High scanning speed along dendrites and spines in 2D by FemtoSmart Galvo

Flexible ROI scanning possibilities

FemtoSmart Galvo

The adaptable X and Y mirrors of the galvanometric scanner, coupled with the special electronic boards from Femtonics, can follow the tortuous protrusions of the dendritic arbor precisely using the 2D random-access point, multiple-line and folded frame scanning methods.

random-access point

multiple line

folded frame

The fastest in 2D

2D multiple-line scanning

The adaptable X and Y mirrors of the galvanometric scanner, coupled with the special electronic boards from Femtonics, can follow the tortuous protrusions of the dendritic arbor precisely using the 2D random-access point, multiple-line and folded frame scanning methods. By limiting the scanning to the interesting dendritic segments carrying spines and omitting the space between them, both the scanning speed (up to 2 kHz) and the SNR can be increased using 2D random-access point and multiple-line scanning modes. The advantage is multiple-line scanning is the imaging without interruption at multiple dendritic branches, it is the cost-effective, alternative solution compared to the acousto-optic scanner based line scanning methods.

Measurement of dendritic and spine activity by multiple-line scanning

Spontaneous Ca2+responses along the lines

Imaging dendrites and spines with motion correction

Folded frame scanning

Using folded frame scanning, an area along a pre-selected line can be imaged. The selected regions can take many shapes, 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 also be advantageous for dendritic measurements in behaving animal models where motion artefacts are a common problem. The images are corrected for motion offline by the control software, as long as the dendrite remains in the scanned area.

Measurement of dendritic activity by folded frame scanning

Folded frame scanning along a dendrite during motions

After motion correction

AP induced Ca2+ signals from 6 dendritic regions and 12 neighboring spines of a Fluo-SF loaded pyramidal cell measured by 3D trajectory scanning.

Follow dendrites in 3D

3D trajectory scanning

The microscope equipped with piezo objective positioner and controlled by Roller Coaster software module enable the user to follow the arborization in the 3D samplte by performing 3D trajectory scanning. 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.

We want to open new perspectives in the microscopic imaging with ultimate 3D scanning solutions

Femto3D AcoustoOptic working together with our measurement and analysis software package enables the scientist to reveal 3D neural network and dendritic activities in the brain of awake animal models during behavior.

The fastest imaging of 3D dendritic arborization by Femto3D AcoustoOptic

3D ROI scanning methods

Femto3D AcoustoOptic

With the Femto3D AcoustoOptic microscopes events less than a millisecond apart can be separated, and therefore propagation speed of regenerative activity determined at multiple sites of the dendritic tree. 3D random-access point scanning and its advanced versions support all kinds of functional dendritic imaging.

Capture hundreds of spines of a neuron

3D multiple-line scanning

The 3D multiple-line scanning method is similar to the 3D trajectory scanning, however it was developed for imaging spines during motions. Thanks to the scanning along short lines, the spines are still covered by the lines if the animal model is moving. In the figure, each scanning line is associated with one spine in a layer II/III pyramidal cell labeled with GCaMP6. The direction of the drift is set to meet the average trajectories calculated from brain motion, helping to eliminate the motion artefacts. 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

Dendritic and spine activity during small motions

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. Figures show 3D ribbons encompassing seven dendritic segments with their spines of a GCaMP6-labeled layer II/III pyramidal neuron measured 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.

RIBBONS COVERED DENDRITES
WITH THEIR SPINES IN VIVO

BEFORE MOTION CORRECTION

AFTER MOTION CORRECTION

CA2+ RESPONSES FROM
SELECTED SPINES

Dendritic and spine activity during large motions

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.

SNAKE SCANNING

PROJECTIONS OF THE DENDRITE
AFTER MOTION CORRECTION

CA2+ RESPONSES FROM SELECTED SPINES

Imaging along the entire length of dendritic arbor

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 lower 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. Motion compensation enables us to record fluorescent signals and responses to visual stimuli while the animal is running on a treadmill. See also Szalay et al., Neuron, 2016.