IMMUNOFLUORESCENCE

Cells in color

Fungi (Mycena chlorophos) found at Hachijojima botanical park. These organisms have a distinct green glow because of their ability to biofluoresce.

Contents

1 How does it work?

      1.1 Primary vs. Secondary Antibody


      1.2 Fluorophores

2 More Reading

Cells are incredibly small. So small, in fact, that it is nearly impossible to see any of their characteristics with the naked eye or even with a standard light microscope. That is why in recent years, researchers have begun turning to fluorescence microscopy to visually isolate and highlight individual cell components, like DNA or proteins. In particular, researchers employ a technique termed immunofluorescence, which uses specially designed proteins called antibodies, in conjunction with dyes known as fluorophores, to illuminate specific areas of the cell. Through immunofluorescence, scientists have been able to observe how cells behave under different circumstances, and they have been able to gain valuable insight into many of the diseases that still plague society today.

How does it work?

Immunofluorescence can be thought of as a complex game of hide-and-seek. First, you have your hiders, or as biologists like to call them, antigens. These represent the targets that scientists are interested in studying and can be anything from proteins to DNA to sugars like glucose.

Then you have your seekers. In our biological game of hide-and-seek, our seekers are Y-shaped proteins called antibodies. Unlike (hopefully) the seekers in your typical childhood game, however, these seekers are able to locate a very specific (and only a very specific) target, or antigen. How specific you may ask? Imagine if the person playing "It" was

only able to find four-foot, brown-haired hiders who had a small cut on their left elbow. Exactly. Antibodies can locate very specific hiders, which is terrible for an actual game of hide-and-seek, but extremely useful for scientists as, often, scientists only want to study one very specific thing at a time.

So what actually happens when an antibody locates an antigen? Does it signal the discovery by yelling, "Found you"? Well, not quite. Instead of yelling out, an antibody will release a brilliant colored light, which can then be detected through special equipment such as a fluorescence microscope. This is possible because the antibodies used in immunofluorescence are actually attached to a special fluorescent substance called a fluorophore. This is the same type of substance that causes the bright bands to appear on US currency when viewed under a black light. Depending on the specific fluorophore used, a red or green or other colored light can be seen under the fluorescence microscope. In this way, scientists are able to pinpoint the exact moment and location in which an antibody finds an antigen. Impressive, right? But there's a bit more to the story, particular because antibodies can actually come in two flavors-primary and secondary.

Primary vs. Secondary

In our game of hide-and-seek, antibodies are the seekers. However, when it comes to immunofluorescence, seekers actually exist in two varieties. The first are the primary antibodies. These are you run-of-the-mill antibodies that function as previously described. They are capable of locating a very specific antigen, be it a protein, DNA or other cell component. However, then you have your secondary antibodies. These you can think of as seekers that seek out other seekers. A mouthful, yes? But that is essentially the function of secondary antibodies. Secondary antibodies target other (primary) antibodies rather than antigens like DNA.

At this point, you may be thinking to yourself, what possible use could there be for an antibody that locates other antibodies? Well, the answer is two-fold and perhaps best illustrated with this example. Suppose that a scientist, let's just call him George, is interested in three different cell components A, B and C. In order to study A, B and C, he will need at minimum three different primary antibodies (remember, each antibody can only locate a single type of target). But he will also need to attach a fluorescent substance to each of the three primary antibodies as well in order for his antibodies to be visible under a fluorescence microscope. And herein lies the problem. Each primary antibody George has to label costs time and money, and the process is very expensive in both resources. But imagine if George could just have a single antibody, one that found other antibodies, and attached a fluorescent substance to it instead. Suddenly, he only needs to do the attachment process once rather than three times, and that single antibody can be used in all three situations A, B and C. Now George has saved a lot of time and money.

The second benefit of using secondary antibodies is that the response to the discovery of an antigen is much stronger. Think back to the hide-and-seek example. For a primary antibody, you would only get one person yelling, "Found you", upon locating a hider. With secondary antibodies, you not only have the original seeker yelling, "Found you", you have all the seekers who found that original seeker yelling as well. In this situation, the yelling, or released light, is amplified by however many secondary antibodies located the first primary antibody. In many cases, this can easily be a 10 to 100 fold increase in brightness.

. . .

Fluorophores

The one subject that we have largely glossed over until now is that of fluorophores. Recall that this is the fluorescent substance attached to antibodies which allow them to emit different colors of light. But how does a fluorophore actual work? Well, you can think of a fluorophore as a really small battery. Like a battery, a fluorophore can be charged, or excited when given energy. Then like a battery, the fluorophore will turn around and discharge that stored energy. Unlike a battery, however, which gives off electricity, a fluorophore will release its energy in the form of visible light (red, blue, green etc.). The actual color of light that is released depends on the specific fluorophore and how much energy it stores before discharging. Below, you can see links to some common fluorophores.

Fluorescein

GFP

DAPI

Content url

https://www.aatbio.com/articles/immunofluorescence/?gclid=CMvHuvKs4NYCFQZ9vQod1BcFNQ

FOLLOW AND VOTE.....