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A dark image illuminated by splashes of deep blue, vibrant green spindles, and red bubbles. What could you be looking at? One of the most fascinating intersections of physics, chemistry, and biology.
Fluorescence microscopy produces some of the world’s most wondrous images that are far too minuscule to be seen by the human eye. Everyone’s heard of traditional microscopes that use lenses to magnify images for observation, but fluorescence microscopy relies on light sources to identify and capture specific elements of microscopic objects using--you guessed it--fluorescence.
So what exactly is fluorescence? We can answer this question at the level of molecular chemistry. Every atom is surrounded by a cloud of electrons, some that tend to hover near the nucleus, and others farther away. When molecules absorb energy in the form of light, their electrons may enter a brief “excited state,” in which the electrons gain enough energy to further resist the pull of the nucleus and thus may travel further from it. However, this state lasts for only a moment as the energy decays through the emission of light at a different, longer wavelength than what was used to excite the electrons. This very process is called fluorescence.
A variety of light sources are used to excite molecules in fluorescent dyes. The most common is a broadband source that creates white light which is actually an amalgamation of all colors on the spectrum. On the other hand, scientists may also target specific wavelengths using lasers or light-emitting diodes (better known as LEDs).
Many of these techniques involve high exposure to powerful light sources, which must be used with caution. If a fluorophore--a molecule or atom that fluoresces--is exposed to a light source for too long at a high intensity, photobleaching may occur. Photobleaching is the process where fluorophores begin to degrade. Their saturation or brightness rapidly decreases until the fluorescent glow disappears.
But how exactly do scientists take these glowing particles and image them for interpretation? That’s where the physics and properties of light come in.
Microscopes utilize a set of special mirrors that allow certain wavelengths of light to pass through them while reflecting others. As depicted below, a dichroic beamsplitter allows longer wavelengths to be transmitted. Oftentimes, images are taken with an epifluorescence microscope which illuminates the entire sample all at once. Microscopes use filters to help isolate wavelengths to be viewed. Many other imaging techniques exist, such as laser scanning confocal microscopy and brightfield microscopy.
All these techniques are used to examine a variety of biological subjects, concepts, and properties. Certain fluorescent dyes may be used to observe cell nuclei, cytoskeletons, and other organelles. Fluorescence microscopy truly represents the possibilities and beauty of science.
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