Construction and performance of a custom-built two-photon laser scanning system

Abstract

Two-photon microscopy, compared with conventional wide-field or laser scanning microscopy, offers several advantages which arise from the near-infrared excitation and the confinement of two-photon excitation to a tiny focal volume. Therefore, there is a considerable interest in optimizing the performance of two-photon laser scanning microscopes (TPLSMs). Despite the existence of several commercially available devices, there are many reasons to start ab initio. Accordingly, we set-up a TPLSM from single components, and in this report construction details of our custom-built system are given. The system was designed for simultaneous optical and electrophysiological recordings and the illumination path was optimized in view of power-delivery and laser pulse-broadening. For this purpose, a solid-state pumped, mode-locked Ti : sapphire laser was directly coupled into a modified upright microscope. The scan unit was built around commercial scanners and a Zeiss scan lens. Fluorescence was detected in non-descanned mode by a photomultiplier tube. Many mechanical parts and the software for system control and image acquisition were developed in our lab and can be readily modified according to special needs of experiments. All components are easily accessible and can be upgraded according to optical requirements. The performance is comparable to available commercial systems, but our TPLSM is superior in many aspects of cost, flexibility and versatility.