Using Two-Photon Lasers to Perform Accurate Network Imaging

The dynamic activity patterns of neural networks have encoded within them all sensations and behaviors. To put it another way, complex networks of many individual neurons respond to visual or auditory stimuli, environmental features, reward or punishment, etc. Neural networks reach over 3D space and most cross lots of cortical layers within the brain.

It is now possible, thanks to two-photon microscopy, for neuroscientists to extend their analysis to the previously-unexplored deep regions of the brain (down to 850 µm), and in so doing assess and, by using many technical solutions, analyze them at a high spatiotemporal resolution.

Using Two-Photon Lasers to Perform Accurate Network Imaging


Femto3D Atlas


The FEMTO3D Atlas microscope has an unparalleled ability to enable the resolution of precise spatial and real-time complexity of neuronal coding, by scanning a large number of cells distributed in a near cubic millimeter 3D volume.

The Imaging of Cell Populations in 3D is the Fastest with FEMTO3D Atlas

3D random access point scanning, Cell3DFinder

By 3D random-access point scanning, several thousand cells can be analyzed and measured near-simultaneously (30 kHz). This is done by limiting the imaging to sub-regions of the 3D volume- and the resulting rapid scanning speed and signal-to-noise ratio. Automatically, the Cell3DFinder software module can find cell centers in 3D image stacks, and can then depict them as a set of points. The figure (A-D) demonstrates the changes in Ca2+ concentration of 2000 cells from the visual cortex of a GCaMP-expressing mouse, shown as a function of time. See more: Wertz et al., Science, 2015; Katona et al., Nat Meth, 2012.

Using Two-Photon Lasers to Perform Accurate Network Imaging


Imaging of Multiple Somata Distributed in 3D in Behaving Animals

3D chessboard scanning using 3D Anti-motion technology

Chessboard scanning is the term for a planar extension of random-access point scanning when using the Anti-motion technology. In Chessboard scanning, the laser beam is drifted which allows for random-access points to be extended to small squares. These squares can be located anywhere in a near cubic millimeter volume, and they include the somata with the surrounding area. In this way, up to 300 somata can be measured at the same time.

The ‘chessboard’ name derives from the arrangement of the squares, which helps to visualize somata, to assess and analyze their activity, and correct for motion artifacts. The accompanying video depicts in vivo neuronal activity, Ca2+ transients from 100 neuronal somata from the mouse V1 region, labeled with GCaMP6. See also Szalay et al., Neuron, 2016.


Imaging of Multiple Somata During Large Motions

3D multi-cube scanning using 3D Anti-motion technology

There is a spatially-extended mode of chessboard scanning, called multi-cube scanning. In multi-cube scanning, once the squares have been created, a Z dimension is added to the squares to cover the entirety of the somata. This thereby preserves all fluorescent information, even during huge amplitude movements.

3D multi-cube scanning using 3D Anti-motion technology


FEMTOSmart provides extensive and widely applicable imaging capabilities, and, thanks to its modularity, the study of neuronal networks from wide variations of perspectives is facilitated, allowing them to save time during the experimental work.



Imaging of somata as ROIs in 2D

FEMTOSmart Galvo with flexible ROI scanning possibilities

The FEMTOSmart Galvo has flexible scanning patterns, including 2D multiple-line, 2D random-access point, and folded-frame scanning. Thanks to these functionalities, it supports the manual selection of individual cells in a 2D plane. It is possible, by skipping measurement of the entire field, to reach a rapid scanning speed and to maintain a high signal-to-noise ratio.

It is also possible, when using the TravellingSalesman additional software module, to establish the shortest pathway whilst visiting defined points, arbitrarily dispersed on the field of view. A higher measurement repetition speed results from the short round-trip time; up to 100 Hz for about 30 cells.

Multiple-line scanning

Imaging of somata as ROIs in 2D


Fast Scanning of the Entire FOV or an Entire Volume

FEMTOSmart Resonant equipped with Piezo objective positioner kit

A well-known and established approach for studying three-dimensional neural networks is fast-frame scanning by FEMTOSmart Resonant, when combined with fast Z-focusing performed by a Piezo objective positioner kit. Indeed, here the entire field of view is continuously imaged by the fast scanner, whilst the objective positioner moves between planes. This may be used in the recording of different cortical layers, or the frames may be assembled to volumes which would produce a four-dimensional dataset.


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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