OVERVIEW

FemtoSmart is the first member of our next-generation two-photon microscope series. In the design, the main goal was to create a totally customizable microscope for in vivo studies with a distinct, charismatic appearance. The special feature of this microscope is the elevated body, which allows extremely free positioning of the sample below the objective.

POSITIONING FREEDOM

The column-based X-Y-Z moving body houses the scanner unit, control circuits, and internal light path. The footprint of the leg is only 250 mm x 250 mm and there is a large space below the objective. The design enables the objective to move in a 50 mm range in the Z direction and the microscope can move in an XY direction around the base in 50 mm range. The tilting objective can further increase mobility.

IN VIVO STUDIES

The large space under the objective, where you can place virtual reality, movable tables, electrophysiological devices, is extremely useful for behavioral studies and supports functional studies of model organisms from C. elegans to non-human primates.

THE HIGHEST LEVEL OF MODULARITY

The modular nature of the microscope allows us to assemble the components, recombine and upgrade the system perfectly fitting the customer’s needs. One or two laser beams can be coupled to the microscope, a femtosecond, a two-photon laser for imaging and a secondary laser, either for imaging or photoactivation. Galvano or resonant scanner directs the laser beam, and dual scanner configuration is available too. The detection is established by multialkali and/or high-sensitivity GaAsP photomultipliers. The system can be extended with a lot of special devices such as a piezo objective positioner and a focus tunable liquid lens for fast Z-scanning, SmartBridge for increased mobility, etc. We offer special solutions for optogenetics and uncaging studies. Tell us the concept, we create it.

PROPERTIES OF IMAGING

The design of this new microscope body is based on the established technology of Femtonics. As a result, the fine-tuned optical construction allows imaging in 850 µm depth on a wide field of view. The shortest optical path (patented) between the sample and the detectors ensures maximal photon collection efficiency and highest SNR.

TECHNOLOGY

OVERVIEW

The FemtoS-Galvo is a galvanometric scanner based two-photon microscope which allows functional imaging focusing on the region-of-interest (ROI). Scanning only the ROI and skipping the background establish fast imaging speed to follow rapid changes and high signal-to-noise ratio to reveal more signals.

FLEXIBLE SCANNING MODES FOR IMAGING AND STIMULATION

The galvanometric scanner – commonly called "galvo" – consists of two galvanometer based motor driven mirrors allowing arbitrary positioning of the focal spot. High accuracy and this positioning freedom support flexible approaches for ROI creation.


MULTIPLE LINE SCANNING

Our patented multiple line scanning mode allows unique enhancement of the signal-to-noise ratio in multisite measurements. The scanner spends the most of time by collecting signals from the ROIs while the intermediate sections are skipped within 60-100 µs. A typical 3-4 fold improvement of the SNR can be achieved compared to frame scanning.

FOLDED FRAME SCANNING

This approach allows imaging of a confined area along a line. The shape of the selected regions can be straight or curved while the position is arbitrary. This advanced scanning method is useful for imaging just cell bodies in the different places of the specimen or following events along winding dendrites with their protrusions.

HIGH SIGNAL-TO-NOISE RATIO

Revealing subtle changes of the evoked signals based on the following achievements:
  • scanning only the interesting part of the FOV and skipping the background allows to reach high signal-to-noise ratio,
  • the shortest possible optical path allows enhanced photon collection efficiency thanks to our patented travelling detector system,
  • using the highest sensitivity GaAsP photomultipliers (quantum efficiency >40%) optimized for scattered photons,
  • carefully chosen high quality filtersets.

SOFTWARE

OVERVIEW

FemtoS-Galvo microscope is controlled and works together with our modular measurement control and analysis software package called MES. MES is written in the MATLAB environment enabling rapid code development and opens the data to the users. It is designed with the day-to-day lab experiences in the field of cellular and network imaging. MES supports 2D ROI selection and immediate ROI activity analysis necessary for high throughput measurements.

LABMASTER

MES fully integrates the control of all hardware units in the microscope:
  • galvo scanner, PMTs, light path actuators, electrical devices,
  • focusing, camera handling,
  • auxiliary digital and analog channels to interface stimulators,
  • behavior control devices,
  • sample stage or patch pipette devices.
MES stores all metadata of the aboves in a complex file containing multiple measurement units along with your comments.

ROI SELECTION

Numerous software tools support all kinds of 2D scanning pattern generation: points, straight or curved lines and areas with various shapes can be selected improving the efficiency of imaging.

