The base of the imaging is the new scanning technique, called 3D Anti-mOtion scanning. This patented scanning method extends the points of the random-access point scanning method by drifting the laser to fast scanned 3D lines. Based on these lines, scanning is executed on 3D lines, surface or volume elements with maintained temporal resolution. The scanned parts cover not only the pre-selected ROIs but also the neighbouring areas or volume elements. This drifting scanning allows fast imaging simultaneously multiple locations. When measuring from awake, behaving animals relatively big motion artefacts can occur caused by vessel pulsing, respiration or locomotion. Extending the ROIs allows line motion compensation and the elimination of most of the motion artefacts.
3D Multi-line scanning
For following neuronal activity in spines
For scanning spines at high speed in vivo, points of 3D random-access point scanning are extended by drifting the focal point along short lines without increasing the overall scanning time. The first step is to select points based on a z-stack along a dendritic segment or any cellular structure than simply define the 3D orientation and extent of the 3D drifts to the main direction of motion. Finally, the average trajectories are calculated cancelling effect of the brain motion. 3D Multi-Line scanning enables functional recording of over 150 spines simultaneously in a 500 µm x 500 µm x 650 µm volume.
3D Ribbon scanning
For 3D dendritic imaging
Ribbon scanning is an extended trajectory scanning which also captures the neighbouring area around the trajectory of dendrites to preserve fluorescent information during motions. The neighbouring area is scanned by generating drifts either parallel or orthogonal to the trajectory. 3D Ribbon scanning can follow the 3D curvature of one or more dendrites at the same time, for example, it enables functional recording with up to 3 kHz on a 50 µm long dendritic segment or imaging of activity simultaneously in over 12 spiny dendritic segments.
Figure shows 3D ribbons encompassing seven dendritic segments. Fluorescent transients were recorded simultaneously along the ribbons and data were projected into a 2D image ordering dendrites above each other. Recorded activity from selected spines and dendrites were visualised in the form of classical calcium transients and raster plots.
3D Snake scanning
For imaging dendrites in a 3D volume during large amplitude motions
3D Snake scanning is a volume extension of the ribbon scanning that contains the entire 3D environment of the dendrite. In larger animals or at certain surgery or behavioural protocols, the amplitude of motion can be larger. To sufficiently preserve fluorescence signals even in these cases, the surface elements can be extended to tortuous cuboids by using this scanning mode. Figure shows fast snake scanning performed at 10 Hz in the selected dendritic region of a V1 pyramidal neuron. Fluorescence data were maximal intensity projected to a straightened 2D image. The representative spontaneous calcium responses are measured from the selected volume elements and the transients are shown following 3D motion correction.
3D Multi-layer scanning
For imaging along the entire length of dendritic arbor
The 650 µm deep z-scanning range of the Femto3D-AcoustoOptic microscope gives possibility to image cells spanning across many layers, e.g. cortical pyramidal cells all the way up from basal to apical dendritic arbors. Imaging of multiple frames with different sizes and at any position in the scanning volume can be used to follow all events propagating along the cell. Scanning of the neighbourhood of the processes allows preserving all fluorescent signals and calcium transients in spite of the brain’s motion. The multi-layer scanning method is not limited to a single dendrite or axon, but even multiple neurons can be simultaneously imaged with their dendritic arbor. Figure demonstrates multi-layer imaging of the activity of a layer V neuron in an awake animal at 41 different depth levels over a 500 µm z range at 30 Hz. Motion artefacts along the x and y-axes were eliminated and calcium transients were recorded for each ROI.
3D Chessboard scanning
For in vivo imaging of hundreds of soma simultaneously in 3D
In Chessboard scanning, scanning points are extended to small squares containing the somata and surrounding area. The special flexibility of the 3D scanning capability allows simultaneous imaging along multiple small squares placed in arbitrary locations in 3D. The name, chessboard is derived from the layout which is generated by arranging side-by-side all the squares containing the selected regions. This pattern allows simultaneous visualisation of the activity of the somata, handling and storing the data and, importantly, to correct for motions. As a result, it makes possible to recover all high speed three-dimensional fluorescence data during the animal’s movements, therefore to follow neuronal network activity in behaving animals.
3D Multi-cube scanning
Imaging somata during large amplitude motions
Multi-cube scanning is an extended mode of chessboard scanning where a z dimension is added to cover the z-extent of the somata to preserve all somatic fluorescence points during motions. The somata as ROIs are ordered as cubes next to each other for visualisation and calcium transients are recorded from each cube corrected to motions. With this method for example, 50 somata can be recorded at 50 Hz when using cubes made of 50 x 10 x 5 voxels.
ast 3D Imaging of Spine, Dendritic, and Neuronal Assemblies in Behaving Animals. G Szalay, L Judak, G Katona, K Ocsai, G Juhasz, M Veress, Z Szadai, A Feher, T Tompa, B Chiovini, P Maak, B Rozsa, Neuron (2016)