Fluorescence microscopy allows you to look at the insides of cells & analyzes the fluorescence from a sample. It uses a beam of light such as UV light, which excites electrons in molecules of certain compounds and causes them to emit light . Some reasons or advantages of using fluorescence are: It allows you to look at cells -dead or alive; it provides high contrast, high signal; allows to label specific components with multiple colors, & using ccd cameras along with fluorescence microscope allows for counting the numbers of cells. For example, live cell imaging can be done with fluorescent proteins which lets you see specific processes in cells- example - an S2 cell during mitosis.
Fluorescence has only been known for 150 years and was first studied by British scientist John F.W. Herschel in 1854, when studying the bark of a tree used to treat malaria. He made an extract of the sample, and noticed that under sunlight the sample glowed blue. The compound in the sample that fluoresced in the UV rays is quinine. The excited quinine molecules absorbed UV light, which has high energy, low wavelength, and quinine molecules emitted blue light which has lower energy and higher wavelength. Plot of wavelength on x axis vs. excitation/absorption and intensity of emitted fluorescence on the y axis shows spectra with maxima, difference between the excitation max and emission max is called Stoke shift, in general , the rule is going from higher energy/low wavelength to lower energy/high wavelength with some exceptions. Fluorescent dyes are classified by large or small Stokes shift . Jablonsky diagrams show energy states- photons that absorb light are in higher energy states, photons that emit light go to lower energy states, the ratio of absorb/emitted photons = quantum efficiency. Dyes with high quantum efficiency and very bright ( high absorption coefficient ) are preferred. A fluorescent microscope has the following parts: Light source - lasers, common- LED; excitation filter - which allows excitation light thru; Dichroic mirror; Objective lens, an emission filter which blocks excitation light and allows emission light thru. The excitation and emission filters and dichroic mirror are packaged into a the fluorescence cube, & a motorized turette can be used to move the different cubes to get different lights. Faster wavelength selection can be done with multi band pass filters and filter wheels. When setting up experiment, the spectra of dyes and filters need to be determined and can be done with references.
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