Dendritic imaging

3D Trajectory scanning

With the Femto3D Atlas events less than a millisecond apart can be separated, and therefore propagation speed of regenerative activity determined at multiple sites of the dendritic tree. Random-access point scanning is a usable method also for measuring dendritic activity, however, random-access point scanning extended by drifting the focal point along short 3D trajectories allows imaging without interruption at multiple dendritic branches. The sampling is continuous during the drift, so this scanning mode gives a more detailed spatial resolution without changing the overall scanning time: this remains as high as during the point scanning. As a result, the function of thin dendritic segments, or even spines and single action potentials can be revealed. See also Katona et al., Nat Meth, 2012; Chiovini et al., Neuron, 2014.

3D Ribbon scanning

An extension of the 3D multiple-line scanning performed by Anti-motion technology is 3D ribbon scanning, which makes it possible to image ribbon shaped surfaces containing dendrites and the neighboring areas. Figure shows 3D ribbons encompassing seven dendritic segments with their spines of a GCaMP6-labeled layer II/III pyramidal neuron measured at 3 kHz within the brain of a living mouse. The seven ribbons were projected into a 2D image ordering dendrites above each other for better visualization, and activity was recorded from 40 selected spines and visualized in the form of classical Ca2+ transients. See also Szalay et al., Neuron, 2016.

RIBBONS COVERED DENDRITES
WITH THEIR SPINES IN VIVO

BEFORE MOTION CORRECTION

AFTER MOTION CORRECTION

CA2+ RESPONSES FROM
SELECTED SPINES

3D Snake scanning

3D snake scanning is a volume extension of ribbon scanning and contains the entire 3D environment of the dendrite. It therefore supports imaging of dendrites in larger animals, or behavioral protocols, where the amplitude of motion can be large. 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. See also Szalay et al., Neuron, 2016.

SNAKE SCANNING

PROJECTIONS OF THE DENDRITE
AFTER MOTION CORRECTION

CA2+ RESPONSES FROM SELECTED SPINES

3D Multiple-line scanning

3D multiple line scanning is similar to the 3D trajectory scanning, but while trajectory scanning was developed for imaging micrometers long structures, that 3D multiple line scanning was developed for imaging of hundreds of spines in awake, behaving animal models. The first step is to select points based on a Z-stack along a dendritic segment or any cellular structure then 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. In the figure, each scanning line is associated with one spine in a layer II/III pyramidal cell labeled with GCaMP6. A total of 100 pre-selected spines were examined simultaneously, and four representative Ca2+ transients are shown before and after motion correction, demonstrating the improved SNR.

BRAIN MOVEMENT

3D MULTI-LINE SCANNING

ACTIVITY OF SPINES

CA2+ TRANSIENTS FROM FOUR SPINES WITHOUT MOTION CORRECTION

AFTER MOTION
CORRECTION

3D Multi-layer scanning

Imaging multiple frames with different sizes at any position in the scanning volume can be used to follow all events propagating along the cell. The figure shows imaging of the entire length of a pyramidal neuron in vivo, where the small scanned rectangles cover the apical dendrite across multiple layers.

Complex systems for behavioral studies

New acousto-optic based scanning methods, especially those that use Anti-motion technology, are excellent for measurements during an animal model performs tasks such as visuomotor learning, memory retrieval, associative learning or spatial navigation. We offer further special hardware and software tools supporting behavioral studies.