OVERVIEW

Femto3D-AcoustoOptic microscope is the first fast, 3D, two-photon microscope on the market. The microscope is capable of scanning neuronal, dendritic, and other neuropil activities about one million faster as compared to previous realizations within a large (about cubic millimeter) scanning volume with preserved two-photon resolution.

The microscope, using electrically tunable crystals, can focus the excitation point with up to 53 kHz speed to any 3D location under the objective without mechanical restrictions reaching sub-millisecond temporal resolution in a millimeter z-dimension scanning range.

„3D AO imaging will open new horizons in the field of neuroscience”


VIRTUAL PRODUCT WALK AROUND

FEMTONICS IS THE FIRST
IN 3D TWO-PHOTON MICROSCOPY

Femto3D-AcoustoOptic microscope combines an intoxicating blend of high-tech science, engineering, refinement in 3D measurements, and technology. Our team developed and published the first 3D AO microscope in the world in 2003. During the more than ten year period we have spent in the field of 3D microscope development we realized and patented many revolutionary new technical solutions to provide users with a 3D system that has incredibly good parameters (such as large scanning volume, high speed, and preserved good spatial resolution) for an affordable price.


REVOLUTION
IN 3D MICROSCOPE
TECHNOLOGY

In the past ten-year period we have developed and patented many new technologies to provide our customers with the best 3D microscope in the world. Among other features, our microscopes now have:
  • optimal arrangement of optical elements based on diffraction-based modeling (over 10× more effective 3D scan head)
  • optimized angular dispersion compensation
  • functionally separated arrangement of AO deflectors for the highest performance
  • dynamic optical error compensation
  • optimized deflector driver signals, deflector geometry, manufacturing, bandwidth and crystal orientation
  • new AO deflector technology for longer wavelengths (for GECIs)
  • minimized optical path length to maximize detection (travelling detector system) with ultrasensitive GaAsP PMTs (>40% quantum efficiency)
  • integrated measurement control and analysis software
  • 4× more effective radio-frequency drives

NEW SCANNING METHODS

The use of fast, electrically controlled lenses in combination with an easy to use 3D data acquisition software package provides maximal flexibility in fast selection of regions of interests with high speed and controlling of measurements.

"Having the third dimension in control and full flexibility in XY scanning gives the ultimate freedom to the experimenter in selecting regions of interests and tuning signal-to-noise ratio to the theoretical limits."

FULL SPECIFICATION

REFERENCES

3D IMAGING OF NEURONAL NETWORKS

The spatial and temporal complexity of neuronal coding requires recording of information flow and processing, not only from a single point or plane, but at the level of large networks distributed in 3D, in large volumes. Therefore several new optical methods have been developed recently for the fast readout of neuronal network activity in 3D. However, only a few of them is based on two-photon excitation which allows deep brain imaging. Among these methods, 3D AO scanning provides the largest increase in the signal-to-noise ratio and measurement speed, more quantitatively:

CHESSBOARD SCANNING
For in vivo imaging of hundreds of soma simultaneously in 3D

The microscope is now capable of using the novel 3D Anti-mOtion scanning technique allowing fast 3D imaging of neuronal network in behaving animals. In Chessboard scanning, scanning points are extended to small squares containing the somata and surrounding area. The special flexibility of the 3D scanning capability allows simultaneous imaging along multiple small squares placed in arbitrary locations in 3D. The name, chessboard is derived from the layout which is generated by arranging side-by-side all the squares containing the selected regions. This pattern allows simultaneous visualization of the activity of the somata, handling and storing the data and, importantly, to correct for motions. As a result, it makes possible to recover all high speed three-dimensional fluorescence data during the animal’s movements, therefore to follow neuronal network activity in behaving animals.
Figure shows chessboard scanning of neuronal somata and the measured Ca+2 transients resulted by neuronal activity. Mouse V1 region in vivo, labeled with GCaMP6 fluorescent sensor.

3D MULTI-CUBE SCANNING
Imaging somata during large amplitude motions

Multi-cube scanning is an extended mode of chessboard scanning where a z dimension is added to cover the z extent of the somata to preserve all somatic fluorescence points during motions. The somata as ROIs are ordered as cubes next to each other for visualization and calcium transients are recorded from each cube corrected to motions. With this method for example 50 somata can be recorded at 50 Hz when using cubes made of 50 x 10 x 5 voxels. Figure shows simultaneous measurements of ten GCaMP6 labeled somata.

UPGRADE KIT FOR DEEP BRAIN IMAGING

We developed seven novel methods to improve deep penetration and to extend the entire 3D z-scanning range from the surface down to over 500 µm depth in vivo.



Several thousands of cells can be measured simultaneously. The most responsive ones can be subselected and measured with higher temporal resolution. Neuronal responses can be visualized as traces or color coded images.

3D IMAGING OF LARGE ASSEMBLIES OF NEURONS

PRESERVED SINGLE AP RESOLUTION

Single BAPs could be resolved in distinguishable Ca2+ transients induced by a train of three APs used AO z focusing in 1200 µm scanning range and along the x axis (760 µm).

