Hopefully now you have a good grasp of the basics of fluorescence- if not, you can remind yourself by reading our general introduction here. Now that you have grasped that, it’s time to talk about how it is used as a tool in microscopy. The short version is: fluorescence microscopy is possible due to fluorophores. Chemical fluorophore moieties have been synthesized for over one hundred years, but their efficiency and adaptability necessarily improved along with the ways in which they could be detected- namely, fluorescence microscopes.
Twentieth Century Fluorophores
Fluorescent stains for single-celled organisms led to fluorescent markers for tissues and cells. In the mid-twentieth century, fluorophores were first attached to antibodies to allow for the now-standard technique of immunofluorescence.
A Veritable Crayola Crayon Case!
But I’m getting ahead of myself. The first question is: what are these fluorophores? As a reminder from the introduction, a fluorophore is a molecule which absorbs light at a certain wavelength, bounces around that energy within the electrons in its chemical structure, and then emits the extra absorbed energy as light at a higher wavelength. Fluorophores come in all shapes, sizes and colors- a veritable Crayola crayon case of colorful tools that can allow you to visualize structures!
Seeing Nucleic Acids
‘Probes’ refer to dyes or other molecules that can bind or intercalate directly to the structure you’re interested in detecting. For example, ethidium bromide, 4?,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) all intercalate into nucleic acids, allowing investigators a direct way to visualize DNA and RNA.
Proteins- location, movement, quantity
Fluorescent moieties can also be covalently attached (or ‘conjugated’) to other molecules. These fluorophores are typically derivatives of acradine, arylmethine, coumarin, cyanine, mapththalene, oxazine, pyrene, tetrapyrrole, or xanthene. Each fluorescent compound must, in addition to the fluorophore, contain a functional group which allows it to covalently bind to the macromolecule you want to visualize. In immunofluorescence, the fluorescent molecule is conjugated to an antibody. That antibody binds to your protein of interest, and voila, you can see where your protein is, how it moves, and how much of it there is.
A Green Glowing Nobel Prize
Proteins themselves can have intrinsic fluorescence if they contain a fluorophore within their structure. The most famous of these, due to its ubiquitous use in the laboratory and for the Nobel Prize awarded to those who adapted it for laboratory use, is green fluorescent protein (GFP). GFP is a naturally occurring protein first isolated from the jellyfish Aequorea victoria. It emits green fluorescence, hence its name. GFP has been mutated in the lab to change its excitation and emission spectra for use in a wide range of fluorescent applications.
At the end of the Rainbow…
There are now colored fluorescent proteins from red to violet and everywhere along the rainbow in between. These fluorescent proteins are cloned into a system as a fusion with your protein of interest or are cloned to be driven by a promoter region of interest.
You can read about the Nobel Prize winning team and their work here;