  • LineMagic allows easy placing of predefined shapes, such as spirals,
  • Cell3DFinder locates cells for ROI scanning,
  • TravellingSalesman creates the shortest pathway between the scanning points increasing the velocity of the scanning,
  • ROIs can be imaged in the three-dimensional space by RollerCoaster software and hardware package.

REAL-TIME DISPLAY
AND ANALYSIS

Integrated, quantitative intensity-based calculations allow following real-time fluorescence changes (e.g. ∆F/F calculation) and simultaneous analysis. To extend the function, MES can adapt electrophysiological recordings too displaying Ca2+ and electrical signals side-by-side.

BATCH PROCESSING

The analysis module of MES contains efficient tools to analyze entire multi-ROI measurement sets conveniently. Sophisticated curve analysis tool allows fast exploration of the measurement data. Having direct access to the data, any user created analysis methods can be developed.

EXTENSION MODULES

MES can be extended with many software modules to support various measurement protocols. It involves import and export functions with other programs such as MS Excel, Imaris, Amira, Huygens. It can integrate the control of numerous devices (Luigs&Neumann, PipettePanipulation). MES can adapt electrophysiological recordings.

APPLICATIONS

DEEP BRAIN IMAGING

Thanks to two-photon laser technology and our optical developments, you can study distinguishable cell bodies and dendrites down to 850 µm depth in behaving animals without photodamage.


CALCIUM-IMAGING

To measure changes in Ca2+-levels is a powerful method for monitoring the activity of many cells in the brain. Calcium-indicators such as the OGB-1 fluorescent dye or the GCaMP fluorescent protein family respond to the binding of Ca2+-ions by changing their fluorescence properties. The fast scanning rate imaging of well-defined, accurate separated regions ensures precise and repeatable measurements to follow the rapid events in distinct neurons and their dendrites.

PARALLEL ELECTROPHYSIOLOGY

Hardware and software tools help parallel two-photon imaging and electrophysiological recordings providing different aspects to study the neuronal cell and network activity.

PHOTOSTIMULATION

The accuracy of the excitation point and the high flexible scanning patterns enables the FemtoS-Galvo to be used for different photostimulating techniques such as optogenetics and uncaging. We support special upgrade modules extending the FemtoS-Galvo to a complete solutions for uncaging and optogenetics.

INTRAVITAL IMAGING

The infrared excitation laser applied in two-photon microscopy can penetrate thick specimens, enabling the visualization of living cell behavior in intact tissues and organs for extended periods without phototoxicity. Moreover, the high precision galvo scanner ensures high resolution for morphological studies.

VERSATILITY

SPECIFICATION

REFERENCES

Combined two-photon imaging, electrophysiological, and anatomical investigation of the human neocortex in vitro. Balint Peter Kerekes, Kinga Toth, Attila Kaszas, Balazs Chiovini, Zoltan Szadai, Gergely Szalay, Denes Palfi, Attila Bago, Klaudia Spitzer, Balazs Rozsa, Istvan Ulbert, Lucia Wittner, Neurophotonics (2014)

Enhanced Dendritic Action Potential Backpropagation in Parvalbumin-positive Basket Cells During Sharp Wave Activity. B. Chiovini, GF. Turi, G. Katona, A. Kaszas, F. Erdelyi, G. Szabo, H. Monyer, A. Csakanyi, ES. Vizi, B. Rozsa, Neurochemical Research (2010)

Local Postsynaptic Voltage-Gated Sodium Channel Activation in Dendritic Spines of Olfactory Bulb Granule Cells. Wolfgang G. Bywalez, Dinu Patirniche, Vanessa Rupprecht, Martin Stemmler, Andreas V.M. Herz, Denes Palfi, Balazs Rozsa, Veronica Egger, Neuron (2015)

MRZ-99030 - A novel modulator of Aβ aggregation: II - Reversal of Aβ oligomer-induced deficits in long-term potentiation (LTP) and cognitive performance in rats and mice. Rammes G, Gravius A, Ruitenberg M, Wegener N, Chambon C, Sroka-Saidi K, Jeggo R, Staniaszek L, Spanswick D, O'Hare E, Palmer P, Kim EM, Bywalez W, Egger V, Parsons CG, Neuropharmacology (2015)

Matching Cell Type to Function in Cortical Circuits. Luc Estebanez, Jens Kremkow, James F.A. Poulet, Neuron (2015)

Plasticity of intrinsic excitability in mature granule cells of the dentate gyrus. Jeffrey Lopez-Rojas, Martin Heine & Michael R. Kreutz, Nature (2016)

TECHNOLGY

OVERVIEW

The FemtoS equipped with resonant based scanner is the most appropriate choice for imaging of the entire field of view with high frame rates. In this microscope Femtonics combines the high speed and high sensitivity imaging of living tissues. Resonant scanner based raster scanning is ~5-fold faster for fast acquisition of the entire field-of-view compared to galvanometric based scanning.

