Performing Accurate Dendrite Imaging Using Two-Photon Lasers

Dendrites and dendritic spine are particularly complex to study, given that they are made up of fine, thin and vulnerable processes. Using two-photon laser technology, however, has offered the opportunity of their examination.

This is possible by collecting signals from femtoliter volumes of deeper regions of the brain, while simultaneously avoiding phototoxicity. In addition, the spatially confined scanning of axons, dendrites and spines as ROIs permits the detection of even subthreshold signals which have a high signal-to-noise ratio (SNR). Dendritic arborization can be visualized with several configurations, in which we can perform functional measurements under in vivo and in vitro conditions.

 

Events less than one millisecond apart can be separated with the FEMTO3D Atlas microscope, and therefore it is possible to determine  propagation speed of regenerative activity at multiple sites of the dendritic tree. All kinds of functional dendritic imaging are supported through 3D random-access point scanning and these advanced versions.

Femto3D Atlas

 

Fast Drift Along the Dendrite

3D trajectory scanning

By drifting the focal point along short 3D trajectories, 3D random-access point scanning is extended, which therefore permits for imaging without interruption at multiple, long dendritic branches. During the drift, this sampling is continuous and so this scanning mode offers a more detailed spatial resolution without resulting in a change of the overall scanning time.

This remains at the same level as during the point scanning. The result of this is that the function of thin dendritic segments, spines and single action potentials can be uncovered. See more in Chiovini et al., Neuron, 2014.

 

Capture Hundreds of Spines of a Neuron

3D multiple-line scanning

This 3D trajectory scanning method is similar to the 3D multiple-line scanning method. The latter was developed specifically to facilitate the imaging of spines during motions. The spines are still covered by the lines if the animal model is moving, thanks to the process of scanning along short lines.

As depicted by the figure below, each scanning line is linked to one spine in a layer II/III pyramidal cell labeled with GCaMP6. The average trajectories calculated from brain motion are used to set the direction of the drift, which thus helps to eliminate the motion artifacts. The simultaneous examination of 100 pre-selected spines is undertaken, and four representative Ca2+ transients are depicted before and after motion correction, which reveals the improved SNR.

Performing Accurate Dendrite Imaging Using Two-Photon Lasers

 

Dendritic and Spine Activity During Small Motions

3D ribbon scanning

3D ribbon scanning is an extension of the 3D multiple-line scanning, performed by Anti-motion technology. 3D ribbon scanning facilitates the imaging of ribbon-shaped surfaces containing dendrites and the surrounding areas. The figure below depicts 3D ribbons encompassing seven dendritic segments with their spines of a GCaMP6-labeled layer II/III pyramidal neuron, with all measurements taken from the brain of a living mouse. For better visualization, the seven ribbons were projected into a 2D image ordering dendrites above each other, and from 40 selected spines, activity was recorded and subsequently visualized in the form of classical Ca2+ transients.

Performing Accurate Dendrite Imaging Using Two-Photon Lasers

 

Dendritic and Spine Activity During Large Motions

3D snake scanning using 3D Anti-motion technology

A volume extension of ribbon scanning, 3D snake scanning contains the entire 3D environment of the dendrite. Therefore, 3D snake scanning supports the quest to image dendrites in larger animals, or behavioral protocols, wherein there can be a large amplitude of motion. The figure below depicts fast snake scanning, as performed at 10 Hz in the chosen dendritic region of a V1 pyramidal neuron.

3D snake scanning using 3D Anti-motion technology

 

Imaging Along the Entire Length of Dendritic Arbor

3D multi-layer scanning

One can use the imaging of multiple frames with different sizes at any position in the scanning volume in order to follow all events propagating along the cell. Imaging of the entire length of a pyramidal neuron in vivo is depicted by the lower image, wherein the small scanned rectangles cover the apical dendrite across multiple layers. We are additionally able to use motion compensation to record fluorescent signals and responses to visual stimuli during the process of the animal running on a treadmill. See also Szalay et al., Neuron, 2016.

3D multi-layer scanning

 

References and Further Reading

About Femtonics Ltd.

Femtonics focuses on the research and development of two-photon laser scanning microscopes for the booming area of cutting-edge brain research and pharmaceutical development. Our specialty is represented by the acousto-optical scanner-based Femto3D Atlas microscope which takes the ability to scan the three-dimensional sample with astonishing speed and thereby it is unique on the market.

In the field of traditional galvanometric and resonant scanner-based systems, we present our customers the flexibility and freedom to customize their own products according to their vision and objective.

The high-valuable measurement and analysis solutions of our MES control software enable scientists to perform a wide variety of experiments. A well-selected microscope working together with the appropriate software modules shapes the customer’s idea into a remarkable product.

Our technology is pioneering and innovative in the field of microscopic imaging. It has been awarded at high levels namely by winning a number of grants and professional prizes and confirmed by our publications in the most prestigious international scientific journals and our presentations at the major conferences.

However, the main achievements for us are customer satisfaction and the scientific results and breakthroughs produced by our microscopes. We are proud of the fact that many results on the field of neuroscience have been published in the highest quality international scientific journals with the tools that we have designed.


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Last updated: Feb 6, 2020 at 9:27 AM

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