3D DENDRITIC IMAGING

The main advantage of 3D AO scanning is that the high resolution characteristic of two-photon microscopy is preserved therefore dendritic imaging with spine resolution is possible in the center (300 µm × 300 µm × 200 µm volume in the middle of the entire scanning volume).

FAST DENDRITIC IMAGING with 3D POINT SCANNING

Random-access point scanning is the fastest method to read-out neuronal activity in large volumes with high resolution in 3D. The method allows simultaneous imaging of multiple points, or line segments, at multiple dendritic branches.

3D MULTI-LINE SCANNING
For following neuronal activity in spines

For scanning spines at high speed in vivo, points of 3D random-access point scanning are extended by drifting the focal point along short lines without increasing the overall scanning time. The first step is to select points based on a z-stack along a dendritic segment or any cellular structure then simply define the 3D orientation and extent of the 3D drifts to the main direction of motion. Finally, the average trajectories are calculated cancelling effect of the brain motion. 3D Multi-Line scanning enables functional recording of over 150 spines simultaneously in a 500 x 500 x 650 µm3 volume.

3D RIBBON SCANNING
For 3D dendritic imaging

Ribbon scanning is an extended trajectory scanning using 3D Anti-mOtion technology which also captures the neighboring area around the trajectory of dendrites to preserve fluorescent information during motions. The neighboring area is scanned by generating drifts either parallel or orthogonal to the trajectory. 3D Ribbon scanning can follow the 3D curvature of one or more dendrites at the same time, for example it enables functional recording with up to 3 kHz on a 50 µm long dendritic segment or imaging of activity simultaneously in over 12 spiny dendritic segments.
Figure shows 3D ribbons encompassing seven dendritic segments. Fluorescent transients were recorded simultaneously along the ribbons and data were projected into a 2D image ordering dendrites above each other. Recorded activity from selected spines and dendrites were visualized in the form of classical Ca2+ transients and raster plots.

3D SNAKE SCANNING
For imaging dendrites in a 3D volume during large amplitude motions

3D Snake scanning is a volume extension of the ribbon scanning that contains the entire 3D environment of the dendrite. In larger animals or at certain surgery or behavioral protocols, the amplitude of motion can be larger. To sufficiently preserve fluorescence signals even in these cases, the surface elements can be extended to tortuous cuboids by using this scanning mode. Figure shows fast snake scanning performed at 10 Hz in the selected dendritic region of a V1 pyramidal neuron. Fluorescence data were maximal intensity projected to a straightened 2D image. The representative spontaneous calcium responses are measured from the selected volume elements and the transients are shown following 3D motion correction.

3D MULTI-LAYER SCANNING
For imaging along the entire length of dendritic arbor

Albeit the majority of neuronal computation occurs in long neuronal processes situating in multiple layers in the brain, previous imaging methods were not able to readout these 3D activity patterns. However, Femto3D-AO microscopy combining the low photo-toxicity of low-power temporal oversampling (LOTOS) with the maximal flexibility of electric 3D scanning allows imaging of cells spanning across many layers, e.g. cortical pyramidal cells all the way up from basal to apical dendritic arbors. Imaging of multiple frames with different sizes and at any position in the scanning volume can be used to follow all events propagating along the cell. Scanning of the neighborhood of the processes allows preserving all fluorescent signals and calcium transients in spite of the brain’s motion. The multi-layer scanning method is not limited to a single dendrite or axon, but even multiple neurons can be simultaneously imaged with their dendritic arbor. Figure demonstrates multi-layer imaging of the activity of a layer V neuron in an awake animal at 41 different depth levels over a 500 µm z range at 30 Hz. Motion artifacts along the x and y axes were eliminated and calcium transients were recorded for each ROI.

PROPAGATION SPEED OF FAST REGENERATIVE EVENTS

The 2014 Nobel Prize in Chemistry was awarded to three scientists, Eric Betzig, Stefan W. Hell and William E. Moerner for spatial super-resolution microscopy. Our Femto3D-AO microscope provides a novel super resolution method for microscopy: the temporal super-resolution to separate Ca2+ responses of neighboring subcellular compartments with over 50 microsecond precision (temporal superresolution microscopy described for the first time by Katona et al. 2012).

INERTIA FREE 3D SCANNING

Acousto-optical scanning does not rely on mirrors and doesn’t contain any moving parts, so positioning the focal spot of the two-photon microscope is performed extremely fast and – importantly – independent of the travelled distance. This makes it suitable for 3D random-access scanning modes.

LARGE FIELD OF VIEW AND
EXTENDED Z-SCANNING RANGE

Thanks to diffraction-based optical modelling, large aperture AO deflectors, an improved radio-frequency drive, angular dispersion compensation, and many other technological steps, now we can provide large field of view (800 µm x 800 µm) in over a 1400 µm z-scanning range.

