Classical fluorphores have been widely utilized in biological research, and have been extensively modified to promote particular qualities. Here we review practical methods of optimizing fluorescence signals for microscopy, discuss experimental solutions to common laboratory applications of fluorescent probes, and describe advantages of the sulfonated rhodamines.

Fluorescence output of dyes The usefulness of a fluorescent probe depends critically on how bright it is and on its ability to resist photobleaching.

The brightness of a dye depends on two factors: first, its efficiency in capturing excitation photons, called the molar extinction coefficient, and second, on the number of photons emitted for each photon absorbed, called the quantum yield. The product of these two factors describes the brightness of a dye. Both the molar extinction coefficient and the quantum yield are constants under specific environmental conditions. Consequently, this is a measure that is useful to compare the brightness of dyes.

In many experimental situations there is some three dimensional particle that is of interest to the microscopist. To obtain morphometric information about the particle, the microscopist must section some reference space that contains the particle or component of interest, but after sectioning, the reference space and component are reduced to a 2-D sample. This sample no longer contains 3-D structures but only 2-D profiles of the reference space and component.

Stereology can be used to obtain 3-D information from 2-D samples. These samples can be micrographs or digital images, or they can even be the cut surface of the 3-D reference space. The four most basic parameters measured using stereological techniques are volume (3-D), area (2-D), length (1-D), and number (0-D). When measuring these parameters it is often advisable to first measure the density of the component within the reference space, next measure the volume of the reference space, and finally multiply the density by the reference volume to obtain the actual structural parameter.

There are many electron microscope centers around the world and it is well recognized that there is no single “right” way to develop successful research and teaching electron microscope organizations.

The model for this discussion is that of the Center for High Resolution Electron Microscopy (CHREM) at Arizona State University. Several factors, including but not limited to those discussed below, have contributed to the continuing growth and development of this Center.

The Center serves students and faculty from eight separate academic units. It is also accessible to a significant number of microscope users from outside the university. These include researchers from industry as well as other academic institutions. The number of active instrument users typically averages eighty per year. There are ten instruments available to those who successfully complete the requirements for microscope use. It is not necessary to discuss the physical plant which houses the Center's instruments. Although very important, this topic has been adequately discussed elsewhere by experienced researchers.