HIGH SPEED IMAGING

The resonant scanner consists of a fast oscillating mirror for x-axis deflection and a galvanometer mirror for y-axis sweep. Thanks to the 8 kHz oscillating speed of the fast mirror, the microscope is capable of gathering images at 31 frames per second.


HIGH SCANNING RATE
WITHOUT IMAGE DISTORSION

The velocity of resonant scanner is non-linear, the speed is different in the central and the edges of the frame. In the FemtoS-Resonant, Pockells-cell limits scanning range to that portion where scanning velocity is near linear, avoiding photobleaching/photodamage at the two sides of the image. Scan electronics performs dynamic pixel dwelling for data linearization and to cancel image distorsions.

LOW -POWER TEMPORAL OVERSAMPLING (LOTOS)

FemtoS-Resonant scans with high velocity thereby distributing excitation to a larger area resulting in decreased photodamage. This idea, called LOTOS method (Varga et al, PNAS (2011), Chen et al, Nature (2012)) involves imaging at high frame rates at lower excitation energy per pixel. As a result, the microscope allows imaging even small dendritic segments within the brain of behaving animals, e.g. to record individual calcium transients processing whisker stimulation.

3D VOLUME SCANNING

Two new upgrade modules support near real-time, XYZ-T imaging of 3D volumes:

Both solutions combined with the fast frame scanning feature of resonant scanner allow fast, near real time scanning of a selected 3D volume.

INCREASED DETECTION
EFFICIENCY

High sensitivity photon detection allows superior signal-to-noise ratio based on the following achievements:
  • the shortest possible optical path allows enhanced photon collection efficiency thanks to our patented travelling detector system,
  • using the highest sensitivity GaAsP photomultipliers (quantum efficiency >40%), which are optimized for scattered photons,
  • carefully chosen high quality filtersets.

SOFTWARE

OVERVIEW

MESc is a highly modular measurement control and analysis software developed to drive our FemtoS-Resonant microscope. It was designed in C++ programming language with day-to-day lab experience in the field of cellular and network imaging.

INTEGRATION

MESc fully integrates the control of all hardware units in the microscope such as core components (scanner, PMTs), light path actuators, various sample stages, auxiliary digital and analog channels.

FEATURES

  • finely tunable measurement parameters (resolution, duration, frame average),
  • automatic adjustment of dynamic pixel dwell time to avoid image distorsion,
  • real-time data display and analysis,
  • data can be exported to common image and video format.

REAL-TIME DISPLAY AND ANALYSIS

Integrated, quantitative intensity-based calculations allow following real-time fluorescence changes and simultaneous analysis.

APPLICATIONS

DEEP BRAIN IMAGING

Thanks to the two-photon laser technology and our optical developments, you can study nervous system with distinguishable cell bodies and dendrites down to 850 µm depth in the brain of behaving animals without photodamage.


CALCIUM-IMAGING

To measure changes in Ca2+-levels is a powerful method for monitoring the activity of many cells in the brain. Calcium-indicators such as the OGB-1 fluorescent dye or the GCaMP fluorescent protein family respond to the binding of Ca2+-ions by changing their fluorescence properties. The fast frame scanning speed of FemtoS-Resonant allows following rapid changes in neurons and the neuropil.

TIME-LAPSE IMAGING

While two-photon excitation ensures depth penetration and fine spatial resolution, the high frame scanning rate of the resonant scanner ensures temporal resolution allowing observation of rapid events in living cells, neuronal networks or other circuits.

LONG-TIME MEASUREMENTS

The high frame scanning rate and the unlimited video streaming combined with automated measurements support long time studies such as following developmental stadiums of a zebrafish embryo.

3D VOLUME SCANNING

Beside 3D activity changes in morphology of cellular networks or the vasculature can be revealed using 3D volume scanning upgrade (Piezo objective positioner, focus tunable liquid lens). The fast XY-scanning and Z-movement ensure near real time measurement of a 3D volume.
Video shows volume scanning which was performed by our resonant based microscope equipped with Piezo objective positioner.