PRESERVED SPATIAL RESOLUTION

The large aperture of the new AO deflectors, the improved angular dispersion compensation, and the dynamic error compensation provides an improved spatial resolution over the entire scanning volume. The diameter of point spread function (PSF) is minimized along X, Y and Z axes and it remains smaller than the average diameter of neuronal somata in the entire scanning volume. Moreover, we can target the focal spot with 50 nm precision into any point in the entire (800 µm × 800 µm × 1,400 µm) 3D volume, therefore neuropil contamination can be avoided.

3D ANTI-MOTION TECHNOLOGY

Femto3D-AcoustoOptic microscope is now capable of using the novel 3D Anti-mOtion scanning technique allowing fast 3D imaging of spine and neuronal assemblies or multiple dendritic segments in behaving animals. This patented 3D scanning method extends the points of the random-access point scanning method to fast scanned 3D lines. Based on these lines, scanning is executed on 3D lines, surface or volume elements with maintained temporal resolution. The scanned parts cover not only the pre-selected ROIs but also the neighboring areas or volume elements. This gives an opportunity to decrease the motion artifacts by more than one order of magnitude in behaving animals.

SCANNING MODES

NOVEL MEASUREMENT POSSIBILITIES IN AWAKE, BEHAVING ANIMALS


  • Imaging a large network of over 130 neurons at subcellular resolution in a scanning volume of up to 500 × 500 × 650 μm3 with an order of magnitude larger signal-to-noise ratio when motion artifact elimination is on
  • Increased transmission in the range of 850-950 nm for GCaMP6 imaging
  • Simultaneous imaging of large parts of the dendritic arbor and neuronal networks in a z scanning range of over 650 µm
  • Fast 3D recording of over 150 dendritic spines in 3D
  • Fast parallel imaging of activity in over 12 spiny dendritic segments
  • Measurement of neuronal networks in multiple planes with over the speed of resonant scanning.

CHESSBOARD SCANNING
For in vivo imaging of hundreds of soma simultaneously in 3D

In Chessboard scanning, scanning points are extended to small squares containing the somata and surrounding area. The special flexibility of the 3D scanning capability allows simultaneous imaging along multiple small squares placed in arbitrary locations in 3D. The name, chessboard is derived from the layout which is generated by arranging side-by-side all the squares containing the selected regions. This pattern allows simultaneous visualization of the activity of the somata, handling and storing the data and, importantly, to correct for motions. As a result, it makes possible to recover all high speed three-dimensional fluorescence data during the animal’s movements, therefore to follow neuronal network activity in behaving animals.
Figure shows chessboard scanning of neuronal somata and the measured Ca+2 transients resulted by neuronal activity. Mouse V1 region in vivo, labeled with GCaMP6 fluorescent sensor.

3D MULTI-CUBE SCANNING
Imaging somata during large amplitude motions

Multi-cube scanning is an extended mode of chessboard scanning where a z dimension is added to cover the z extent of the somata to preserve all somatic fluorescence points during motions. The somata as ROIs are ordered as cubes next to each other for visualization and calcium transients are recorded from each cube corrected to motions. With this method for example 50 somata can be recorded at 50 Hz when using cubes made of 50 x 10 x 5 voxels. Figure shows simultaneous measurements of ten GCaMP6 labeled somata.

3D MULTI-LAYER SCANNING
For imaging along the entire length of dendritic arbor

The 650 µm deep z-scanning range of the Femto3D-AcoustoOptic microscope gives possibility to image cells spanning across many layers, e.g. cortical pyramidal cells all the way up from basal to apical dendritic arbors. Imaging of multiple frames with different sizes and at any position in the scanning volume can be used to follow all events propagating along the cell. Scanning of the neighborhood of the processes allows preserving all fluorescent signals and calcium transients in spite of the brain’s motion. The multi-layer scanning method is not limited to a single dendrite or axon, but even multiple neurons can be simultaneously imaged with their dendritic arbor. Figure demonstrates multi-layer imaging of the activity of a layer V neuron in an awake animal at 41 different depth levels over a 500 µm z range at 30 Hz. Motion artifacts along the x and y axes were eliminated and calcium transients were recorded for each ROI.

3D RIBBON SCANNING
For 3D dendritic imaging

Ribbon scanning is an extended trajectory scanning which also captures the neighboring area around the trajectory of dendrites to preserve fluorescent information during motions. The neighboring area is scanned by generating drifts either parallel or orthogonal to the trajectory. 3D Ribbon scanning can follow the 3D curvature of one or more dendrites at the same time, for example it enables functional recording with up to 3 kHz on a 50 µm long dendritic segment or imaging of activity simultaneously in over 12 spiny dendritic segments.
Figure shows 3D ribbons encompassing seven dendritic segments. Fluorescent transients were recorded simultaneously along the ribbons and data were projected into a 2D image ordering dendrites above each other. Recorded activity from selected spines and dendrites were visualized in the form of classical Ca2+ transients and raster plots.

