispersion inherent in fast laser beam scanning with acousto-optic deflectors, as well as a set of biological protocols for visualizing nearly diffraction-limited structures and delivering physiological synaptic stimuli. The microscope clearly resolves dendritic spines and evokes electrophysiological transients in single neurons that are similar to endogenous responses. This system enables the study of multisynaptic integration and will assist our understanding of single neuron function and dendritic computation.
Subject(s)
Hippocampus/physiology , Microscopy, Fluorescence, Multiphoton/methods , Neurons/physiology , Neurotransmitter Agents/physiology , Photolysis , Animals , Dendrites/physiology , Male , Models, Neurological , Rats , Rats, Sprague-DawleyABSTRACT
We have developed a fully automated procedure for extracting dendritic morphology from multiple three-dimensional image stacks produced by laser scanning microscopy. By eliminating human intervention, we ensure that the results are objective, quickly generated, and accurate. The software suite accounts for typical experimental conditions by reducing background noise, removing pipette artifacts, and aligning multiple overlapping image stacks. The output morphology is appropriate for simulation in compartmental simulation environments. In this report, we validate the utility of this procedure by comparing its performance on live neurons and test specimens with other fully and semiautomated reconstruction tools.
Subject(s)
Image Processing, Computer-Assisted/methods , Microscopy, Confocal/methods , Neurons/physiology , Neurons/ultrastructure , Animals , Capillaries/physiology , Capillaries/ultrastructure , Computer Simulation , Dendrites/physiology , Dendrites/ultrastructure , Pyramidal Cells/physiology , Pyramidal Cells/ultrastructure , Rats , Rats, Sprague-Dawley , Synapses/physiologyABSTRACT
We are investigating the computational properties of principal neurons in the mammalian brain. To manage the small size and intricate structure of neuronal dendrites, we employ advanced optical imaging techniques in combination with automatic image reconstruction and computational modeling to study their complex spatio-temporal pattern of activity.
Subject(s)
Action Potentials/physiology , Brain Mapping/methods , Brain/physiology , Microscopy, Fluorescence/methods , Models, Neurological , Nerve Net/physiology , Neurons/physiology , Animals , Cells, Cultured , Computer Simulation , Rats , Systems IntegrationABSTRACT
We describe novel approaches for compensating dispersion effects that arise when acousto-optic (AO) beam deflection of ultrafast laser pluses is used for multiphoton laser-scanning microscopy (MPLSM). AO deflection supports quick positioning of a laser beam to random locations, allowing high frame-rate imaging of user-selected sites of interest, in addition to conventional raster scanning. Compared to standard line-scan approaches, this results in improved signal strength (and thus increased signal-to-noise) as well as reduced photobleaching and photodamage. However, 2-D AO scanning has not yet been applied for multiphoton microscopy, largely because ultrafast laser pulses experience significant spatial and temporal dispersion while propagating through AO materials. We describe and quantify spatial dispersion, demonstrating it to be a significant barrier to achieving maximal spatial resolution. We also address temporal dispersion, which is a well-documented effect that limits multiphoton excitation efficacy, and is particularly severe for AO devices. To address both problems, we have developed a single diffraction grating scheme that reduces spatial dispersion more than three-fold throughout the field of view, and a novel four-pass stacked-prism prechirper that fully compensates for temporal dispersion while reducing by two-fold the required physical length relative to commonly employed designs. These developments enable the construction of a 2-D acousto-optic multiphoton laser-scanning microscope system.