PHOTOSTIMULATION

Whole field illumination kit enables stimulation of cell populations in different locations of a specimen while the resonant scanner allows simultaneous imaging of the evoked signals. The selective stimulation of the cells can be ensured by expressing photosensitive molecules such as channelrhodopsin or halorhodopsin (optogenetics).

VERSATILITY

SPECIFICATION

TECHNOLOGY

OVERVIEW

The FemtoS-Dual microscope gives the ability to perform dual scanning using both galvo and resonant scanners in tandem. With galvo scanner you can zoom to tiny structures such as dendritic spines and jump quickly between these zoomed regions. In contrast, with resonant scanner you can capture images with high frame rate to follow rapid changes on the FOV.

COMBINED ADVANTAGES OF GALVO AND RESONANT SCANNERS

Choose the best scanning mode which fits your imaging requirements:
  • resonant scanner ensures high speed image acquisition up to 31 frames per second (fps) over the whole FOV
  • galvo scanner provides flexible ROI scanning opportunities.

MULTIPLE LINE SCANNING

Our patented multiple line scanning mode allows unique enhancement of the signal-to-noise ratio in multisite measurements. The scanner spends the most of time by collecting signals from the ROIs while the intermediate sections are skipped within 60-100 µs. A typical 3-4 fold improvement of the SNR can be achieved compared to frame scanning.

FOLDED FRAME SCANNING

This approach allows imaging of a confined area along a line. The shape of the selected regions can be straight or curved while the position is arbitrary. This advanced scanning method is useful for imaging just cell bodies in the different places of the specimen or following events along winding dendrites with their protrusions.

TIME-LAPSE IMAGING

While two-photon excitation ensures depth penetration and fine spatial resolution, the high frame scanning rate of the resonant scanner ensures temporal resolution allowing observation of rapid events in living cells, neuronal networks or other circuits.

3D VOLUME SCANNING

Two new upgrade modules support near real-time, XYZ-T imaging of 3D volumes:

Both solutions combined with the fast frame scanning feature of resonant scanner allow fast, near real time scanning of a selected 3D volume.
Video shows volume scanning which was performed by our resonant based microscope equipped with Piezo objective positioner.

HIGH SIGNAL-TO-NOISE RATIO

Revealing subtle changes of the evoked signals based on the following achievements:
  • the shortest possible optical path allows enhanced photon collection efficiency thanks to our patented travelling detector system,
  • using the highest sensitivity GaAsP photomultipliers (quantum efficiency >40%) optimized for scattered photons,
  • carefully chosen high quality filtersets.

APPLICATIONS

DEEP BRAIN IMAGING

Thanks to the two-photon laser technology and our optical developments, you can study nervus system with distinguishable cell bodies and dendrites down to 850 µm depth in the brain of behaving animals without photodamage.

CALCIUM IMAGING

To measure changes in Ca2+-levels is a powerful means for monitoring the activity of neural circuits. Calcium-indicators such as OGB-1 fluorescent dye or GCaMP fluorescent molecule can respond to the binding of Ca2+-ions by changing their fluorescence properties. To follow changes, the two kinds of scanners give the following possibilities:
  • resonant scanner allows to perform rapid series of measurements revealing events on a large field,
  • and using galvo scanner, you can measure the Ca2+-level on small, predetermined regions with high signal-to-noise ratio.

PARALLEL ELECTROPHYSIOLOGY

Hardware and software tools help parallel two-photon imaging and electrophysiological recordings, which provides different aspects to study the neuronal cell and network activity.

VERSATILITY

SPECIFICATION

REFERENCES

Microglia protect against brain injury and their selective elimination dysregulates neuronal network activity after stroke. Szalay G, Matinecz B, Lenart B, Kornyei Z, Orsolits B, Judak L, Csaszar E, Fekete R, West BL, Katona G, Rozsa B, Denes A, Nature Communications (2016)

OVERVIEW

FemtoS-Bridge is a special edition of the FemtoSmart microscope which is manufactured to have for extreme freedom in positioning of the body. It involves all advantages of the FemtoSmart series but it is extended to studies which need huge place for the sample or accessories therefore allowing functional brain imaging in head fixed mice navigating in a virtual reality environment.

