Ultrafast Real-Time 3D Imaging in Multiphoton Microscopy - FEMTO3D Atlas

The FEMTO3D Atlas is a three-dimensional (3D), real-time imaging two-photon microscope that integrates advanced science and engineering in 3D measurements. It comes with innovative features—the electrically tunable acousto-optic deflectors (AODs) which enable the extremely fast focusing in X, Y, and Z axes (AO or acousto-optic technology) and 3D random-access scanning possibilities.

The microscope implements and goes beyond the conventional imaging functions based on galvo and resonant scanners. It integrates these functions with a special fast 3D imaging feature, offering a complete solution. Using the FEMTO3D Atlas, users can scan dendritic, neuronal, or other biological processes in 3D, around a million times faster when compared to traditional scanning techniques, at a sustained two-photon resolution.

Users can integrate the FEMTO3D Atlas microscope with a preexisting upright microscope via its adjustable, modular frame. ATLAS has a variable frame that allows coupling the microscope to various host microscopes. The Atlas microscope can also function as a separate system.

Network Imaging

Neural networks contain thousands of neurons distributed in 3D space, usually across several cortical layers of the brain. Using FEMTO3D Atlas to perform 3D random-access point (3D ROI) scanning is an optimal choice to expose the dynamics of the neural networks during data processing. This process allows users to record neuronal activity from cell populations containing up to thousands of cells in vivo.

All the preselected ROIs can be quickly and accurately targeted without wasting time by measuring on unwanted background. This further increases the measurement speed by up to 30 kHz, and also increases the signal-to-noise ratio by several orders of magnitude when compared to traditional raster scanning.

Dendritic Imaging

3D random-access scanning offers maximum flexibility in rapidly choosing 3D ROIs with different sizes and shapes such as trajectories, ribbons, tilted frames and so on. Expanding 3D random-access point scanning by drifting the focal point over short 3D trajectories enables imaging without any interruption at several dendritic branches with maintained scanning speed but with a more comprehensive spatial resolution. 3D ribbon scanning enables following the curvature of dendrites, even during the animal moves.

Moreover, the AO technology allows also random selection of the entire scanning plane which can be positioned in tilted mode, a high-speed raster scan mode offers a scanning speed of 40 fps at 510 x 510 pixels and 500 x 500 µm2.

Behavioral Studies

The 3D anti-motion technology from Femtonics makes it possible to capture cell activity while an animal is carrying out tasks and moving in virtual reality. Scanning points are extended to drifted lines to preserve signals. These lines are accurately fitted to one another, allowing the target object to be enclosed with surface or volume elements.

Such elements cover the pre-selected ROIs as well as the adjacent regions. This provides a chance for motion correction by maintaining all fluorescent data during motions and reducing the artifacts by over one order of magnitude in behaving animals. There are numerous advanced ROI scanning methods specialized to neural network, dendritic and spine imaging.

Optogenetics

Through photostimulation methods, cells can be activated in an accurate and selective fashion. An integrated second laser and the 3D random-access excitation technique of FEMTO3D Atlas facilitates flexible parameters for photostimulation.

Based on the chosen scanning pattern, users can activate dendritic processes or sparsely distributed individual cells in a large volume, with excellent accuracy. By quickly changing between the two laser lines, users can record the activity almost concurrently with photostimulation.