Editing Green fluorescent protein (section)

=== Fluorescence microscopy ===
{{Main|Fluorescence microscope}}
[[Image:GFP Superresolution Christoph Cremer.JPG|thumb|200px|Superresolution with two [[fusion protein]]s (GFP-Snf2H and RFP-H2A), Co-localisation studies (2CLM) in the nucleus of a bone cancer cell. 120.000 localized molecules in a widefield area (470&nbsp;μm<sup>2</sup>).]]
The availability of GFP and its derivatives has thoroughly redefined [[fluorescence microscopy]] and the way it is used in cell biology and other biological disciplines.<ref name=Yutse_2005>{{cite journal | vauthors = Yuste R | title = Fluorescence microscopy today | journal = Nature Methods | volume = 2 | issue = 12 | pages = 902–4 | date = Dec 2005 | pmid = 16299474 | doi = 10.1038/nmeth1205-902 | s2cid = 205418407 }}</ref> While most small fluorescent molecules such as [[Fluorescein|FITC]] (fluorescein isothiocyanate) are strongly [[phototoxic]] when used in live cells, fluorescent proteins such as GFP are usually much less harmful when illuminated in living cells. This has triggered the development of highly automated live-cell fluorescence microscopy systems, which can be used to observe cells over time expressing one or more proteins tagged with fluorescent proteins.

There are many techniques to utilize GFP in a live cell imaging experiment. The most direct way of utilizing GFP is to directly attach it to a protein of interest. For example, GFP can be included in a plasmid expressing other genes to indicate a successful transfection of a gene of interest. Another method is to use a GFP that contains a mutation where the fluorescence will change from green to yellow over time, which is referred to as a fluorescent timer. With the fluorescent timer, researchers can study the state of protein production such as recently activated, continuously activated, or recently deactivated based on the color reported by the fluorescent protein.<ref>{{cite journal | vauthors = Terskikh A, Fradkov A, Ermakova G, Zaraisky A, Tan P, Kajava AV, Zhao X, Lukyanov S, Matz M, Kim S, Weissman I, Siebert P | title = "Fluorescent timer": protein that changes color with time | journal = Science | volume = 290 | issue = 5496 | pages = 1585–8 | date = November 2000 | pmid = 11090358 | doi = 10.1126/science.290.5496.1585 | bibcode = 2000Sci...290.1585T }}</ref> In yet another example, scientists have modified GFP to become active only after exposure to irradiation giving researchers a tool to selectively activate certain portions of a cell and observe where proteins tagged with the GFP move from the starting location.<ref>{{cite journal | vauthors = Patterson GH, Lippincott-Schwartz J | title = A photoactivatable GFP for selective photolabeling of proteins and cells | journal = Science | volume = 297 | issue = 5588 | pages = 1873–7 | date = September 2002 | pmid = 12228718 | doi = 10.1126/science.1074952 | bibcode = 2002Sci...297.1873P | s2cid = 45058411 }}</ref> These are only two examples in a burgeoning field of fluorescent microcopy and a more complete review of biosensors utilizing GFP and other fluorescent proteins can be found here <ref>{{cite journal | vauthors = Lin W, Mehta S, Zhang J | title = Genetically encoded fluorescent biosensors illuminate kinase signaling in cancer | journal = The Journal of Biological Chemistry | volume = 294 | issue = 40 | pages = 14814–14822 | date = October 2019 | pmid = 31434714 | pmc = 6779441 | doi = 10.1074/jbc.REV119.006177 | doi-access = free }}</ref>

For example, GFP had been widely used in labelling the [[Spermatozoon|spermatozoa]] of various organisms for identification purposes as in ''[[Drosophila melanogaster]]'', where expression of GFP can be used as a marker for a particular characteristic. GFP can also be expressed in different structures enabling morphological distinction. In such cases, the gene for the production of GFP is incorporated into the genome of the organism in the region of the DNA that codes for the target proteins and that is controlled by the same [[regulatory sequence]]; that is, the gene's regulatory sequence now controls the production of GFP, in addition to the tagged protein(s). In cells where the gene is expressed, and the tagged proteins are produced, GFP is produced at the same time. Thus, only those cells in which the tagged gene is expressed, or the target proteins are produced, will fluoresce when observed under fluorescence microscopy. Analysis of such time lapse movies has redefined the understanding of many biological processes including protein folding, protein transport, and RNA dynamics, which in the past had been studied using fixed (i.e., dead) material. Obtained data are also used to calibrate mathematical models of intracellular systems and to estimate rates of gene expression.<ref name="pmid20550887">{{cite journal | vauthors = Komorowski M, Finkenstädt B, Rand D | title = Using a single fluorescent reporter gene to infer half-life of extrinsic noise and other parameters of gene expression | journal = Biophysical Journal | volume = 98 | issue = 12 | pages = 2759–2769 | date = Jun 2010 | pmid = 20550887 | pmc = 2884236 | doi = 10.1016/j.bpj.2010.03.032 | bibcode = 2010BpJ....98.2759K }}</ref> Similarly, GFP can be used as an indicator of protein expression in heterologous systems. In this scenario, fusion proteins containing GFP are introduced indirectly, using RNA of the construct, or directly, with the tagged protein itself. This method is useful for studying structural and functional characteristics of the tagged protein on a macromolecular or single-molecule scale with fluorescence microscopy.

