RESUMO
Two-photon scanning microscopy (TPSM) is a powerful tool for imaging deep inside living tissues with sub-cellular resolution. The temporal resolution of TPSM is however strongly limited by the galvanometric mirrors used to steer the laser beam. Fast physiological events can therefore only be followed by scanning repeatedly a single line within the field of view. Because acousto-optic deflectors (AODs) are non-mechanical devices, they allow access at any point within the field of view on a microsecond time scale and are therefore excellent candidates to improve the temporal resolution of TPSM. However, the use of AOD-based scanners with femtosecond pulses raises several technical difficulties. In this paper, we describe an all-digital TPSM setup based on two crossed AODs. It includes in particular an acousto-optic modulator (AOM) placed at 45 degrees with respect to the AODs to pre-compensate for the large spatial distortions of femtosecond pulses occurring in the AODs, in order to optimize the spatial resolution and the fluorescence excitation. Our setup allows recording from freely selectable point-of-interest at high speed (1kHz). By maximizing the time spent on points of interest, random-access TPSM (RA-TPSM) constitutes a promising method for multiunit recordings with millisecond resolution in biological tissues.
Assuntos
Diagnóstico por Imagem/instrumentação , Microscopia Confocal/instrumentação , Microscopia de Fluorescência/instrumentação , Potenciais de Ação/fisiologia , Algoritmos , Animais , Sinalização do Cálcio/fisiologia , Células Cultivadas , Hipocampo/citologia , Hipocampo/fisiologia , Lasers , Modelos Teóricos , Neurônios/fisiologia , Células Piramidais/fisiologia , Ratos , Processamento de Sinais Assistido por ComputadorRESUMO
Optogenetics is the combination of optical tools to monitor (i.e. "reporters") or interfere (i.e. "actuators") with neural activity, and genetic techniques to restrain the expression of these reporters and actuators in the neuronal populations of interest. Such combination of optical and genetic tools, together with the emergence of new animal models such as the zebrafish larva, has proven extremely valuable is dissecting neural circuits. Optogenetics provide a new framework to address issues that are fundamentally dynamic processes, such as sensorimotor integration in the vertebrate spinal cord. By shifting from spatially targeted electrical stimulation to genetically targeted optical stimulation, optogenetic also opens new avenues for innovative neurorehabilitative strategies, in particular after spinal cord injury.
Assuntos
Monitorização Fisiológica/métodos , Condução Nervosa/fisiologia , Neurônios/fisiologia , Optogenética , Animais , Genes Reporter , Humanos , Medula Espinal/citologia , Traumatismos da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/reabilitação , Peixe-ZebraRESUMO
The brains of Alzheimer's disease patients contain extracellular Abeta amyloid deposits (senile plaques). Although genetic evidence causally links Abeta deposition to the disease, the mechanism by which Abeta disrupts cortical function is unknown. Using triple immunofluorescent confocal microscopy and three-dimensional reconstructions, we found that neuronal processes that cross through an Abeta deposit are likely to have a radically changed morphology. We modeled the electrophysiological effect of this changed morphology and found a predicted delay of several milliseconds over an average plaque. We propose that this type of delay, played out among thousands of plaques throughout neocortical areas, disrupts the precise temporal firing patterns of action potentials, contributing directly to neural system failure and dementia.