3D SNAKE SCANNING
For imaging dendrites in a 3D volume during large amplitude motions

3D Snake scanning is a volume extension of the ribbon scanning that contains the entire 3D environment of the dendrite. In larger animals or at certain surgery or behavioral protocols, the amplitude of motion can be larger. To sufficiently preserve fluorescence signals even in these cases, the surface elements can be extended to tortuous cuboids by using this scanning mode. Figure shows fast snake scanning performed at 10 Hz in the selected dendritic region of a V1 pyramidal neuron. Fluorescence data were maximal intensity projected to a straightened 2D image. The representative spontaneous calcium responses are measured from the selected volume elements and the transients are shown following 3D motion correction.

3D MULTI-LINE SCANNING
For following neuronal activity in spines

For scanning spines at high speed in vivo, points of 3D random-access point scanning are extended by drifting the focal point along short lines without increasing the overall scanning time. The first step is to select points based on a z-stack along a dendritic segment or any cellular structure then simply define the 3D orientation and extent of the 3D drifts to the main direction of motion. Finally, the average trajectories are calculated cancelling effect of the brain motion. 3D Multi-Line scanning enables functional recording of over 150 spines simultaneously in a 500 x 500 x 650 µm3 volume.

RELATED PUBLICATION

Fast 3D Imaging of Spine, Dendritic, and Neuronal Assemblies in Behaving Animals. Szalay G, Judak L, Katona G, Ocsai K, Juhasz G, Veress M, Szadai Z, Feher A, Tompa T, Chiovini B, Maak P, Rozsa B, Neuron (2016)

OVERVIEW

Femto3D-AcoustoOptic microscope is supported with our modular measurement control and analysis software package called MES. MES was designed with the day-to-day lab experience in the field of cellular and network imaging. MES supports 3D ROI selection and immediate ROI activity analysis necessary for high throughput measurements.

3D ROI SELECTION

New software tools with custom designed user interface support the most flexible 3D scanning pattern selection of multiple points, multiple areas or 3D trajectories as the basis of the advanced scanning modes. Various software tools help selection, such as 3D cell localization, automatic pattern generation and XYZ adjustment.

EXPERT IN AO DRIVING

The scan control software incorporates the latest AO driving theory allowing the special SNR optimizing scanning methods, and also enables dynamic error compensation ensuring excellent optical properties of this complex optical system.

INTEGRATION

MES – besides performing all standard microscopy functions – fully integrates the control of all hardware units in the microscope:
  • focusing, PMTs, light path actuators
  • AcoustoOptic scanners
  • camera handling
  • auxiliary digital and analog channels to interface stimulators or behavior control devices
  • sample stage and patch pipette devices

FLEXIBLE ENVIRONMENT

The MES software is written in the MATLAB high level programming environment. This enables rapid code development and opens the data to the users to the deepest levels. Data can be interactively accessed at the MATLAB command prompt or through self-developed script or GUI tools beyond the wide opportunities of the existing program package.

3D DATA VISUALISATION

Fluent software interface allows real-time visualization of the recorded 3D fluorescent data. Using direct data access and MATLAB’s 3D rendering capabilities, any custom 3D visualization or animation can be easily constructed.

EXTENSION MODULES

MES can be extended by various features with additional modules:
  • batch analysis
  • electrophysiology importer
  • 3D cell localization
  • excel interface for analysis
  • visual stimulation / intrinsic imaging
  • sample stage and pipette manipulation
  • sample stabilization
  • ratio imaging calculations
  • electrophysiology
  • measurement automation

3D AO SCAN HEAD
UPGRADE KIT

The AO upgrade kit is a cost effective solution which can be easily added to many existing upright and inverted microscopes.

UPGRADE KIT FOR YOUR EXISTING MICROSCOPES

Extend your existing microscope to 3D imaging.

The 3D microscope building kit consists of:
  • a 3D AO scan head
  • a digital automatic beam alignment system which preserves the diffraction limited resolution of the whole system
  • detectors
  • a controller box
  • software; PC
  • two mirrors with six mechanical freedom allows precise alignment of the scanned beam relative to the objective optical axis
  • adjustable high-tensile aluminium legs which can flexible tuned to custom made two-photon microscopes

UPGRADE KIT
FOR MICROSCOPES
DEVELOPED IN YOUR LAB

We support the upgrade and redesign of custom-made two-photon microscopes to build a 3D imaging system.

For more detailed information on optical, mechanical, electrical, software features and design, please info [at] femtonics.eu (contact us).

BUDAPEST, HUNGARY

BASEL, SWITZERLAND

MARSEILLES, FRANCE

SZEGED, HUNGARY

FLORIDA, USA

Group Leader: David Fitzpatrick
Functional Archtitecture and Development of Cerebral Cortex
Max Planck Institute
sales [at] femtonics.eu (Contact)

UMEA, SWEDEN

Group Leader: Paolo Medini
Department of Molecular Biology
Umea University
sales [at] femtonics.eu (Contact)

OVERVIEW

OVERVIEW

Femto3D-AcoustoOptic microscope is the first fast, 3D, two-photon microscope on the market. The microscope is capable of scanning neuronal, dendritic, and other neuropil activities about one million faster as compared to previous realizations within a large (about cubic millimeter) scanning volume with preserved two-photon resolution.

