Non-microscopy applications for fluorescent proteins

Here, I wanted to briefly discuss other applications of fluorescent molecules in biology, other than microscopy.

One simple use for fluorescent molecules is measuring the amount of emitted light by a fluorimeter. This can be used with different florescent dyes that respond to different biological aspects (e.g. calcium levels, cell viability, mitochondrial function, DNA replication, protease function etc…) and fluorescent or phosphorescent proteins (luciferase, GFP) to measure the amount of the expression of said protein fused to a protein or promoter of interest. This cannot be used at the single cell level but on cell culture, or cell lysate.

Fluorescence activated cell sorting (FACS) – this method uses flow cytometry, combined with fluorescence detection to sort cells based on different properties. These properties include cell size and morphology (based on light diffraction) and fluorescent properties, as designed by the researcher. In short, the cells flow through a very narrow tube and pass through several light and laser beams. The light scatter and the fluorescence of molecules excited by the lasers is detected by detectors found along the tube. Thus, one can count the number of cells that have this or that property. For instance, it is possible to count the number of cells that express GFP, and even divide them into groups based on fluorescence intensity (which is assumed to be equivalent to expression level of the GFP). For some applications, the cells are thrown away; for other applications, the machine can collect the cells with the required properties defined by the researcher (e.g. high fluorescence) so that the researcher can sort out undesired cell populations, and continue to work with only the desired cell population. Several years ago, I used such a set up to isolate cell clones that express high levels of a drug, which was correlated to high GFP expression.

Recently, a new technology was developed that combine FACS with microscopy, called imaging FACS. With this system you can take pictures of the cells that you are sorting. The resolution is high, but there are several useful applications.

Another use is as a biomarker to differentiate between different biological states. For instance, GFP could be fused to a promoter that is activated by high salinity in a certain plant. If that plant will sense high-salinity conditions, it will glow green.

A new commercial use is glowing fish. Other pets might follow…

Finally, GFP can be used as a publicity stunt to show biological capabilities (hence glowing fish, mice, rabbits, cats, pigs and monkeys. Soon – fluorescent humans).

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