Principle of operation
The sample is fixed on top of a small rod that can be moved up and down to place the sample in the right position in front of a horizontal placed objective. The complete rod-sample system is located in a chamber which can be filled with water or buffer solution to image samples such as zebra-fish embryos in their natural environment (also in air). This configuration allows side-viewing of the sample. The excitation laser beam is then deflected by standard galvanometric or resonant scanners and scans over the sample. By moving the objective horizontal, stacks perpendicular to the rotation axes can be recorded. The fluorescence light is detected in backward and forward direction with up to six PMTs.
Horizontal 2-Photon Microscope
LaVision BioTec’s Horizontal 2-Photon Microscope is a new 2-photon laser scanning microscope that allows sample rotation by 360°. Sample rotation enables the user not only to image the specimen from all sides but also offers various advantages:
- Increased imaging depth
- Good resolution in all dimensions
- Tomographic reconstruction
- Imaging of freestanding samples
- Multi-color imaging
Many different kinds of specimen can be imaged with this new microscope: from zebra-fish embryos and plant structures to fly larvae.
The Horizontal 2-Photon Microscope exists in two versions: as a stand-alone device including excitation source, scan optics, and detectors and as an add-on for the TriM Scope II. As an add-on, it shares the sample excitation source and scan optics like the standard Trim Scope II but has its own detection part
Zebra-fish embryo imaged in backward direction with the Horizontal 2-Photon Microscope. The sample was rotated and imaged by 180°, showing the embryo development over 8 hours. The membrane was labeled with eGFP and the nuclei with mCherry. Courtesy of Nadine Peyriéras, Director BioEmergences, Gif sur Yvette, France.
Integration into the TriM Scope II
The Horizontal 2-Photon Microscope is an upgrade for LaVision BioTec’s TriMScope II utilizing the original TriM Scope II scan optics. Switching between both imaging modalities can be done within seconds. The user only has to push one slider without any re-alignment of the microscope
Imaging chamber of the H2-P integrated into the TriM Scope II (above).
The Horizontal 2-Photon microscope as a stand-alone device
The Horizontal 2-Photon microscope is also available as a stand-alone device. Like the TriM Scope II it is really modular in the combination of laser sources, scanners, and detectors. For excitation, up to two Ti:Sapphire laser beams and one OPO can be combined. Also lasers with a tunable range (700-1300nm) and with a dual beam can be integrated into the system. The Horizontal 2-Photon microscope can be equipped with standard galvanometric and resonant scanners. For detection in backward and forward direction, up to 6 PMTs can be chosen.
- Beam-Optimizer to combine up to two Ti:Sapphire laser beams and one OPO beam
- Scanhead: Two axis galvanometric XY-scanner (2.5 frames/sec @ 512x512 pixel resolution)
- Resonant Scanner (>30 frames/sec @ 512x512 pixel resolution)
- Measurement chamber can be filled with buffer solutions
- Detectors: Standard PMTs, GaAsP PMTs, GaAs PMTs (NIR)