Femtonics has introduced the FEMTOSmart two-photon microscope series whose unique feature is the elevated body. The body can move in X, Y, and Z directions and offering sufficient room under the objective to position the samples optimally.
This aspect makes the two-photon microscopes perfect for conducting in vivo studies in a variety of model organisms, such as non-human primates, zebrafish larvae, and mice navigating in virtual reality.
FEMTOSmart can be fully customized, so each of them is always fit optimally based on customers’ needs.
With its three members (FEMTOSmart Galvo, Resonant, Dual), the FEMTOSmart series represents conventional two-photon imaging systems that are capable of performing functional imaging based on galvo and resonant scanners.
Network and Dendritic Imaging
Flexible scanning patterns, developed by FEMTONICS, support the selection and activity measurements of each cells and following the tortuous protrusions of the dendritic arbor in a precise way in a 2D plane. This ROI scanning methods result in a high signal-to-noise ratio and high scanning speed. Fast-frame scanning method of FEMTOSmart Resonant combined with fast Z-focusing (liquid lens objective kit and piezo-objective positioner) is a popular method for analyzing 2D and 3D neural networks or the dendritic arborization in 3D.
Noninvasive in-depth functional imaging is facilitated by three-photon (3P) microscopy, which allows users to visualize cells present in deep tissues, with better contrast and a high spatial resolution when compared to two-photon excitation. For 3P imaging, the FEMTOSmart Galvo can be fitted with a 3P range microscopy optional module.
CCells or subcellular components can be stimulated by using an LED light source to quickly steer the laser beam through the entire FOV, or by using multiple beam path in optimized scanning patterns, where the path can take a line, point, zigzag, or spiral shape.
Focusing on Regions of Interest for High Speed and SNR
Integrated with Femtonics’ intelligent control software, the galvo scanner allows users to choose various scanning patterns that cover broadly distributed ROIs. Femtonics’ software includes features such as analysis functions, real-time display, built-in parallel data acquisition of electrical recordings, and ∆F/F calculations, allowing users to focus and understand the physiological procedures in their research.
Multiple Line Scanning
In this scanning mode, the laser beam is guided by the X and Y mirrors in a flexible way along complex curves or straight lines. The scanner spends a large part of its time by gathering signals from these lines, avoiding intermediate sections that exist between them.
As a result, the scanning speed and the signal-to-noise ratio (SNR) of the signals produced by multiple sites of ROIs increase by three- to four-fold when compared to frame scanning.
Folded Frame Scanning
Folded frame scanning is a patented technology that allows users to image a limited area along a line, where the chosen regions can have a curved or straight shape. Using this sophisticated scanning technique, single-cell bodies in different areas of the specimen can be imaged. This technique can also be used to image by following events that occur along the winding dendrites with their protrusions, in the tissues of moving animals as well.
In addition, photostimulation techniques such as uncaging and optogenetics are supported by Femtonics’ exclusive scanning patterns and their combinations. Stimulation in femtoliter volumes can be achieved using random-access point scanning, especially close to dendritic spines, where the time taken for the stimulation can be set from microseconds to seconds. The triggered signals can be followed along the dendrite by performing line scanning almost concurrently with photostimulation.
High-Speed Imaging in a Wide Field of View
Resonant-scanner-based raster scanning by FEMTOSmart Resonant obtains images for hours at a speed of 31 frames per second, which is approximately five times faster when compared to galvanometer-based scanning in the entire field of view.
Long-Term Measurements or Time-Lapse Imaging
A high frame scanning rate and an unrestricted video streaming, along with the possibility to perform automated measurements, support long-term research such as memory retrieval, following learning processes, development of model organisms, associative learning, and so on.
The video below displays an early stage of development of a zebrafish embryo, the ontogenesis of which was monitored and recorded for more than a day, from a low cell state.
3D Volume Scanning
A liquid lens objective or a piezo-objective positioner can be used to carry out the movement in the Z plane. There are two scanning techniques such as in-frame ramping, and frame-by-frame scanning. Together with a rapid movement in the Z plane, fast XY-scanning guarantees near real-time volume measurement in 3D. This helps in investigating activity changes that occur in the morphology of organs or the 3D cellular networks.
Using the resonant microscope fitted with accurately timed LED light sources, cells distributed in one or more layers can be activated and imaged with excellent speed.
The combined use of a Pockels cell and gated detectors helps achieve the microsecond-scale switching time between imaging and stimulation.
Implements the Advantages of the Galvo and Resonant
Both resonant scanner and galvanometric scanner are integrated into the FEMTOSmart Dual microscope, which offers all the benefits of the two imaging methods. The combined use of the functions of both scanners offers an ideal solution for photostimulation.
The galvanometric scanner selectively guides the laser to cells or subcellular components. This selectivity is guaranteed by scanning along random line patterns, located on dendritic segments or cell bodies.
Through the resonant scanner, users can track the cellular changes of a neural network, acquiring imaging data at the same time by scanning the surrounding area at high-speed frames.
Protocols developed on the easy-to-use GUI, regulate the timing of photostimulation and detector gating. Being an optional module, the Tilting objective can increase the objective’s accessibility to the sample.