THE ELEVATOR

In this FemtoS-Bridge microscope, the foot is replaced with a lifting apparatus that provides an adjustable space between the microscope base and the top lens of the objective. The role of the modified elevating foot is to move the head along vertical direction. The moving range is 50 cm, the coarse Z-adjustment along the column is 1 mm. This elevator is situated outside of the workplace allowing an enlarged place below the Smart head. We provide the optimal combination of multiple height and working distance choices to address most animal models.

ACCESSORIES FOR FINE MOTIONS

Beside of the large movement established by the elevator, the fine motions can be supported
  • by the objective holding arm in a 50 mm range of Z direction,
  • piezo objective positioner for moving the objective with fast Z positioning in several hundred micrometers ranges,
  • an XY actuator can move the body relative to the column in X and Y direction (+/-25 mm range, step size: 1 um),
  • finally our tilting objective can further increase the accessibility to the side parts of the sample.

VERSATILITY

The FemtoS-Bridge offers the larges choice of height permutations that addresses applications from virtual reality, treadmills both line and spherical versions to electrophysiology hardware. If you have special clearance needs, please contact us with the dimensions so we can customize the travel mechanism.

OVERVIEW

OVERVIEW

FemtoSmart is the first member of our next-generation two-photon microscope series. In the design, the main goal was to create a totally customizable microscope for in vivo studies with a distinct, charismatic appearance. The special feature of this microscope is the elevated body, which allows extremely free positioning of the sample below the objective.

POSITIONING FREEDOM

The column-based X-Y-Z moving body houses the scanner unit, control circuits, and internal light path. The footprint of the leg is only 250 mm x 250 mm and there is a large space below the objective. The design enables the objective to move in a 50 mm range in the Z direction and the microscope can move in an XY direction around the base in 50 mm range. The tilting objective can further increase mobility.

IN VIVO STUDIES

The large space under the objective, where you can place virtual reality, movable tables, electrophysiological devices, is extremely useful for behavioral studies and supports functional studies of model organisms from C. elegans to non-human primates.

THE HIGHEST LEVEL OF MODULARITY

The modular nature of the microscope allows us to assemble the components, recombine and upgrade the system perfectly fitting the customer’s needs. One or two laser beams can be coupled to the microscope, a femtosecond, a two-photon laser for imaging and a secondary laser, either for imaging or photoactivation. Galvano or resonant scanner directs the laser beam, and dual scanner configuration is available too. The detection is established by multialkali and/or high-sensitivity GaAsP photomultipliers. The system can be extended with a lot of special devices such as a piezo objective positioner and a focus tunable liquid lens for fast Z-scanning, SmartBridge for increased mobility, etc. We offer special solutions for optogenetics and uncaging studies.

PROPERTIES OF IMAGING

The design of this new microscope body is based on the established technology of Femtonics. As a result, the fine-tuned optical construction allows imaging in 850 µm depth on a wide field of view. The shortest optical path (patented) between the sample and the detectors ensures maximal photon collection efficiency and highest SNR. All scanning modes and excellent optical properties of the Femto2D-Galvo and Femto2D-Resonant microscopes are met in this microscope (see detailed specification on these pages).

FEMTO-S BRIDGE

OVERVIEW

FemtoS Bridge is a special edition of the FemtoSmart microscope which is manufactured to have for extreme freedom in positioning of the body. It involves all advantages of the FemtoSmart series but it is extended to studies which need huge place for the sample or accessories therefore allowing functional brain imaging in head fixed mice navigating in a virtual reality environment.

THE ELEVATOR

In this FemtoS Bridge microscope, the foot is replaced with a lifting apparatus that provides an adjustable space between the microscope base and the top lens of the objective. The role of the modified elevating foot is to move the head along vertical direction. The moving range is 50 cm, the coarse Z-adjustment along the column is 1 mm. This elevator is situated outside of the workplace allowing an enlarged place below the Smart head. We provide the optimal combination of multiple height and working distance choices to address most animal models.

ACCESSORIES FOR FINE MOTIONS

Beside of the large movement established by the elevator, the fine motions can be supported
  • by the objective holding arm in a 50 mm range of Z direction,
  • piezo objective positioner for moving the objective with fast Z positioning in several hundred micrometers ranges,
  • an XY actuator can move the body relative to the column in X and Y direction (+/-25 mm range, step size: 1 um),
  • finally our tilting objective can further increase the accessibility to the side parts of the sample.

VERSATILITY

The FemtoS bridge offers the larges choice of height permutations that addresses applications from virtual reality, treadmills both line and spherical versions to electrophysiology hardware. If you have special clearance needs, please contact us with the dimensions so we can customize the travel mechanism.

METRICS