The microscope, using electrically tunable crystals, can focus the excitation point with up to 53 kHz speed to any 3D location under the objective without mechanical restrictions reaching sub-millisecond temporal resolution in a millimeter z-dimension scanning range.

„3D AO imaging will open new horizons in the field of neuroscience”


VIRTUAL PRODUCT WALK AROUND

FEMTONICS IS THE FIRST
IN 3D TWO-PHOTON MICROSCOPY

Femto3D-AcoustoOptic microscope combines an intoxicating blend of high-tech science, engineering, refinement in 3D measurements, and technology. Our team developed and published the first 3D AO microscope in the world in 2003. During the more than ten year period we have spent in the field of 3D microscope development we realized and patented many revolutionary new technical solutions to provide users with a 3D system that has incredibly good parameters (such as large scanning volume, high speed, and preserved good spatial resolution) for an affordable price.


REVOLUTION
IN 3D MICROSCOPE
TECHNOLOGY

In the past ten-year period we have developed and patented many new technologies to provide our customers with the best 3D microscope in the world. Among other features, our microscopes now have:
  • optimal arrangement of optical elements based on diffraction-based modeling (over 10× more effective 3D scan head)
  • optimized angular dispersion compensation
  • functionally separated arrangement of AO deflectors for the highest performance
  • dynamic optical error compensation
  • optimized deflector driver signals, deflector geometry, manufacturing, bandwidth and crystal orientation
  • new AO deflector technology for longer wavelengths (for GECIs)
  • minimized optical path length to maximize detection (travelling detector system) with ultrasensitive GaAsP PMTs (>40% quantum efficiency)
  • integrated measurement control and analysis software
  • 4× more effective radio-frequency drives

NEW SCANNING METHODS

The use of fast, electrically controlled lenses in combination with an easy to use 3D data acquisition software package provides maximal flexibility in fast selection of regions of interests with high speed and controlling of measurements.

"Having the third dimension in control and full flexibility in XY scanning gives the ultimate freedom to the experimenter in selecting regions of interests and tuning signal-to-noise ratio to the theoretical limits."

FULL SPECIFICATION

REFERENCES

NETWORK IMAGING

3D IMAGING OF NEURONAL NETWORKS

The spatial and temporal complexity of neuronal coding requires recording of information flow and processing, not only from a single point or plane, but at the level of large networks distributed in 3D, in large volumes. Therefore several new optical methods have been developed recently for the fast readout of neuronal network activity in 3D. However, only a few of them is based on two-photon excitation which allows deep brain imaging. Among these methods, 3D AO scanning provides the largest increase in the signal-to-noise ratio and measurement speed, more quantitatively:

CHESSBOARD SCANNING
For in vivo imaging of hundreds of soma simultaneously in 3D

The microscope is now capable of using the novel 3D Anti-mOtion scanning technique allowing fast 3D imaging of neuronal network in behaving animals. In Chessboard scanning, scanning points are extended to small squares containing the somata and surrounding area. The special flexibility of the 3D scanning capability allows simultaneous imaging along multiple small squares placed in arbitrary locations in 3D. The name, chessboard is derived from the layout which is generated by arranging side-by-side all the squares containing the selected regions. This pattern allows simultaneous visualization of the activity of the somata, handling and storing the data and, importantly, to correct for motions. As a result, it makes possible to recover all high speed three-dimensional fluorescence data during the animal’s movements, therefore to follow neuronal network activity in behaving animals.
Figure shows chessboard scanning of neuronal somata and the measured Ca+2 transients resulted by neuronal activity. Mouse V1 region in vivo, labeled with GCaMP6 fluorescent sensor.

3D MULTI-CUBE SCANNING
Imaging somata during large amplitude motions

Multi-cube scanning is an extended mode of chessboard scanning where a z dimension is added to cover the z extent of the somata to preserve all somatic fluorescence points during motions. The somata as ROIs are ordered as cubes next to each other for visualization and calcium transients are recorded from each cube corrected to motions. With this method for example 50 somata can be recorded at 50 Hz when using cubes made of 50 x 10 x 5 voxels. Figure shows simultaneous measurements of ten GCaMP6 labeled somata.

UPGRADE KIT FOR DEEP BRAIN IMAGING

We developed seven novel methods to improve deep penetration and to extend the entire 3D z-scanning range from the surface down to over 500 µm depth in vivo.



Several thousands of cells can be measured simultaneously. The most responsive ones can be subselected and measured with higher temporal resolution. Neuronal responses can be visualized as traces or color coded images.

3D IMAGING OF LARGE ASSEMBLIES OF NEURONS

PRESERVED SINGLE AP RESOLUTION

Single BAPs could be resolved in distinguishable Ca2+ transients induced by a train of three APs used AO z focusing in 1200 µm scanning range and along the x axis (760 µm).

