Basic Confocal Microscopy

Confocal scanning laser microscopy has become a primary method in many laboratories for visualizing microscopic structure in the third and fourth dimensions. However, in many cases individual users are expected to understand and operate complex confocal microscopes without being given the opportunity to learn basic principals. This text will target beginning and intermediate users of confocal microscopy and will include information that students, technologists and researchers need to know to efficiently collect and analyze data and to obtain publication quality images. The first part of the volume will provide information on a number of topics relevant to confocal microscopy, including the basics of fluorescence and fluorescent probes, biological specimen preparation (fixation, staining, optical properties and mounting materials), the basic components of a confocal microscope (lasers, dichroic mirrors, microscope objectives, photomultiplier tubes, etc.) and an overview of some applications of confocal microscopy. Proper selection of user adjustable parameters to optimize image collection will also be addressed and demonstrated.