The [[Vertico SMI]] microscope using the SPDM Phymod technology uses the so-called "reversible photobleaching" effect of fluorescent dyes like GFP and its derivatives to localize them as single molecules in an optical resolution of 10&nbsp;nm. This can also be performed as a co-localization of two GFP derivatives (2CLM).<ref name="pmid19548231">{{cite journal | vauthors = Gunkel M, Erdel F, Rippe K, Lemmer P, Kaufmann R, Hörmann C, Amberger R, Cremer C | title = Dual color localization microscopy of cellular nanostructures | journal = Biotechnology Journal | volume = 4 | issue = 6 | pages = 927–38 | date = Jun 2009 | pmid = 19548231 | doi = 10.1002/biot.200900005 | s2cid = 18162278 | url = https://hal.archives-ouvertes.fr/hal-00494027/document }}</ref>

Another powerful use of GFP is to express the protein in small sets of specific cells. This allows researchers to optically detect specific types of cells ''[[in vitro]]'' (in a dish), or even ''[[in vivo]]'' (in the living organism).<ref name=Chudakov_2005>{{cite journal | vauthors = Chudakov DM, Lukyanov S, Lukyanov KA | title = Fluorescent proteins as a toolkit for in vivo imaging | journal = Trends in Biotechnology | volume = 23 | issue = 12 | pages = 605–13 | date = Dec 2005 | pmid = 16269193 | doi = 10.1016/j.tibtech.2005.10.005 }}</ref> GFP is considered to be a reliable reporter of gene expression in eukaryotic cells when the fluorescence is measured by flow cytometry.<ref>{{cite journal | vauthors = Soboleski MR, Oaks J, Halford WP | title = Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells | journal = FASEB Journal | volume = 19 | issue = 3 | pages = 440–442 | date = March 2005 | pmid = 15640280 | pmc = 1242169 | doi = 10.1096/fj.04-3180fje | doi-access = free }}</ref> Genetically combining several spectral variants of GFP is a useful trick for the analysis of brain circuitry ([[Brainbow]]).<ref name="pmid17972876">{{cite journal | vauthors = Livet J, Weissman TA, Kang H, Draft RW, Lu J, Bennis RA, Sanes JR, Lichtman JW | title = Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system | journal = Nature | volume = 450 | issue = 7166 | pages = 56–62 | date = Nov 2007 | pmid = 17972876 | doi = 10.1038/nature06293 | bibcode = 2007Natur.450...56L | s2cid = 4402093 }}</ref> Other interesting uses of fluorescent proteins in the literature include using FPs as sensors of [[neuron]] [[membrane potential]],<ref name="pmid18679801">{{cite journal | vauthors = Baker BJ, Mutoh H, Dimitrov D, Akemann W, Perron A, Iwamoto Y, Jin L, Cohen LB, Isacoff EY, Pieribone VA, Hughes T, Knöpfel T | title = Genetically encoded fluorescent sensors of membrane potential | journal = Brain Cell Biology | volume = 36 | issue = 1–4 | pages = 53–67 | date = Aug 2008 | pmid = 18679801 | pmc = 2775812 | doi = 10.1007/s11068-008-9026-7 }}</ref> tracking of [[AMPA]] receptors on cell membranes,<ref name="pmid16364901">{{cite journal | vauthors = Adesnik H, Nicoll RA, England PM | title = Photoinactivation of native AMPA receptors reveals their real-time trafficking | journal = Neuron | volume = 48 | issue = 6 | pages = 977–85 | date = Dec 2005 | pmid = 16364901 | doi = 10.1016/j.neuron.2005.11.030 | doi-access = free }}</ref> [[viral entry]] and the infection of individual [[influenza]] viruses and lentiviral viruses,<ref name="pmid12883000">{{cite journal | vauthors = Lakadamyali M, Rust MJ, Babcock HP, Zhuang X | title = Visualizing infection of individual influenza viruses | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 100 | issue = 16 | pages = 9280–5 | date = Aug 2003 | pmid = 12883000 | pmc = 170909 | doi = 10.1073/pnas.0832269100 | bibcode = 2003PNAS..100.9280L | doi-access = free }}</ref><ref name="pmid18480844">{{cite journal | vauthors = Joo KI, Wang P | title = Visualization of targeted transduction by engineered lentiviral vectors | journal = Gene Therapy | volume = 15 | issue = 20 | pages = 1384–96 | date = Oct 2008 | pmid = 18480844 | pmc = 2575058 | doi = 10.1038/gt.2008.87 }}</ref> etc.

