Three-dimensional imaging and photostimulation by remote-focusing and holographic light patterning

Abstract

Access to three-dimensional structures in the brain is fundamental to probe signal processing at multiple levels, from integration of synaptic inputs to network activity mapping. Here, we present an optical method for independent three-dimensional photoactivation and imaging by combination of digital holography with remote-focusing. We experimentally demonstrate compensation of spherical aberration for out-of-focus imaging in a range of at least 300 μm, as well as scanless imaging along oblique planes. We apply this method to perform functional imaging along tilted dendrites of hippocampal pyramidal neurons in brain slices, after photostimulation by multiple spots glutamate uncaging. By bringing extended portions of tilted dendrites simultaneously in-focus, we monitor the spatial extent of dendritic calcium signals, showing a shift from a widespread to a spatially confined response upon blockage of voltage-gated Na channels. B rain architecture, from the cellular up to the anatomical level, develops in three dimensions (3D): Dendritic and axonal trees often spread in extensive spatial patterns, organizing neuronal circuits into complex volumes. As an essential step in understanding how information is processed in the brain and studying the relationship between structure and function in cerebral circuits, techniques to stimulate and record neuronal signals in 3D are required. This need is particularly evident in research fields such as the study of dendritic integration and plasticity (1) or of neuronal network activity mapping (2, 3). Optical methods for stimulation and imaging enable targeting of a wide range of structures-from subcellular compartments up to multiple cells-within intact neuronal circuits, with millisecond and submicron resolution. Caged neurotransmitters and, more recently, optogenetic tools such as channelrhodopsin and halorhodopsin provide a diverse toolbox for neuronal excitation and inhibition (4, 5), whereas optical reporters such as calcium and voltagesensitive dyes allow recording neuronal activity (6, 7).Both imaging and photostimulation have been performed in 3D by using scanning or parallel excitation methods (8-12). However, to reach a full optical control of 3D structures, these approaches need to be combined into a unique optical system. One of the main challenges, in this respect, is to axially decouple the imaging and stimulation planes when both optical pathways are combined (as it is often the case) into the same microscope objective. Until now, simultaneous imaging and photostimulation have been limited to restricted areas, for example to the relatively short portion of a dendrite which extends in the focal plane of the microscope (13, 14), or, in the case of functional mapping, to neuronal bodies located on the same plane (15).To overcome these limitations, we propose here a unique scanless optical system for simultaneous imaging and stimulation in 3D. Wide-field imaging on arbitrary oriented planes is achieved by using a remote-focusing technique adapted from Botcherby et a...

All-Optical Interrogation of Neural Circuits

“…Early efforts to combine imaging and one-photon (1P) optogenetic manipulation involved the use of nongenetic indicators of activity such as the calcium sensors fura-2 (Zhang et al, 2007) or Fluo-5F (Zhang and Oertner, 2007), or the voltage sensor RH-155 (Airan et al, 2007). This strategy was more recently implemented in vivo to examine the functional properties of interneuron networks (Wilson et al, 2012), map interhemispheric and intrahemispheric connectivity (Lim et al, 2012), and probe motor pattern generation during behavior (Fajardo et al, 2013).…”

High-resolution real-time dual-view imaging with multiple point of view microscopy

“…Since both views are imaged side by side on a single camera, the field of view of each view is half that of conventional microscopy using the same camera. In our implementation, the angle between the front and side views was 60 degrees, but this can be modified by using another micromirror design or by tilting the mirror in front of the refocusing objective [13,18]. It is important to note that this setup is straightforward and can be readily incorporated in an existing fluorescence microscope, without the need for expensive or custom optics.…”

“…Moreover, since usually only two planes are imaged side by side on a single camera, a relatively large field of view can be obtained. Oblique imaging also allows the direct observation of the object along the axial axis of the microscope objective, which can be beneficial for some applications [13,14]; in contrast, obtaining the same view with conventional microscopy requires sequential imaging of the object along the axial axis, which is slow.…”

“…In the original version of refocusing, the Wilson group proposed to use two facing objectives (the first objective to refocus the object and the second objective to image the refocused object, or to use a single objective facing a mirror to achieve the same goal [15]. For oblique imaging, the combination of two facing objective has been modified by tilting the second -imaging -objective with respect to the refocusing objective [17], whereas in the configuration using a microscope objective facing a mirror, the mirror is tilted [13,18]. One limitation of these methods is that the information contained in the pupil of the imaging objective is clipped when oblique planes are refocused [18].…”

Superresolving dendritic spine morphology with STED microscopy under holographic photostimulation

“…Consequently, CGH enables simultaneous excitation with arbitrary patterns, overcoming the sequential targeting imposed by the use of galvanometric mirrors. 41 CGH was used to address dendritic integration on apical dendrites in acute brain slices 37,42,43 and to control the kinetics of glutamate-evoked currents. 40 However, holographic stimulation and superresolution imaging have never been combined.…”