Different Ways to Add Fluorescent Labels

Using fluorescence to detect your target

Using fluorescence provides greater contrast compared to viewing your samples with brightfield microscopy alone. Labeling various targets with separate fluorescent colors allows you to visualize different structures or proteins within a cell in the same experiment. Ways to fluorescently label your target include fluorescent dyes, immunolabeling, and fluorescent fusion proteins—all of which can provide a means to selectively mark structures and proteins within the cell, allowing you to see them more easily when you image.

Some dyes can be used in live cells, while others have uses in fixed and permeabilized cells. As with any technique, using fluorescent dyes to label your target has limitations. When fluorescent dyes are used in live cells, they can be phototoxic. Sometimes a fluorescent dye that labels exactly what you want to see is not available. If a specific dye is not available, you can try using immunolabeling in fixed cells or fluorescent fusion proteins in fixed or live cells to visualize your target.

One fluorescent dye molecule, many different selectivities

Many fluorescent tools for cell biology are essentially fluorophores that have been modified in different ways or conjugated to various molecules to give them a certain function or allow them to bind to specific organelles or proteins.

Through chemical modifications, a single fluorophore can be produced in a number of variant forms, each with a different specificity. For example, the green-fluorescent Alexa Fluor® 488 dye molecule can be modified to target immunolabeling (B), or can act as a whole cell stain (C).

Figure 1. A single fluorophore can be modified to carry out any number of labeling jobs, including functionalized forms for labeling cell structure components such as actin (A) and tubulin (B) and salt forms for whole-cell staining (C).

Fluorescent labeling methods can vary

As mentioned above, fluorescent labels usually comprise a fluorophore that is modified in a way that gives them specificity. In addition to linking fluorophores to various molecules, other types of modifications can give fluorophores novel characteristics.

Linking a fluorophore to a specific molecule, for example, an antibody, can give it selectivity for its target, in this case, an antigen. The advantage to this approach is that the fluorophore is bound to a target, and you wash away any unbound or excess fluorescent dye, resulting in high signal to background and improved contrast.

Fluorogenic dyes start off with dim emissions, but conditions or activity inside the cell trigger an increase in their brightness. For example:

• Increased fluorescence after binding to target—many nuclear stains like SYTOX® Green and Hoechst show little fluorescence on their own. When these stains bind to nucleic acids, however, the fluorescence intensity is greatly increased, resulting in a very bright signal.
• Increased fluorescence after intracellular modification—other stains contain a chemically reactive group that becomes modified by cellular activity. For example, calcein AM contains an ester group. Inside live cells, in the presence of active esterases, the ester group is cleaved, which converts nonfluorescent calcein AM to green-fluorescent calcein.

Fluorogenic dyes typically have high signal to background, since the unbound/uncleaved dye has a dim signal compared to the dramatic increase in fluorescence observed when they are bound or activated. Because the unmodified fluorogenic dye is not very bright, you usually don’t have to wash it out.

Using fluorescent dyes as functional indicators

Sometimes you don’t just want to label something; you want to know if your cells are healthy, or if they are functioning the way they should be. You may want to know: are my cells alive? are they apoptotic? or are they stressed out? There are many fluorescent dyes available that can act as indicators for various cellular functions and answer these questions.

For example, the fluorescent dye tetramethylrhodamine, methyl ester (TMRM) specifically labels mitochondria, and it can also indicate if the mitochondria are healthy. Active mitochondria in healthy cells will maintain a mitochondrial membrane potential, and TMRM is brightly fluorescent in these mitochondria. As mitochondrial membrane potential is lost (in sick or dying cells), TMRM signal is diminished.

Figure 2. Panel A shows TMRM staining in healthy HeLa cells; panel B shows the loss of TMRM signal concurrent with treatment to destroy the mitochondrial membrane potential.

Important characteristics of fluorescent dyes

When people talk about fluorophores, or fluorescent dyes, you may hear them use words like extinction coefficient and quantum yield. These terms are physical properties of the fluorescent dye molecules themselves, and they give chemists an idea of how bright the dyes are. However, there’s more to fluorescent dyes than the extinction coefficient and quantum yield when it comes to biological investigations. For scientists who need to actually use these fluorescent labels in a biological system, there are many other characteristics that should be considered when designing an imaging experiment.

1. Selectivity—you want your fluorescent label to be confined to the molecule or activity you’re interested in. For example, if you want to label actin, you want a fluorescent label that will target only actin.
2. Signal to background—you want something that will give you bright fluorescence with low nonspecific background signal. A high signal with a low background will give you the greatest contrast between what you are interested in seeing and everything else. See Background Fluorescence. 
3. Photostability—photostability is an indicator of how well the fluorescent signal is maintained with repeated exposures to illumination light. It is difficult to work with fluorescent dyes that are not photostable, as you can lose your signal in the time that it takes to focus the microscope. Photostability is also important if you want to perform time-lapse imaging.
4. Excitation/emission properties—you want to choose a fluorescent dye that has excitation and emission properties that are compatible with the filter set you are going to use. See Using Filters to Capture Your Signal.
5. Environmental stability—although sometimes inevitable, it can be difficult to work with dyes that are environmentally unstable. Some fluorescent dyes are sensitive to air, light, or temperature, and you’ll want to consider those parameters before you get started.