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Femtonics configuration     Femto2D-GalvoFemto2D-Resonant     Femto2D-Dual
Scanner: XYGalvo   XY vector scan mirror for high resolution imaging or photostimulation; able   to have 1P & 2P laser for a single Galvo scannerResonant   XY raster scan mirror for high speed imagingFor   2P system only: Galvo + Resonant or Galvo + Galvo; 1 for imaging & 1 for   photostimulation
Scanner:  ZMicroscope Z motorStandard Z stack image   capture
Tunable focus liquid lensThe   liquid lens utilizes electrically controlled shape-changing membrane allowing   fast switching between focal planes. In combination with our 2D two-photon   microscopes, it provides scanning with high temporal resolution in   three-dimensional sample and near real time volume scanning can be performed.
Piezo objective positionerRollerCoasterR   (patented scanning technology) module enables a new measurement mode called   RollerCoaster with which one can perform high speed 3D line-scanning

The Femto2D-Galvo is a galvanometric scanner based two-photon microscope which allows functional imaging and photo-stimulation of 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.

Femto2D-Resonant microscope is the most appropriate choice for imaging of the entire field of view with high frame rates. Resonant scanner based raster scanning is ~5-fold faster for fast acquisition of the entire field-of-view compared to galvanometric based scanning.

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.

1. Field scan

2in1 solution two-photon microscope with single photon confocal mode: the confocal unit of Femtonics is designed for researchers who need to extend their two-photon microscopy research also with confocal imaging. The main advantage of our confocal microscope is that it is developed to perform measurements in living tissues. Flexible software tools support different stimulus protocols, parallel electrophysiological recordings and synchronization of other lab instruments necessary for physiological measurements. Fast photo activation using one or two parallel laser lines is also possible during fluorescent measurements without damaging the high sensitivity GaAsP detectors.

2. ROI Scan

The possibility to select certain regions of interest (ROI) in the confocal images makes it possible to apply multiple ROI (region of interesting) scan can be configured on the same field with different excitation light on and off for overall background image and different ROIs. Scanning of the region-of-interest (ROI) increases both the speed of measurement and signal-to-noise ratio (SNR). The scanner spends most of the time on the studied area and the photons are derived only from the exciting regions while the not interested parts are skipped. Both the SNR and the imaging speed can be enhanced with choosing the proper scanning method which fits tightly to the interesting region. ROI scan also benefits in making point bleaching experiment, FRAP (fluorescence recovery after photo bleaching) and FRET (fluorescent resonance energy transfer) very easy to be implemented.

The advantage of Femto2D-Galvo scanner system is the flexible scanning modes, accuracy, speed and a constant velocity for superior image quality.

3. Uncaging

Uncaging: activation of biochemically masked molecules via photolysis mimics physiological release of bioactive compounds. Using two-photon photostimulation, precise release can be elicited in miniature volumes.

The accuracy of the uncaging and imaging is established by the followings:

  • the stimulating laser is focused on femtoliter excitation volume

  • the galvanometric scanner ensures microsecond-precision

  • MES control software allows flexible spatiotemporal scanning

Application fields

  • neurotransmitter signaling

  • ion channels

  • receptor mapping

  • signal transduction

4. Cleared Brain

Scale and CLARITY methods render biological samples optically transparent but completely preserves fluorescent signals in the structures. In the treated mouse brain, neurons labeled with genetically encoded fluorescent proteins, can be visualized at an unprecedented depth in millimeter-scale networks and at subcellular resolution. The improved depth and scale of imaging permits comprehensive three-dimensional reconstructions of brain sections such as cortex, corpus callosum or hippocampus. The extent was limited only by the working distance of the objective lenses. These methods are useful for light microscopy-based connectomics of cellular networks in brain and other tissues

Using the appropriate process, transparent brain tissue is suitable for deep imaging with the Olympus Scaleview objective family to up to a depth of more than 4 mm (illuminated by a two-photon laser and using GaAsP detectors).

5. Network Imaging

All sensations and behaviors are encoded in dynamic activity patters of the neural networks. Studying of these dynamic spatiotemporal patterns at cellular level of the living brain is crucial to get closer to understanding the function of the nervous system. Thanks to two-photon laser scanning microscopy, we are able to reach the deeper regions of the brain (down to 850 µm) in behaving animals and to study the function of neuron population resolving single cells, dendrites and spines. The signal that shows the neuronal activity could be the intracellular Ca2+ and its detection is accomplished by using different Ca2+ indicators like OGB-1 fluorescent dye or GCaMP protein. To study the activity of hundreds of labeled cells with sufficient temporal resolution, there are the following solutions for the simultaneous sampling of neuronal calcium signals.

To study the neural network, the Femto2D-Resonant is an applicable microscope thanks for the fast frame scanning speed (31 fps). The microscope equipped with piezo objective positioner or fast focusing liquid lens is able to move in z-direction with 10 ms switching time between imaging of the focal planes and to collect signals resolving activity in 3D samples (3D volume scanning).

6. GCaMP6 imaging

Neural activity causes rapid changes in intracellular Ca2+. Calcium imaging experiments have relied on this principle to track the activity of neuronal populations and to probe excitation of small neurons and neuronal micro compartments. Genetically encoded protein sensors can be targeted to specific cell types for non-invasive imaging of identified neurons and neuronal compartments over chronic timescales. The new family of ultrasensitive protein calcium sensors (GCaMP6 or higher versions) outperforms other sensors in cultured neurons and in zebra fish, flies and mice in vivo.

Femtonics has developed new hardware and software tools to record GCaMP6 activity during in vivo conditions from up to several hundred neurons with excellent signal-to-noise ratio. Our method allows real-time visualization of the response of the neuronal assemblies. Simultaneous analysis of multiple cell activity with the simultaneously recorded electrophysiological data is also possible.

7. Axonal Measurements

Measuring the faintest and most phototoxicity sensitive processes in the brain. The axonal arborization made from fine and thin processes is extremely hard to fill with fluorescent dyes. On the other hand, these structures are extremely sensitive, and phototoxicity arrives in an unexpected way: it first appears to affect ultrastructure, and only at larger extents alter functional mesaurements: Ca2+ transients. See supplementary data of Noemi Holderith, Andrea Lorincz, Gergely Katona, Balázs Rózsa, Akos Kulik, Masahiko Watanabe & Zoltan Nusser Release probability of hippocampal glutamatergic terminals scales with the size of the active zone, Nature Neuroscience (2012). As a result, to image structures faint and sensitive like axons and boutons, topmost quality detection optics and detectors are necessary. Our travelling detector assemblies equipped with GaAsP detector modules reach to the physical limits of detection efficiency.

8. Functional correlation and EM Micrograph

Using Femtonics microscopes and careful procedures it is possible to correlate functional properties measured by calcium imaging with the ultrastructure obtained by electron microscopy. Most functional studies suffer from the lack of precise anatomical characterization of the cell or tissue. The high spatial resolution of Femtonics microscopes enables easier identification of the functionally characterized structure in electron microscopy in order to correlate anatomical structure with functional properties.

An elegant example has been shown by Holderith and colleges: Authors included biocytin in the intracellular solution in addition to the fluorescent dyes allowing post hoc light microscopic and subsequent electron microscopic visualization of the imaged areas. Their results showed clear correlation between the functional and the ultra-structural properties.

9. Second/Third Harmonic Generation (SHG/THG) combined with 2-Photon Fluorescence (2PF)


11. 3D trajectory scan

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