Fluorescence and wide field microscopy

Transmitted light microscopy is a classical technique to observe microscopic structures. Stained sections are widely used and give valuable information in many studies. Other transmitted light microscopy approaches, including techniques such as phase-contrast, differential interference contrast (DIC), and polarized microscopy, enhance the inherent contrast of samples, including living specimens, to make them more visible.
Fluorescence microscopy, using a variety of fluorescent indicators or probes, specific for targets such as proteins, lipids, or ions allows watching cell physiology. The number of available probes specific for different cell components or physiological indicators increases constantly. Other fluorescent staining or fluorescent properties of many materials gives valuable information in other fields out of biological specimens. In wide field fluorescence microscopy, a parallel beam of light simultaneously illuminates the whole specimen (or wide-field of view) to excite (via the filter block) the fluorophore(s) it contains. Excitation light is provided by a mercury or xenon high-pressure bulb and the required wavelengths are selected with custom optical filters. The resulting fluorescence in the specimen is viewed by eye or captured electronically with a camera, as all parts of the specimen are viewed simultaneously. This constitutes an advantage in many cases. However, in others, diffraction-limited optics and the unavoidable projection of out-of-focus light onto the single image plane of the camera, can result in images of lower contrast and spatial resolution. Nonetheless, the wide-field fluorescence microscope is commonly used and can be successfully used with thin sections or layers of single cells.