OVERVIEW

Femto2D product line involves our firstly developed microscopes that have been on the market for more than 10 years. It involves conventional, modular galvanometer and resonant based two-photon microscopes which are used for both in vivo and in vitro studies. Since 2007, more than 60 Femto2D systems have been installed and being worked around the world.

CLASSICAL MICROSCOPE DESIGN

Femto2D series contains three basic two-photon microscopes: Femto2D-Galvo, Femto2D-Resonant and Femto2D-Dual. They have standard Olympus frame which can be equipped with any kind of extensions selected from FemtoS and Femto2D modules’ portfolio, including in vitro extension. All of the in vivo imaging capability demonstrated in the case of the FemtoS is implemented to these systems, all scanning modes and optical properties are the same as in the FemtoS members.

FEMTO2D-GALVO

All galvanometrer based scanning modes and excellent optical properties of the FemtoS-Galvo had been established in this microscope. High accuracy and positioning freedom of the scanner are combined with the intelligent MES control software supporting flexible approaches for ROI creation.

FEMTO2D-RESONANT

The Femto2D-Resonant is the twin of the FemtoS-Resonant. Femtonics combines the high speed and high sensitivity imaging of living tissues in this scope, where the imaging speed reaches the 31 frames per second. The optomechanical quality is ensured by the established Femtonics’ technology.

FEMTO2D-DUAL

The Femto2D-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.

DETECTION

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.

OVERVIEW

Femto3D-RC is a Piezo objective positioner equipped galvanometric scanner based microscope. This advanced system controlled by special RollerCoaster software module of MES ensures scanning along trajectories in the three-dimensional space.

DENDRITIC IMAGING

Femto3D-RC is capable of collecting signals along several hundreds micrometers long dendrites from different depths fast enough to follow rapid events in the 3D biological sample.

  • imaging long dendritic segments (up to 250-300 µm)
  • resolution of individual spines
  • travelling range up to 400 µm
  • resonant frequency of the loaded piezo up to 500 Hz
  • millisecond step-and-settle

TECHNOLOGY

The 3D scanning modes of the Femto3D-RC microscope are controlled by the special RollerCoaster software. RollerCoaster controlled scanning is patented by Femtonics and uses fast sinusoidal driving of the objective positioner and it is insensitive to distortions of the actual objective path. The XY free line-scanning with galvanometric mirrors is specially synchronized to the nonlinear resonance of the z-axis movement of the objective. This approach yields a 3D trajectory matching the path of dendritic shafts and spines of neurons without limiting pixel dwell time. Available z-range depends on the resonanting frequency of piezo objective postioner (~scanning speed).

G. Katona, A. Kaszas, G. F. Turi, N. Hajos, G. Tamas, E.S. Vizi, B. Rozsa
Roller Coaster Scanning reveals spontaneous triggering of dendritic spikes in CA1 interneurons, PNAS (2011)

ADVANCED SCANNING MODES

UPGRADED FEMTO2D-GALVO

Upgrade your Femto2D-Galvo microscope with our RC hardware and software tools. The new configuration will contain and extend further all advantages of the Femto2D-Galvo microscope.

FULL SPECIFICATION

OVERVIEW

OVERVIEW

The Femto2D-Galvo is a galvanometric scanner based two-photon microscope which allows functional imaging focusing on the region-of-interest. 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.


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.

MODULARITY

The modular nature of the microscope supports to extend it with many upgrade elements ensuring a future proof performance in all kinds of optical-physiology measurements (dendritic imaging, uncaging, optogenetics, parallel electrophysiology, etc.). Tell us the concept, we create it.

FULL SPECIFICATION

REFERENCES

TECHNOLOGY

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.

3D ROLLER COASTER SCANNING

The Femto2D-Galvo enhanced with RollerCoaster upgrade is capable of collecting signals from different depths fast enough to follow events and resolve biological functionality all through the three-dimensional space.

Roller Coaster Scanning reveals spontaneous triggering of dendritic spikes in CA1 interneurons. G. Katona, A. Kaszas, G. F. Turi, N. Hajos, G. Tamas, E.S. Vizi, B. Rozsa, PNAS (2011)

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 Femto2D-Galvo to be used for different photostimulating techniques such as optogenetics and uncaging. We support special upgrade modules extending the Femto2D-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.

SOFTWARE

OVERVIEW

Femto2D-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.

TRAVELLING SALESMAN

TravellingSalesman creates the shortest pathway visiting user defined points for galvo scanning by approximating the traveling salesman problem. The short roundtrip time results in a high measurement speed reaching 100 Hz for a number of points arbitrarily dispersed in the field of view. Recommended for 2D optimized random access point scanning and high S/N network measurements.

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.

UPGRADE