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