Fredrick W. Herschel discovered fluorescence in 1845. He observed that UV light can excite quinine solution, resulting in the emission of blue light. Sir George G. Stokes built upon this observation, noting that fluorescence emissions were of longer wavelengths than the original UV light used to excite them. In the early 1900s, fluorophores were first used to stain tissues, bacteria and other pathogens for biological studies. Carl Zeiss and Carl Reichert later developed these observations into fluorescence microscopy.
Ellinger and Hirt developed fluorescence labelling in the early 1940s, but it was not until the 1990s that the cloning of green fluorescent protein (GFP) was achieved. Once it was, however, its application in fluorescence microscopy was immediately solidified.
How Fluorescence Techniques Evolved
Fluorescence microscopy can be applied in a very controlled manner, thanks to the many experiments and studies around the excitation and emission properties of a variety of substances. The results are now quite reliable and extremely detailed.
The introduction of pinhole apertures to the beam path resulted in much resolution improvement during early fluorescence microscopy. One was added in front of the light source, and a second one in front of the detector. These pinholes helped to focus the light – both emission and excitation – and their use came to be known as the double focusing system. This technique led to the development of modern confocal microscopes.
A further advancement on confocal microscopy is spinning disc microscopy, which reduces the image acquisition time and the exposure time of the sample to laser light. The laser passes through a rotating disc containing multiple pinhole apertures, which illuminates several spots on the sample simultaneously, increasing the possible frame rates.
F-photon microscopy is another variation of fluorescence microscopy, in which two photons are absorbed, simultaneously, instead of just one. This increases the excitation wavelength to a point higher than the emission wavelength.
No out of focus light is produced in two-photon microscopy, reducing the chances and risk of photobleaching and other light-related damage.
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