Journal of Biomedical Optics • Open AccessFluorescence Microscopy: From Principles to Biological Applications
Microscopes are tools invented by humans to study specimens at spatial and temporal resolution that exceed the human eye. Today the researcher has a wide range of microscopes to employ in order to answer carefully poised experimental questions. The key is to match the research questions with the appropriate microscope. In order to optimize the success it is necessary to understand the physical principles of the microscope, how to properly align the instrument, and how to correctly use the instrument. The object of the investigation or measurement is also a major consideration as well as the preparation of the specimen, the interaction of the specimen, and the radiation from the microscope. Often the words “quantitative microscopy” appear in publications; but all measurements contain errors, and the critical investigator must be able to understand the sources of error in a measurement and, if possible, mitigate the magnitude of the errors. Validation of microscopic measurements across disparate types of microscopes is a useful approach. The critical elements in optical microscopy include: optical aberrations, sampling errors, nonphysiological specimen preparation, such as over expression of genetic fluorescent proteins, the deleterious effects of the light source on biological specimens, nonlinear effects in the detector and amplifiers, and artifacts of image analysis and image interpretation.