Setting the Confocal Microscope Operating Parameters

Abstract

The goal of this chapter is to systematically look at each decision made in obtaining images from a sample stained with single or multiple fluorochromes. While in this chapter most of the specific examples of choices made in setting instrument parameters are given by using images collected on a Leica SP8 confocal microscope equipped with the following lasers, 488 and 552 nm optically pumped semiconductor lasers (OPSLs), a 638 nm diode laser, and a Coherent tunable IR laser (680 to 1080 nm), for multiphoton and second harmonic generation imaging, the basic choices made are the same regardless of the manufacturer of the system being used and the available hardware. When operating various confocal instruments, it is often simply a matter of identifying the terminology used, for example, pinhole versus iris, the name of a specific lookup table (LUT), or finding where a particular software function is hidden in the multiple menus necessary to operate the system. Once the basic principles are understood, it is possible to quickly learn to operate any confocal microscope and to obtain publication quality images.