Dendrites and dendritic spines are made of fine, thin and vulnerable processes therefore they are difficult to study. However, using two-photon laser technology, we are able to collect signals from femtoliter volumes of deeper regions of the brain, while avoiding phototoxicity at the same time. Beside of this, spatially confined scanning of axons, dendrites and spines as ROIs allows detecting even subthreshold signals with high signal-to-noise ratio (SNR). We have several configurations with which we are able to visualize dendritic arborization, and perform functional measurements under in vivo and in vitro conditions.
With the highly flexible X and Y mirrors of the galvanometric scanners coupled with the special electronic boards of Femtonics it became possible to scan along any arbitrary scanning pattern. Thus this panel can precisely follow the dendritic arbor of interest using i.e. multiple line scanning method. By limiting the scanning to these regions of high information content while omitting the space between the scanning regions and the background area, both the scanning speed (up to 2 kHz) and the SNR can be increased. See more at FemtoS-Galvo.
Folded frame scanning
Folded frame scanning allows imaging an area along a pre-selected line. The shape of the selected regions are flexible, from areas around straight lines to complex bent curves. This advanced scanning method is useful for following events along curved dendrites with spines and can be advantageous during dendritic measurements in behaving animals as well. In the latter case motion artefacts can be compensated offline as long as the dendrite remains in the scanned area.
Femto2D-Galvo equipped by piezo objective positioner
3D trajectory scanning
3D trajectory scanning is a spatial extension of galvanometric line scanning mode for which the microscope has been equipped with Piezo objective positioner and controlled Roller coaster software module. This advanced scanning mode enables collection of signals from i.e. 25 µm range with 150 Hz which is fast enough to follow changes in the Ca2+-level and to resolve biological functionality through the 3D space.
3D random-access point scanning
Random-access scanning is the fastest method on the field of two-photon microscopy to read out neuronal activity from multiple points or line segments in 3D allowing the measurement form multiple dendritic branches, dendritic spines, or even axonal segments. With fast point scanning, events less than a millisecond apart can be distinguished, or propagation speed along tens of micrometers long dendritic segments can be determined. See more at Femto3D-AcoustoOptic. Video shows dendritic imaging with parallel electrophysiology: spheres represent the fluorescence changes in the determined points, red graph represents the evoked action potential.
3D Multiple trajectory scannig
In the 3D Multiple trajectory scanning, the scanning points of 3D random-access scanning are extended by drifting the focal point along lines (Anti-mOtion technology), which method increases the scanning speed when measuring single trajectories along dendritic segments. With this method, scanning speed can be increased up to 50 fold.
3D Multiple-line scanning
The Anti-mOtion technology can be used not only for fast dendritic scanning, but also imaging spines in awake, behaving animals. The direction of the drift are set to meet the average trajectories calculated from brain motion, this helps to eliminate the motion artefacts. 3D Multi-line scanning enables functional recording of over 150 spines simultaneously in a 500 x 500 x 650 µm3 volume with 200 Hz.
3D Ribbon and Snake scanning
Ribbon scanning is a planar extension, snake scanning is a volume extension of the 3D trajectory scanning mode performed by Anti-mOtion technology. In these cases, the neighboring area around the trajectory is captured by generating drifts either parallel or orthogonal to the trajectory. These two scanning tactics are able to follow the 3D curvature of one or more dendrites at the same time and capture the neighboring areas in a way we can preserve the fluorescent information during motions in behaving animals. Ribbon scanning can run with maintained scanning speed compared to trajectory scanning, therefore it is advantageous when there is no significant z-motion, while snake scanning should be used when the extent of the animal’s motion is larger.
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 visualized in the form of classical Ca2+ transients and raster plots.