Quantitative Microscopy

While light microscopy is almost 400 years old, developments of the past decade have offered a variety of new mechanisms for examination of biological and material samples. These developments include exploitation of techniques such as confocal microscopy, scanning near field microscopy, standing wave microscopy, fluorescence lifetime microscopy, and two-photon microscopy. In biology, advances in molecular biology and biochemistry have made it possible to selectively tag (and thus make visible) specific parts of cells, such as actin molecules, or sequences of DNA of 1000 base pairs or longer. In sensor technology, modern charge-coupled device (CCD) cameras are capable of achieving high spatial resolution and high sensitivity measurements of signals in the optical microscope. Modern CCD camera systems are limited by the fundamental quantum fluctuations of photons, which cannot be eliminated by "better" design. Further, proper choice of the sampling density involves not only an understanding of classic linear system theory-the Nyquist theorem-but also the equally stringent requirements of digital measurement theory. Experimental procedures that rely on the CV can be used to evaluate the quality of one's quantitative microscope systems and to identify which components are the "weakest link". Typical values of relatively straightforward parameters such as size can easily be measured to CVs around 1%.