Two-Photon Laser Scanning Microscope
Our TPLSM started as fully custom-built system consisting of a solid-state pumped Ti:sapphire laser (Tsunami/Millenia Vs; Spectra Physics) and a modified upright microscope (Axioskop FS, Zeiss).
General system overview
The scanhead consists of the two galvanometer driven mirrors, scanner controls (both General Scanning Inc.), scan lens (Zeiss), and beam expander. It is designed as a two-mirror system with two closely spaced scann mirrors (General Scanning Inc) and the conjugate focal plane in between the mirrors.
Exposed scan unit positioned at the back of the microscope
Close-up view of scan mirrors and beam expander
The high precision Z-stage was custom made from massive stainless steel. For combined optical and electrophysiological recordings it carries two micromanipulators. Driven by two piezo actuators it can be moved up and down by a total of 30 µm at an accuracy of <10 nm. With its weight of 14 kg it perfectly resists thermal fluctuations and mechanical disturbances.
Z-stage arrangement with micro-manipulators and experimental bath
Based on the orignal system layout, we constantly improved and optimized our TPLSM to extend its imaging capabilities. A second upright microscope was installed, which is equipped with two detection channels and single-photon counting PMTs. This created the foundation for emission ratiometric and FRET analyzes as well as colocalization studies.
System upgrade by a 2nd upright microscope and highly sensitive dual-channel detection
More recently, a state of the art laser system (Mai Tai eHP DS, Spectra-Physics) was installed. Furthermore, the second upright microscope was equipped with a commercial can head plus control software (TriM Scope II, LaVision BioTec), a computerized intravital stage, and a fast FLIM detector.
Current system status with commerical scan head and control software