DENDRITIC IMAGING

3D DENDRITIC IMAGING

The main advantage of 3D AO scanning is that the high resolution characteristic of two-photon microscopy is preserved therefore dendritic imaging with spine resolution is possible in the center (300 µm × 300 µm × 200 µm volume in the middle of the entire scanning volume).

FAST DENDRITIC IMAGING with 3D POINT SCANNING

Random-access point scanning is the fastest method to read-out neuronal activity in large volumes with high resolution in 3D. The method allows simultaneous imaging of multiple points, or line segments, at multiple dendritic branches.

3D MULTI-LINE SCANNING
For following neuronal activity in spines

For scanning spines at high speed in vivo, points of 3D random-access point scanning are extended by drifting the focal point along short lines without increasing the overall scanning time. The first step is to select points based on a z-stack along a dendritic segment or any cellular structure then simply define the 3D orientation and extent of the 3D drifts to the main direction of motion. Finally, the average trajectories are calculated cancelling effect of the brain motion. 3D Multi-Line scanning enables functional recording of over 150 spines simultaneously in a 500 x 500 x 650 µm3 volume.

3D RIBBON SCANNING
For 3D dendritic imaging

Ribbon scanning is an extended trajectory scanning using 3D Anti-mOtion technology which also captures the neighboring area around the trajectory of dendrites to preserve fluorescent information during motions. The neighboring area is scanned by generating drifts either parallel or orthogonal to the trajectory. 3D Ribbon scanning can follow the 3D curvature of one or more dendrites at the same time, for example it enables functional recording with up to 3 kHz on a 50 µm long dendritic segment or imaging of activity simultaneously in over 12 spiny dendritic segments.
Figure shows 3D ribbons encompassing seven dendritic segments. Fluorescent transients were recorded simultaneously along the ribbons and data were projected into a 2D image ordering dendrites above each other. Recorded activity from selected spines and dendrites were visualized in the form of classical Ca2+ transients and raster plots.

3D SNAKE SCANNING
For imaging dendrites in a 3D volume during large amplitude motions

3D Snake scanning is a volume extension of the ribbon scanning that contains the entire 3D environment of the dendrite. In larger animals or at certain surgery or behavioral protocols, the amplitude of motion can be larger. To sufficiently preserve fluorescence signals even in these cases, the surface elements can be extended to tortuous cuboids by using this scanning mode. Figure shows fast snake scanning performed at 10 Hz in the selected dendritic region of a V1 pyramidal neuron. Fluorescence data were maximal intensity projected to a straightened 2D image. The representative spontaneous calcium responses are measured from the selected volume elements and the transients are shown following 3D motion correction.

3D MULTI-LAYER SCANNING
For imaging along the entire length of dendritic arbor

Albeit the majority of neuronal computation occurs in long neuronal processes situating in multiple layers in the brain, previous imaging methods were not able to readout these 3D activity patterns. However, Femto3D-AO microscopy combining the low photo-toxicity of low-power temporal oversampling (LOTOS) with the maximal flexibility of electric 3D scanning allows imaging of cells spanning across many layers, e.g. cortical pyramidal cells all the way up from basal to apical dendritic arbors. Imaging of multiple frames with different sizes and at any position in the scanning volume can be used to follow all events propagating along the cell. Scanning of the neighborhood of the processes allows preserving all fluorescent signals and calcium transients in spite of the brain’s motion. The multi-layer scanning method is not limited to a single dendrite or axon, but even multiple neurons can be simultaneously imaged with their dendritic arbor. Figure demonstrates multi-layer imaging of the activity of a layer V neuron in an awake animal at 41 different depth levels over a 500 µm z range at 30 Hz. Motion artifacts along the x and y axes were eliminated and calcium transients were recorded for each ROI.

PROPAGATION SPEED OF FAST REGENERATIVE EVENTS

The 2014 Nobel Prize in Chemistry was awarded to three scientists, Eric Betzig, Stefan W. Hell and William E. Moerner for spatial super-resolution microscopy. Our Femto3D-AO microscope provides a novel super resolution method for microscopy: the temporal super-resolution to separate Ca2+ responses of neighboring subcellular compartments with over 50 microsecond precision (temporal superresolution microscopy described for the first time by Katona et al. 2012).

TECHNOLOGY

INERTIA FREE 3D SCANNING

Acousto-optical scanning does not rely on mirrors and doesn’t contain any moving parts, so positioning the focal spot of the two-photon microscope is performed extremely fast and – importantly – independent of the travelled distance. This makes it suitable for 3D random-access scanning modes.

LARGE FIELD OF VIEW AND
EXTENDED Z-SCANNING RANGE

Thanks to diffraction-based optical modelling, large aperture AO deflectors, an improved radio-frequency drive, angular dispersion compensation, and many other technological steps, now we can provide large field of view (800 µm x 800 µm) in over a 1400 µm z-scanning range.

