Fluorescence microscopy
Introduction
Fluorescence microscopy is a type of light microscopy that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to study properties of organic or inorganic substances. The "fluorescent microscope" was first developed by Carl Zeiss in the early 20th century, and has since been extensively refined.
Principle of Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation. The most striking examples of fluorescence occur when the absorbed radiation is in the ultraviolet region of the spectrum, and thus invisible to the human eye, and the emitted light is in the visible region.
Fluorescence Microscopy
Fluorescence microscopy is a major tool that scientists use to examine the structure and function of internal cellular organelles and other biological specimens. It works on the principle of fluorescence, i.e., the ability of certain compounds to emit light when they are excited by more energetic light.
Types of Fluorescence Microscopy
There are several types of fluorescence microscopy, including epifluorescence, confocal, two-photon, and super-resolution microscopy.
Epifluorescence Microscopy
In epifluorescence microscopy, the excitation light is passed through the objective lens from the same side of the specimen as the detecting lens. This is in contrast to the traditional transmitted light microscope, where the excitation light comes from the opposite side of the specimen.
Confocal Microscopy
Confocal microscopy, a more advanced fluorescence imaging technique, uses a laser to excite a single plane of fluorescent molecules within a cell. This allows for more detailed imaging of structures within the cell.
Two-Photon Microscopy
Two-photon microscopy is a fluorescence imaging technique that allows imaging of living tissue up to about one millimeter in depth. It differs from traditional fluorescence microscopy, in which the excitation wavelength is shorter than the emission wavelength, as the wavelengths of the two exciting photons are longer than the wavelength of the resulting emitted light.
Super-Resolution Microscopy
Super-resolution microscopy is a form of light microscopy that allows for imaging of objects at a much higher resolution than that traditionally allowed by the diffraction limit of light. This is achieved by the use of fluorescent molecules and specific techniques to effectively 'switch on' and 'switch off' these molecules.
Applications
Fluorescence microscopy has become an essential tool in biology and the medical sciences, as well as in materials science due to its high sensitivity and high specificity. It can be used to study organic molecules, cells and tissues, thin coatings and films, and even certain inorganic materials.