• Femto3D AcoustoOptic

    Novel acousto-optic scanner-based microscopes for 3D imaging

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Femto3D AcoustoOptic Product line

THE FLAGSHIP PRODUCTS

3D imaging

Astonishing speed, real-time measurements

The novel features of these microscopes are the electrically tunable acousto-optic (AO) deflectors, which do not contain scanning mirrors or any other slowly moving mechanical components, so the positioning of the focal spot is fast, flexible, stable and independent of the travelling distance. This position- ing freedom results an extremely high scanning speed, up to 30 kHz at any 3D location under the objective in a cubic millimeter volume.

Acousto-optic deflectors

The highest technology

In contrast to a traditional scanner, the Femto3D AcoustoOptic microscopes contain two pairs of AO deflectors (AODs) which are responsible for the X, Y scanning, and Z focusing. The AODs control the optical beam spatially, utilizing the interaction between sound and light waves. In imaging, AODs diffract laser beam through ultrasonically generated gratings, and sound waves induce pressure fluctuation in the AOD crystal. By changing the sound frequency, the focus point also changes. The operation of the AOD is silent, it does not disrupt the experiment and the animal sample. The integrated electric stabili- zation of the 3D scanner provides long-term stability in measurements without the need for maintenance.

Focusing on the ROIs

Flexible 3D scanning patterns

Using the AO scanning technology in combination with an easy-to-use 3D data acquisition software package provides maximal flexibility in fast selection of ROIs. The preselected ROIs can be precisely and rapidly targeted without wasting measurement time on unnecessary background areas or volume ele- ments increasing further the measurement speed and the signal-to-noise ratio by several orders of magnitude in comparison to classical raster scanning. The extremely high scanning speed enables to collect data near simultaneously from 2000 ROIs distributed in 3D by random access point scanning, and many other 3D scanning patterns and a vast array of measurement possibilities, online and offline analysis functions are available.

Fast scanning with high SNR

The highest technology

If we compare the relative gains in measurement speed (vgain) and signal-to-noise ratio (SNRgain) for 3D AO scanning relative to traditional raster scanning of the same sample volume, we can say that the vgain × (SNRgain)2 is equivalent to the ratio of the total image volume to the volume covered by the pre-se- lected scanning points. This ratio can be very large, up to over 106 per ROI, which makes 3D AO scanning suitable for precise multisite activity measure- ments, especially when ROIs are sparsely dispersed in the 3D volume..

Scanning volume

The highest technology

Thanks to large aperture AO deflectors, angular dispersion compensation and diffraction-based optical modelling, in the case of in vivo measurement, the size of the scanning volume is 500 x 500 x 700 μm3, while it is 650 x 650 x 700 μm3 under in vitro conditions. The transmission of the AODs have been im- proved for longer, 850-950 nm wavelengths supporting effective GCaMP imaging in the deep tissues. The imaging depth is different in the cases of the types of the biological labelling and it is depends also on the power of the laser source.

Preserved spatial resolution

The large aperture of the new AODs, the improved angular dispersion compensation, and the dynamic error compensation provide the expected spatial resolution over the entire scanning volume. The precision of positioning the laser remains <100 nm, the diameter of point spread function (PSF) is remains below 500 nm along X, Y and below 2.5 μm Z axes in the center which size is smaller than the average diameter of neuronal somata.

Astonishing speed for imaging of cell populations

Near real-time measurements

3D random-access point scanning is the fastest method to read-out neuronal activity because it enables multiple points, distributed in 3D, to be imaged si- multaneously. Within a large scanning volume, it is approximately one million times faster than other frame-by-frame scanning methods. This imaging speed means that thousands of individual neurons (e.g. in different cortical layers) can be measured with microsecond resolution simultaneously, revealing the dynamics of neuronal networks.

Fast 3D dendritic imaging

Follow the action potential

3D random-access point scanning is the fastest method to read-out neuronal activity because it enables multiple points, distributed in 3D, to be imaged si- multaneously. Within a large scanning volume, it is approximately one million times faster than other frame-by-frame scanning methods. This imaging speed means that thousands of individual neurons (e.g. in different cortical layers) can be measured with microsecond resolution simultaneously, revealing the dynamics of neuronal networks.

Anti-motion technology

For motion correction

The microscope control software incorporating the latest AO driving theory enables the AODs to drift the focal spot in 3D in any specified direction. This drifting technology allows extending the individual scanning points to small lines, surface or even volume elements. These elements can cover not only the interesting regions but also the neighboring areas and they can be set parallel to the average direction of brain movement, thereby you can preserve fluo- rescence information during motions caused by vessel pulsing, respiration, locomotion, or behavior and using the data for motion correction.