PRESERVED SPATIAL RESOLUTION

The large aperture of the new AO deflectors, the improved angular dispersion compensation, and the dynamic error compensation provides an improved spatial resolution over the entire scanning volume. The diameter of point spread function (PSF) is minimized along X, Y and Z axes and it remains smaller than the average diameter of neuronal somata in the entire scanning volume. Moreover, we can target the focal spot with 50 nm precision into any point in the entire (800 µm × 800 µm × 1,400 µm) 3D volume, therefore neuropil contamination can be avoided.

ANTI-MOTION

3D ANTI-MOTION TECHNOLOGY

Femto3D-AcoustoOptic microscope is now capable of using the novel 3D Anti-mOtion scanning technique allowing fast 3D imaging of spine and neuronal assemblies or multiple dendritic segments in behaving animals. This patented 3D scanning method extends the points of the random-access point scanning method to fast scanned 3D lines. Based on these lines, scanning is executed on 3D lines, surface or volume elements with maintained temporal resolution. The scanned parts cover not only the pre-selected ROIs but also the neighboring areas or volume elements. This gives an opportunity to decrease the motion artifacts by more than one order of magnitude in behaving animals.

SCANNING MODES

NOVEL MEASUREMENT POSSIBILITIES IN AWAKE, BEHAVING ANIMALS


  • Imaging a large network of over 130 neurons at subcellular resolution in a scanning volume of up to 500 × 500 × 650 μm3 with an order of magnitude larger signal-to-noise ratio when motion artifact elimination is on
  • Increased transmission in the range of 850-950 nm for GCaMP6 imaging
  • Simultaneous imaging of large parts of the dendritic arbor and neuronal networks in a z scanning range of over 650 µm
  • Fast 3D recording of over 150 dendritic spines in 3D
  • Fast parallel imaging of activity in over 12 spiny dendritic segments
  • Measurement of neuronal networks in multiple planes with over the speed of resonant scanning.

CHESSBOARD SCANNING
For in vivo imaging of hundreds of soma simultaneously in 3D

In Chessboard scanning, scanning points are extended to small squares containing the somata and surrounding area. The special flexibility of the 3D scanning capability allows simultaneous imaging along multiple small squares placed in arbitrary locations in 3D. The name, chessboard is derived from the layout which is generated by arranging side-by-side all the squares containing the selected regions. This pattern allows simultaneous visualization of the activity of the somata, handling and storing the data and, importantly, to correct for motions. As a result, it makes possible to recover all high speed three-dimensional fluorescence data during the animal’s movements, therefore to follow neuronal network activity in behaving animals.
Figure shows chessboard scanning of neuronal somata and the measured Ca+2 transients resulted by neuronal activity. Mouse V1 region in vivo, labeled with GCaMP6 fluorescent sensor.

3D MULTI-CUBE SCANNING
Imaging somata during large amplitude motions

Multi-cube scanning is an extended mode of chessboard scanning where a z dimension is added to cover the z extent of the somata to preserve all somatic fluorescence points during motions. The somata as ROIs are ordered as cubes next to each other for visualization and calcium transients are recorded from each cube corrected to motions. With this method for example 50 somata can be recorded at 50 Hz when using cubes made of 50 x 10 x 5 voxels. Figure shows simultaneous measurements of ten GCaMP6 labeled somata.

3D MULTI-LAYER SCANNING
For imaging along the entire length of dendritic arbor

The 650 µm deep z-scanning range of the Femto3D-AcoustoOptic microscope gives possibility to image cells spanning across many layers, e.g. cortical pyramidal cells all the way up from basal to apical dendritic arbors. Imaging of multiple frames with different sizes and at any position in the scanning volume can be used to follow all events propagating along the cell. Scanning of the neighborhood of the processes allows preserving all fluorescent signals and calcium transients in spite of the brain’s motion. The multi-layer scanning method is not limited to a single dendrite or axon, but even multiple neurons can be simultaneously imaged with their dendritic arbor. Figure demonstrates multi-layer imaging of the activity of a layer V neuron in an awake animal at 41 different depth levels over a 500 µm z range at 30 Hz. Motion artifacts along the x and y axes were eliminated and calcium transients were recorded for each ROI.

3D RIBBON SCANNING
For 3D dendritic imaging

Ribbon scanning is an extended trajectory scanning which also captures the neighboring area around the trajectory of dendrites to preserve fluorescent information during motions. The neighboring area is scanned by generating drifts either parallel or orthogonal to the trajectory. 3D Ribbon scanning can follow the 3D curvature of one or more dendrites at the same time, for example it enables functional recording with up to 3 kHz on a 50 µm long dendritic segment or imaging of activity simultaneously in over 12 spiny dendritic segments.
Figure shows 3D ribbons encompassing seven dendritic segments. Fluorescent transients were recorded simultaneously along the ribbons and data were projected into a 2D image ordering dendrites above each other. Recorded activity from selected spines and dendrites were visualized in the form of classical Ca2+ transients and raster plots.

