Put simply, a fluorescence microscope is any microscope that uses fluorescence to study specimens instead of using regular illumination and magnification methods found in most traditional optical microscopes. Fluorescence microscopes are helpful when scientists need to study complex samples that can't be thoroughly studied under a regular microscope. They also can be used with samples present in such low concentrations that a highly-sensitive microscope is necessary to detect them.
How Does a Fluorescence Microscope Work?
In many ways, the construction of a fluorescence microscope is similar to its traditional light counterpart. There are two distinct features that set fluorescence microscopes apart: (1) fluorescence microscopes use a very powerful light source and (2) utilize specialized filters, like the excitation filter and the emission filter. The most common light sources that fluorescence microscopes use include xenon arc lamps and mercury-vapor lamps, though some also use supercontinuum sources, LEDs, or lasers. A mercury-vapor lamp tends to burn anywhere from 10 to 100 times brighter than lamps used in traditional microscopes, and it's able to give off wavelengths that range from infrared to ultraviolet. Meanwhile, the excitation and emission filters control the wavelengths of the microscope's light source to observe the fluorophore under inspection.
A fluorophore is a fluorescent molecule that absorbs light at a particular wavelength and then re-emits it when the fluorophore is ""excited."" In most laboratory settings, the fluorophore is chemically (or biologically) added to the sample being studied to identify proteins, antibodies, or amino acids, depending on the kind of fluorophore used. After the fluorophore is added, the fluorescence microscope filters will separate the surrounding radiation from the sample's fluorescent light so that the fluorescent light is all that the user sees.
In addition to having a powerful light source, an excitation filter, and an emission filter, a fluorescence microscope also uses a dichroic mirror, an objective lens, and a camera system. The dichroic mirror works as a beam splitter, which means that it quite literally splits the beam of light from the light source in two, placed at a 45-degree angle between the excitation filter and the emission filter. The objective lens simply transmits light from the light source to the sample after it first passes through the dichroic mirror, producing an observable image. The microscope's camera system allows the user to capture high-speed, high-resolution images of the sample, which is especially helpful in classroom and research settings, where further study and observation of the specimen is expected.
Fluorescence microscopes can be as simple as an epifluorescence microscope or as complex as a confocal microscope. One kind isn't necessarily better than another, and the type that any person needs will depend on the settings and applications that the microscopes will be used for. The six main types of fluorescence microscopes include epifluorescence, LED, upright, inverted, confocal, and widefield. Epifluorescence microscopes, which are the most common, are almost always used in life science applications to study the structure of organic cells, while the more complicated confocal microscopes are often used in industrial settings to inspect things like semiconductors.
What Is Fluorescence Microscopes Used For?
Because fluorescence microscopes are unique in the way that they're able to help scientists identify cells and cellular components, they're most beneficial in biology and material science applications. A few specific settings in which fluorescence microscopes are used include:
- Histochemistry - This is a field of science that blends biochemistry and histology to study the chemical components of cells and tissues. For example, fluorescence microscopes can detect biogenic amines that serve as neurotransmitters, like dopamine, serotonin, norepinephrine, and epinephrine, which are far too small to be detectable under a traditional light microscope.
- Food chemistry – this specific chemistry field assesses the structural organization and chemical processes of the components within food. Knowing and understanding the microstructure of any food affects its nutritional and physical qualities, so food chemists play a large part in quality control and keeping consumers safe.
- Fluorescence speckle microscopy - A technique that analyzes macromolecular structures, like proteins. The study of these macromolecules can help scientists understand how proteins move and the cycles of protein synthesis and degradation.
- Mineralogy - The scientific study of different minerals' crystal structures, physical properties, and chemistry. Fluorescence is often used to identify minerals, but it can also be used to identify the mineral's place of origin.
- The Textile industry – Fluorescence microscopy is especially beneficial in the analysis of fibers. Confocal fluorescence microscopes are especially helpful in textile applications since they offer such clear, three-dimensional views of fibers and yarns. On the other hand, Epifluorescence microscopes are great for studying materials like paper.
- The study of porosity in ceramics, which allows scientists to measure an object's density, strength, and durability. Generally, the less porosity a piece of ceramics has, the stronger it is, and the weaker, the higher porosity.
One of the most important parts of fluorescence microscopy is to make sure that the samples studied are fluorescent. Some specimens have an intrinsic fluorescence (autofluorescence), which means that they don't require any outside help to be fluorescent, but many other samples will need to be labeled/tagged/stained as we mentioned above. The fluorescence microscope's application will determine which stain will need to be used if any.
One of the risks of fluorescence microscopy is the possibility of photobleaching, which happens when the fluorescent molecules in a sample become chemically damaged from the excited electrons. Over time, this causes the sample in question to lose its fluorescence. Two ways to try and prevent or reduce the effects of photobleaching include minimizing the amount of illumination used and using stronger fluorophores.
In conclusion, the field of fluorescence microscopy is wide, but all fluorescence microscopes have a few things in common: they have a dichroic mirror, excitation, and emission filters, and, most importantly, they rely on fluorescence and phosphorescence to thoroughly observe their samples. Whether you're in the market for a fluorescence microscope or you just enjoy learning new things, we're glad you stopped by.