Phosphorescence and its relation to Fluorescence Microscope
The concept of phosphorescence is very much interrelated to fluorescence. Both fluorescence and phosphorescence are principles of quantum mechanics. And it is rather remarkable to note that these two concepts are being applied to microscopy. A good way to understand these principles better is by using the popular fluorescence and phosphorescence paints that are available in the market today.
These two paints share some similar characteristics, as they both have the ability to react to a certain light source. Under an ultraviolet lamp, fluorescent paint tends to glow very brightly. But as soon as the lamp is turned off, it also stops emitting light. On the other hand, a phosphorescent paint behaves a little differently. The paint keeps glowing for a while even if the light source was already off. Phosphorescence paint has the ability to store light, allowing it to glow for several seconds longer.
Phosphorescence paint is commonly used in watches and other glow-in-the dark items. It is the neon green paint you see on the hands of the watch, showing you what time it is despite the darkness around you. But have you noticed that the glow doesn’t really last long? The luminescence of phosphorescence paints is at its best form a few seconds after the light was turned off. But after sometime, the glow will eventually wears off, to the extent that it will give no hint of light whatsoever.
In microscopy, phosphorescence is used to observe the minute structures of cells and cell products. It is also widely used in observing micro crystals. Organic and inorganic substances can also be observed accurately adapting the phosphorescence concept. And it is in this particular aspect that phosphorescence becomes very similar to fluorescence microscopy.
A fluorescence microscope is a device that is primarily used to study and analyze the structure and properties of both organic and inorganic substances. But aside from entirely adapting the concept of fluorescence, fluorescence microscopes also use phosphorescence, absorption, and reflection along with it.
Basically, what happens is that the specimen to be observed is tagged with fluorophore. Fluorophore can either be fluorescein, DyLight 488, or Green fluorescent protein. It is placed on the actual part of the specimen of primary interest. And when the sample is illuminated, a certain wavelength of light is subjected to it. The whole idea is for these wavelengths to be absorbed by fluorophores. When this happens, a longer wavelength of light is emitted. And usually, it is comes in a certain color that is different from the absorbed light.
To possibly observe phosphorescence using a fluorescence microscope, slight modifications to it are required. The mechanical shutter arrangement of the fluorescence microscope, which usually consists of two shutters, should be altered in such a way the excitation shutter allows for a series of very short pulses of excitation. Also, the emission shutter should be properly synchronized to clear out the viewing pathway throughout each period of excitation. In this case, no barrier filter is required, as the choppers will totally cut off the excitation lights. Rotating shutters may be used alternately, for as long as its phase can be varied.
Time resolution luminescence is technically easier nowadays. As a result, phosphorescence microscopy is starting to become popular and widely used. It is always best to conduct phosphorescence studies at extremely low temperatures. If you need to observe liquid nitrogen, do it at 77 Kelvin or less. Temperature this low increases phosphorescence’s intensity. This condition also suppresses fluorescence because the radiation process is weaker.
Fluorescence microscopes work very similarly to fluorescent bulbs. The gasses inside a fluorescent bulb take in high-energy electrons when power runs through it. And at that point, the gasses start to transmit or reradiate the same energy they absorb at an entirely different frequency. They fluoresce, so to speak.
Phosphorescence, on the other hand, can be likened to a cathode ray tube monitor. The surface of these types of monitors is filled with phosphors. And these phosphors absorb high-energy electrons subjected to them by the system. They would then gradually release the said energy after some time via a visible band.
With all these concepts presented, it is quite easy to see that both fluorescence and phosphorescence are rather complex concepts. They are certainly not like the principles of transmitted and reflected of light, which are very commonly used concepts, not only in microscopy by in a lot of other applications as well.
Different microscope models exhibiting both fluorescence and phosphorescence capabilities are starting to penetrate the market, although these high-end microscopes are not expected to be as popular as traditional light microscopes. To date, only seasoned professionals use these microscopes, as they tend to be very expensive and require extensive knowledge to manipulate.
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