3D SNAKE SCANNING
For imaging dendrites in a 3D volume during large amplitude motions

3D Snake scanning is a volume extension of the ribbon scanning that contains the entire 3D environment of the dendrite. In larger animals or at certain surgery or behavioral protocols, the amplitude of motion can be larger. To sufficiently preserve fluorescence signals even in these cases, the surface elements can be extended to tortuous cuboids by using this scanning mode. Figure shows fast snake scanning performed at 10 Hz in the selected dendritic region of a V1 pyramidal neuron. Fluorescence data were maximal intensity projected to a straightened 2D image. The representative spontaneous calcium responses are measured from the selected volume elements and the transients are shown following 3D motion correction.

3D MULTI-LINE SCANNING
For following neuronal activity in spines

For scanning spines at high speed in vivo, points of 3D random-access point scanning are extended by drifting the focal point along short lines without increasing the overall scanning time. The first step is to select points based on a z-stack along a dendritic segment or any cellular structure then simply define the 3D orientation and extent of the 3D drifts to the main direction of motion. Finally, the average trajectories are calculated cancelling effect of the brain motion. 3D Multi-Line scanning enables functional recording of over 150 spines simultaneously in a 500 x 500 x 650 µm3 volume.

RELATED PUBLICATION

Fast 3D Imaging of Spine, Dendritic, and Neuronal Assemblies in Behaving Animals. Szalay G, Judak L, Katona G, Ocsai K, Juhasz G, Veress M, Szadai Z, Feher A, Tompa T, Chiovini B, Maak P, Rozsa B, Neuron (2016)

SOFTWARE

OVERVIEW

Femto3D-AcoustoOptic microscope is supported with our modular measurement control and analysis software package called MES. MES was designed with the day-to-day lab experience in the field of cellular and network imaging. MES supports 3D ROI selection and immediate ROI activity analysis necessary for high throughput measurements.

3D ROI SELECTION

New software tools with custom designed user interface support the most flexible 3D scanning pattern selection of multiple points, multiple areas or 3D trajectories as the basis of the advanced scanning modes. Various software tools help selection, such as 3D cell localization, automatic pattern generation and XYZ adjustment.

EXPERT IN AO DRIVING

The scan control software incorporates the latest AO driving theory allowing the special SNR optimizing scanning methods, and also enables dynamic error compensation ensuring excellent optical properties of this complex optical system.

INTEGRATION

MES – besides performing all standard microscopy functions – fully integrates the control of all hardware units in the microscope:
  • focusing, PMTs, light path actuators
  • AcoustoOptic scanners
  • camera handling
  • auxiliary digital and analog channels to interface stimulators or behavior control devices
  • sample stage and patch pipette devices

FLEXIBLE ENVIRONMENT

The MES software is written in the MATLAB high level programming environment. This enables rapid code development and opens the data to the users to the deepest levels. Data can be interactively accessed at the MATLAB command prompt or through self-developed script or GUI tools beyond the wide opportunities of the existing program package.

3D DATA VISUALISATION

Fluent software interface allows real-time visualization of the recorded 3D fluorescent data. Using direct data access and MATLAB’s 3D rendering capabilities, any custom 3D visualization or animation can be easily constructed.

EXTENSION MODULES

MES can be extended by various features with additional modules:
  • batch analysis
  • electrophysiology importer
  • 3D cell localization
  • excel interface for analysis
  • visual stimulation / intrinsic imaging
  • sample stage and pipette manipulation
  • sample stabilization
  • ratio imaging calculations
  • electrophysiology
  • measurement automation

UPGRADE

3D AO SCAN HEAD
UPGRADE KIT

The AO upgrade kit is a cost effective solution which can be easily added to many existing upright and inverted microscopes.

UPGRADE KIT FOR YOUR EXISTING MICROSCOPES

Extend your existing microscope to 3D imaging.

The 3D microscope building kit consists of:
  • a 3D AO scan head
  • a digital automatic beam alignment system which preserves the diffraction limited resolution of the whole system
  • detectors
  • a controller box
  • software; PC
  • two mirrors with six mechanical freedom allows precise alignment of the scanned beam relative to the objective optical axis
  • adjustable high-tensile aluminium legs which can flexible tuned to custom made two-photon microscopes

UPGRADE KIT
FOR MICROSCOPES
DEVELOPED IN YOUR LAB

We support the upgrade and redesign of custom-made two-photon microscopes to build a 3D imaging system.

For more detailed information on optical, mechanical, electrical, software features and design, please info [at] femtonics.eu (contact us).

DEMO ROOMS

BUDAPEST, HUNGARY

BASEL, SWITZERLAND

MARSEILLES, FRANCE

SZEGED, HUNGARY

FLORIDA, USA

Group Leader: David Fitzpatrick
Functional Archtitecture and Development of Cerebral Cortex
Max Planck Institute
sales [at] femtonics.eu (Contact)

UMEA, SWEDEN

Group Leader: Paolo Medini
Department of Molecular Biology
Umea University
sales [at] femtonics.eu (Contact)