Making the Most of Microscopy with mNeonGreen

Three years since our flagship fluorescent protein was published in Nature Methods, mNeonGreen is still shining bright. mNeonGreen is a green-yellow fluorescent protein that has been shown to be highly useful in optical assays from general imaging to FRET to super resolution microscopy applications and more.

What makes this fluorescent protein such a hit among researchers?

Since the cloning of green fluorescent protein (GFP) over 20 years ago, fluorescent proteins have become a standard research tool, enabling labeling and imaging of individual proteins within a cell in real time. To make these probes brighter, faster folding, and to cover a wider range of the visible spectrum, new fluorescent proteins are continually being developed. mNeonGreen was engineered by scientists at Allele Biotech from a protein isolated from Branchiostoma lanceolatum, a marine invertebrate, and is the brightest monomeric green-yellow fluorescent protein ever characterized.

Perhaps the most common use of fluorescent proteins today is genetically fusing them to a protein of interest to image protein localization. Unfortunately, many researchers are unaware that many fluorescent proteins – such as GFP – are prone to forming noncovalent dimers, which can lead to significant artifacts. True monomeric fluorescent proteins such as mNeonGreen are less likely to affect the localization, dynamics, or normal behavior when fused to proteins of interest.

For quantitative imaging and live-cell imaging applications, arguably the most critical parameters to consider when choosing fluorescent proteins are brightness and photostability. mNeonGreen has high fluorescence brightness, so less light can be delivered to the cells to collect ample signal intensity, resulting in less phototoxicity. mNeonGreen also has superior photostability, meaning it can undergo many excitation-emission cycles before photobleaching occurs. Because of these properties, mNeonGreen has been shown to be very effective as a fluorophore in fluorescence resonance energy transfer (FRET) – both as an acceptor and a donor.

The community of biologists taking advantage of super-resolution fluorescence microscopy (Nobel Prize in Chemistry 2014) is rapidly growing. But the ability to resolve cellular structural features depends on the chosen fluorophore’s brightness, labeling density, and the stability of the dark state. mNeonGreen is not only extraordinarily bright, but also can be driven into a temporary dark state by light irradiation, making it a useful tag for single-molecule super-resolution imaging of proteins.

Researchers around the world continue to develop novel applications for mNeonGreen. Recently, mNeonGreen was used to create a genetically encoded voltage sensor that can be used to image subcellular changes in electrical activity. Researchers fused mNeonGreen to a light-sensitive ion channel in neurons, linking mNeonGreen fluorescence with the membrane voltage to create an optical readout for neuronal activity with unprecedented speed and accuracy.

Choosing an appropriate fluorescent protein for an assay is often a source of confusion for researchers. In many cases, the selection of a fluorescent protein is motivated by convenience (e.g., availability of the construct) rather than its performance for a given assay. If mNeonGreen seems like the right fluorescent protein for your assay, we at Allele Biotech have made it easy and painless for researchers to get their hands on it. Laboratories can license mNeonGreen for full use at a low cost.

Questions about licensing or whether mNeonGreen is really right for your lab? Contact fp@allelebiotech.com.

References:
“A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum.”
Shaner, N. C., Lambert, G. G., Chammas, A., Ni, Y., Cranfill, P. J., Baird, M. A., Sell, B.R., Allen, J.R., Day, R.N., Davidson, M.W., Wang, J.
2013 Nat Methods, 10(5), 407-409. doi:10.1038/nmeth.2413

“High-speed recording of neural spikes in awake mice and flies with a fluorescent voltage sensor.”
Gong, Y., Huang, C., Li, J. Z., Grewe, B. F., Zhang, Y., Eismann, S., & Schnitzer, M. J.
2015 Science, 350(6266), 1361-1366. doi:10.1126/science.aab0810