Design optimization of a commercial multi-photon laser scanning microscope system

This paper reviews the design process underlying development of a second generation multi-photon laser scanning microscope (MPLSM) system and the methods used to characterize its performance. The purpose is to show how each of several elements of the design present complex design choices which must be resolved in the overall interests of instrument performance combined with ease of use in difficult experiments involving live samples. The paper is intended to stimulate discussion of how close we are to making MPLSM accessible as a routine microscopy method, as opposed to a specialized, user engineered technique. The issue of detector design in MPLSM poses the well known problem of how to collect as much as possible of the emitted fluorescence as it escapes from the sample. The challenge in design of a commercial system is to combine the highest possible detection sensitivity with the requirements of laser safety, ease of use and adaptability to different microscope platforms. This paper will present a 'hybrid' approach to MPLSM detection where the user can select one of several detection strategies according to the nature of the particular sample. Proposals will be presented how detector performance can be compared between systems of different design.