Due to cellular complexity, studying fast signaling in neurons is often limited by: 1. the number of sites that can be simultaneously probed with conventional tools, such as patch pipettes, and 2. the recording speed of imaging tools, such as confocal or multiphoton microscopy. To overcome these spatiotemporal limitations, we develop an addressable confocal microscope that permits concurrent optical recordings from multiple user-selected sites of interest at high frame rates. Our system utilizes acousto-optic deflectors (AODs) for rapid positioning of a focused laser beam and a digital micromirror device (DMD) for addressable spatial filtering to achieve confocality. A registration algorithm synchronizes the AODs and DMD such that point illumination and point detection are always colocalized in conjugate image planes. The current system has an adjustable spatial resolution of ~0.5 to 1 µm. Furthermore, we show that recordings can be made at an aggregate frame rate of ~40 kHz. The system is capable of optical sectioning; this property is used to create 3-D reconstructions of fluorescently labeled test specimens and visualize neurons in brain slices. Additionally, we use the system to record intracellular calcium transients at several sites in hippocampal neurons using the fluorescent calcium indicator Oregon Green BAPTA-1.
Neurons are known to possess active computational properties. To investigate these properties, it is desirable to study the electrical and chemical properties not only at a living neuron's cell body, but also at many sites within its dendritic arborization. However, currently available recording techniques force a tradeoff between spatial and temporal resolution. To overcome these limitations, we have developed a confocal microscope that can make multisite optical recordings at an effective frame rate that is sufficient to measure fast neuronal events, such as action potentials, that occur on a timescale of milliseconds. We accomplished this by combining acousto-optic deflectors for addressable point illumination with a digital micromirror device for addressable point detection. After developing a registration algorithm to ensure synchronicity between point illumination and point detection, we used light-scattering test preparations to demonstrate that our system is capable of optical sectioning and therefore capable of imaging in living brain tissue. Furthermore, we have shown that fluorescence changes can be monitored at an effective frame rate of 25 kHz.