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Fluorescence Imaging
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 Fluorescent proteins

The first evidence for the existence of the first fluorescent protein, green fluorescent protein, came during the purification of the bioluminescent protein aequorin described in the paper below. In addition to aequorin the purification also yielded a companion protein that fluoresced green.

Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan Aequorea.
Shimomura, O., Johnson, F.H. & Saiga, Y.

Twelve years after the first evidence for the existence of green fluorescent protein (GFP), this paper described the first crystallization of GFP.

Intermolecular energy transfer in the bioluminescent system of Aequorea.
Morise, H., Shimomura, O., Johnson, F.H. & Winant, J.

This paper describes the original cloning and sequencing of green fluorescent protein from Aequorea victoria. This set the stage for a revolution in fluorescence methods by providing the sequence needed for the first genetically encoded fluorescent indicators, but it took two years for someone to finally get the gene to produce fluorescent protein in cells.

Primary structure of the Aequorea victoria green-fluorescent protein.
Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G. & Cormier, M.J.

This paper provided the necessary breakthrough for GFP to become a usable fluorescent indicator. Other people had been trying to get the GFP gene to produce fluorescent protein without success, but Martin Chalfie wanted to use only the coding region of the gene without any flanking sequence. They used PCR to isolate just the coding region and put that into an expression vector and produced beautiful green fluorescence in cells upon excitation with UV or blue light.

Green fluorescent protein as a marker for gene expression.
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W. & Prasher D.C.

The following manuscript reports the creation of the first genetically encoded physiological sensor based on fluorescent proteins. Although it was not practically useful, it did pave the way for an extraordinary amount of follow-up work to design useful genetically encoded fluorescent sensors for fluorescence imaging.

Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.
Miyawaki, A., Llopis, J., Heim, R., McCaffery, J.M., Adams, J.A., Ikura, M. & Tsien, R.Y.

This paper is the first report of a fluorescent protein (DsRed) that fluoresced red. This was extremely valuable since most of the existing fluorophores emitted green light, thus limiting the ability to image multiple fluorophores in the same sample. The primary drawback was the fact that it is an obligate tetramer, thus limiting its usefulness in some types of studies, particularly as a fusion tag.

Fluorescent proteins from nonbioluminescent Anthozoa species.
Matz M.V., Fradkov A.F., Labas Y.A., Savitsky A.P., Zaraisky A.G., Markelov M.L. & Lukyanov S.A.

This report describes the directed evolution of DsRed to remove its requirement for tetrameriztion and create a monomeric version that the authors termed mRFP1, thus greatly improving its usability.

A monomeric red fluorescent protein.
Campbell, R.E., Tour, O., Palmer, A.E., Steinbach, P.A., Baird, G.S., Zacharias, D.A. & Tsien, R.Y.

This paper describes the first photoactivatable fluorescent protein whose fluorescent properties could be drastically altered after exposure to high intensity light of a specific wavelength. This variant of GFP increases its fluorescence 100 times after activation and is stable for days.

A photoactivatable GFP for selective photolabeling of proteins and cells.
Patterson, G.H. & Lippincott-Schwartz, J.

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ISSN: 1548-7091
EISSN: 1548-7105
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