INTRODUCTION

All sensations and behaviors are encoded in dynamic activity patterns of neural networks. In other words, complex networks clustering many individual neurons respond to environmental features, such as reward, punishment, visual or auditory features of the environment, etc. Neural networks are extended in 3D space and in most cases they cross many cortical layers of the brain. Thanks to two-photon microscopy and many new scanning methods, we are able to reach the deeper regions of the brain (down to 850 µm) and to study the function of the neuron populations containing hundreds or even more a few thousand cells in 3D. The high spatiotemporal resolution of two-photon imaging technique allows resolving not just single cells, but dendrites and spines in functional brain.

MICROSCOPE CONFIGURATIONS FOR IMAGING OF NEURAL NETWORKS

FemtoS-Galvo

FemtoS-Galvo with the flexible scanning patterns such as multiple line scanning or folded frame scanning supports manual selection of individual cells in a 2D plane without involving the background signal and therefore it is able to maintain high SNR. TravellingSalesman software module of MES gives a possibility to create the shortest pathway visiting defined areas arbitrarily dispersed on the field of view. The short round trip time results in a high measurement speed reaching 100 Hz for a number of points. Using piezo objective positioner or focus tunable liquid lens enables the focal point to be switched between planes with high speed allowing scanning of neuronal populations in a volume.

FemtoS-Resonant

2D fast frame scanning based on resonant scanners (FemtoS-Resonant) combined with fast Z-focusing performed by Piezo objective positioner is a classical approach to study three-dimensional neural networks. In this case, the entire field of view is being imaged continuously by the fast scanner while the objective positioner takes a step between each plane, and the assembled frames give the final measurement volume. The piezo positioner is able to move the objective during the scanning which results slightly tilted planes ensuring higher scanning speed along the volume therefore supporting better temporal resolution.
Video shows volume scanning which was performed by our resonant based microscope equipped with Piezo objective positioner.

Femto3D-AcoustoOptic

3D random-access point scanning

Femto3D-AO enables resolving spatial and temporal complexity of neuronal coding by scanning distributed points with the fastest available speed in a large 3D volume. Several thousands of cells can be measured by random access point scanning with high signal-to-noise ratio by scanning only subregions of the 3D volume. The most responsive cells then can be sub-selected and measured to further increase temporal resolution.

3D chessboard and multicube scanning


Chessboard scanning is using the Anti-mOtion technology, to extend random access points to small squares by drifting the laser beam. These squares can contain the somata with the surrounding area allowing simultaneous imaging of soma in arbitrary 3D locations. The name, chessboard is derived from the layout which is generated by arranging all the squares side-by-side to get a chessboard like pattern containing the selected regions. This pattern allows simultaneous visualization of the soma activity, handling and storing the data and, importantly, to carry out motion correction.
Multi-cube scanning is a spatially extended mode of chessboard scanning where a z dimension is added to the aforementioned squares to cover the whole extent of the somata, therefore to preserve all somatic fluorescence during larger motions.

Cell3DFinder

3D localization of cells in Z-stacks


Cell3DFinder software module of MES helps to find cell centers in 3D image stacks as a set of points automatically instead of selecting the cells manually. It is recommended for the fast selection of a large number of ROIs for network imaging with the Femto3D-AO, RollerCoaster, and TravellingSalesman scanning modes. See more.

Batch analysis

Batch point scan, area scan, and curve analysis functions


This analysis module contains efficient tools to analyze entire multi-ROI measurement sets conveniently. It implements the grouped analysis of imaging data at multiple regions and multiple measurement repetitions, and the handling of the resulting calcium transients, electrophysiology time series, and additional measurement data. See more.

REFERENCES

Single-cell-initiated monosynaptic tracing reveals layer-specific cortical network modules Wertz A, Trenholm S, Yonehara K, Hillier D, Raics Z, Leinweber M, Szalay G, Ghanem A, Keller G, Rozsa B, Conzelmann KK, Roska B, Science (2015)