Advanced scanning modes

For motion correction

The surface scanning methods are optimized for speed, while the methods based on volume imaging are optimized for large amplitude movements, main- taining the 10–1,000 Hz sampling rate necessary to resolve neural activity at the individual ROIs. Each scanning mode is useful for different neurobiologi- cal aims: ribbon scanning, snake scanning, 3D multiple line scanning are optimal for different dendritic measurements, while chessboard scanning and multi-cube scanning are best for somatic recordings.

Technology Overview

Femto3D AcoustoOptic microscopes combine an intoxicat- ing blend of high-tech science, engineering, refinement in 3D measurements and technology. Being unique in the world, these imaging systems represent the fast, 3D two-photon microscopes on the market. They are capable of scanning neuronal, dendritic, and other neuropil activi- ties about one million faster compared to classical scan- ning methods with preserved two-photon resolution. 3D AO imaging opens new horizons in the field of neuro- science.

Features

  • 30-50 kHz/ROI scanning speed in 3D

  • In vitro measurements and in vivo deep tissue imaging over 650 μm

  • Over 500 μm × 500 μm × 650 μm scanning volume in vivo

  • New-generation acousto-optics provides over 4-fold im- proved excitation

  • Automatic beam stabilization

  • Angular dispersion compensation

  • Multiple wavelengths and automatic wavelength tunability

  • Dynamic compensation for optical errorsDynamic compensation for optical errors

  • Intelligent software control

Benefits

  • Novel 3D scanning methods both for in vivo and in vitro

  • Flexible ROI scanning possibilities

  • Fast recording of over 2000 soma distributed in 3D

  • Imaging of dendritic branches without interruption

  • Simultaneous measurements of thousands of spines with high SNR

  • Motion correction in behaving animals

  • Improved in total excitation efficiency for genetically encoded calcium indicators

  • No mechanical vibration induced noise

  • High sensitivity, bright signals

  • A vast of measurement and analysis possibilities

Demo rooms

BUDAPEST, HUNGARY

Group Leader: Balázs Rózsa
Two-Photon Imaging Center
 Institute for Experimental Medicine
Hungarian Academy of Sciences
Contact

BASEL, SWITZERLAND

Group Leader: Botond Roska
Structure and Function of Neural Circuits Group
Friedrich Miescher Institute
Contact

MARSEILLES, FRANCE

Head: Christophe Bernard; Ivo Vanzetta
Physiology & Physiopathology of Neuronal Networks Group
French National Institute of Health and Medical Research
Contact

SZEGED, HUNGARY

Principal Investigator: Gábor Tamás
Research Group for Cortical Microcircuits

Hungarian Academy of Sciences, University of Szeged
Contact

FLORIDA, USA

Group Leader: David Fitzpatrick
Functional Archtitecture and Development of Cerebral Cortex
Max Planck Institute
Contact

UMEA, SWEDEN

Group Leader: Paolo Medini
Department of Molecular Biology
Umea University
Contact

References

Fast 3D Imaging of Spine, Dendritic, and Neuronal Assemblies in Behaving Animals. Gergely Szalay, Linda Judak, Gergely Katona, Katalin Ocsai, Gabor Juhasz, Mate Veress, Zoltan Szadai, Andras Feher, Tamas Tompa, Balazs Chiovini, Pal Maak, Balazs Rozsa, Neuron (2016)

Accurate spike estimation from noisy calcium signals for ultrafast three-dimensional imaging of large neuronal populations in vivo.Thomas Deneux, Attila Kaszas, Gergely Szalay, Gergely Katona, Tamas Lakner, Amiram Grinvald, Balazs Rozsa & Ivo Vanzetta, Nature Communications (2016)

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)

Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes, Gergely Katona, Gergely Szalay, Pál Maak, Attila Kaszas, Mate Veress, Daniel Hillier, Balazs Chiovini, E Sylvester Vizi, Botond Roska & Balazs Rozsa, Nature Methods (2012)

Localized Neuron Stimulation with Organic Electrochemical Transistors on Delaminating Depth Probes. Williamson A, Ferro M, Leleux P, Ismailova E, Kaszas A, Doublet T, Quilichini P, Rivnay J, Rozsa B, Katona G, Bernard C, Malliaras GG., Advanced Materials (2015)

Dendritic spikes induce ripples in parvalbumin interneurons during hippocampal sharp waves. B Chiovini, G F Turi, G Katona, A Kaszas, D Palfi, P Maak, G Szalay, M F Szabo, Z Szadai, Sz Kali and B Rozsa, Neuron (2014)

Sensitization of neonatal rat lumbar motoneuron by the inflammatory pain mediator bradykinin Mouloud Bouhadfane, Attila Kaszas, Balazs Razsa, Ronald M Harris-Warrick, Laurent Vinay, Frederic Brocard, eLife (2015)

Unitary GABAergic volume transmission from individual interneurons to astrocytes in the cerebral cortex. Rozsa M, Baka J, Borde S, Rozsa B, Katona G, Tamas G, Brain Struct Funct. (2017)