It has also been found that new lines of transgenic GFP rats can be relevant for gene therapy as well as regenerative medicine.<ref name="pmid20094912">{{cite journal | vauthors = Remy S, Tesson L, Usal C, Menoret S, Bonnamain V, Nerriere-Daguin V, Rossignol J, Boyer C, Nguyen TH, Naveilhan P, Lescaudron L, Anegon I | title = New lines of GFP transgenic rats relevant for regenerative medicine and gene therapy | journal = Transgenic Research | volume = 19 | issue = 5 | pages = 745–63 | date = Oct 2010 | pmid = 20094912 | doi = 10.1007/s11248-009-9352-2 | s2cid = 42499768 }}</ref> By using "high-expresser" GFP, transgenic rats display high expression in most tissues, and many cells that have not been characterized or have been only poorly characterized in previous GFP-transgenic rats.

GFP has been shown to be useful in [[cryobiology]] as a [[viability assay]]. Correlation of viability as measured by [[trypan blue]] assays were 0.97.<ref>{{cite journal | vauthors = Elliott G, McGrath J, Crockett-Torabi E | title = Green fluorescent protein: A novel viability assay for cryobiological applications | journal = Cryobiology | volume = 40 | issue = 4 | pages = 360–369 | date = Jun 2000 | pmid = 10924267 | doi = 10.1006/cryo.2000.2258 }}</ref> Another application is the use of GFP co-transfection as internal control for transfection efficiency in mammalian cells.<ref name="pmid20064974">{{cite journal | vauthors = Fakhrudin N, Ladurner A, Atanasov AG, Heiss EH, Baumgartner L, Markt P, Schuster D, Ellmerer EP, Wolber G, Rollinger JM, Stuppner H, Dirsch VM | title = Computer-aided discovery, validation, and mechanistic characterization of novel neolignan activators of peroxisome proliferator-activated receptor gamma | journal = Molecular Pharmacology | volume = 77 | issue = 4 | pages = 559–66 | date = Apr 2010 | pmid = 20064974 | pmc = 3523390 | doi = 10.1124/mol.109.062141 }}</ref>

A novel possible use of GFP includes using it as a sensitive monitor of intracellular processes via an eGFP laser system made out of a human embryonic kidney cell line. The first engineered living laser is made by an eGFP expressing cell inside a reflective optical cavity and hitting it with pulses of blue light. At a certain pulse threshold, the eGFP's optical output becomes brighter and completely uniform in color of pure green with a wavelength of 516&nbsp;nm. Before being emitted as laser light, the light bounces back and forth within the resonator cavity and passes the cell numerous times. By studying the changes in optical activity, researchers may better understand cellular processes.<ref>{{cite journal | vauthors = Gather MC, Yun SH | s2cid = 54971962 | title = Single-cell biological lasers | date = 2011 | journal = Nature Photonics | volume = 5 | issue = 7 | pages = 406–410 | doi = 10.1038/nphoton.2011.99 | bibcode = 2011NaPho...5..406G }}</ref><ref>{{cite journal | vauthors = Matson J | date = 2011 | title = Green Fluorescent Protein Makes for Living Lasers | journal = Scientific American | url = http://www.scientificamerican.com/article.cfm?id=biological-laser-cell | access-date = 2011-06-13 }}</ref>

GFP is used widely in cancer research to label and track cancer cells. GFP-labelled cancer cells have been used to model metastasis, the process by which cancer cells spread to distant organs.<ref>{{cite journal | vauthors = Kouros-Mehr H, Bechis SK, Slorach EM, Littlepage LE, Egeblad M, Ewald AJ, Pai SY, Ho IC, Werb Z | title = GATA-3 links tumor differentiation and dissemination in a luminal breast cancer model | journal = Cancer Cell | volume = 13 | issue = 2 | pages = 141–52 | date = Feb 2008 | pmid = 18242514 | pmc = 2262951 | doi = 10.1016/j.ccr.2008.01.011 }}</ref>