Name: measurement of 3-dimensional camp distributions in living cells using 4-dimensional (x, y, z, and &lambda;) hyperspectral fret imaging and analysis
Uploaded: 2020-10-27T13:30:00
Description: Watch this Scientific Journal Video about Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and Î» Hyperspectral FRET Imaging and Analysis at JoVE.com

Yazarlar, dr. Kees Jalink'i (Hollanda Kanser Enstitüsü ve van Leeuwenhoek İleri Mikroskopi Merkezi, Amsterdam, Hollanda) bize H188 cAMP FRET biyosensörü ve Kenny Trinh'i (Güney Alabama Mühendislik Fakültesi) özel olarak geliştirilen programlama komut dosyalarımızı çalıştırmak için geçen süreyi azaltmada teknik yardım sağladığı için kabul etmek istiyor.
Yazarlar fon kaynaklarını kabul etmek istiyor: Amerikan Kalp Derneği (16PRE27130004), Ulusal Bilim Vakfı; (1725937) NIH, S100D020149, S10RR027535, R01HL058506, P01HL066299).

Bu protokol, Güney Alabama Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi tarafından onaylanan prosedürleri izler.
1. Görüntüleme için hücre, numune ve reaktif hazırlığı
<ol><li>Daha önce açıklandığı gibi sıçan pulmoner mikrovasküler endotel hücrelerini (PMVEC) izole edin35. NOT: Hücreler, Güney Alabama Üniversitesi, Mobile, AL'deki Hücre Kültürü Çekirdeği tarafından 100 mm hücre kültürü yemeklerinde izole edildi ve k...

<ol>
	<li>Corbin, J. D., Sugden, P. H., Lincoln, T. M., Keely, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalization+of+adenosine+3':5'-monophosphate+and+adenosine+3':5'-monophosphate-dependent+protein+kinase+in+heart+tissue.">Compartmentalization of adenosine 3':5'-monophosphate and adenosine 3':5'-monophosphate-dependent protein kinase in heart tissue.</a> The Journal of Biological Chemistry. 252, 3854-3861 (1977).</li><li>Terrin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PGE1+stimulation+of+HEK293+cells+generates+multiple+contiguous+domains+with+different+[cAMP]:+role+of+compartmentalized+phosphodiesterases.">PGE1 stimulation of HEK293 cells generates multiple contiguous domains with different [cAMP]: role of compartmentalized phosphodiesterases.</a> The Journal of Cell Biology. 175, 441-451 (2006).</li><li>Bacskai, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+resolved+dynamics+of+cAMP+and+protein+kinase+A+subunits+in+Aplysia+sensory+neurons.">Spatially resolved dynamics of cAMP and protein kinase A subunits in Aplysia sensory neurons.</a> Science. 260, 222-226 (1993).</li><li>Iancu, R. V., Ramamurthy, G., Harvey, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+aspects+of+cAMP+signalling+in+cardiac+myocytes.">Spatial and temporal aspects of cAMP signalling in cardiac myocytes.</a> Clinical and Experimental Pharmacology &amp; Physiology. 35, 1343-1348 (2008).</li><li>Brunton, L. L., Hayes, J. S., Mayer, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+compartmentation+of+cyclic+AMP+and+protein+kinase+in+heart.">Functional compartmentation of cyclic AMP and protein kinase in heart.</a> Advances in Cyclic Nucleotide Research. 14, 391-397 (1981).</li><li>Hohl, C. M., Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentation+of+cAMP+in+adult+canine+ventricular+myocytes.+Relation+to+single-cell+free+Ca2++transients.">Compartmentation of cAMP in adult canine ventricular myocytes. Relation to single-cell free Ca2+ transients.</a> Circulation Research. 69, 1369-1379 (1991).</li><li>Rich, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+uniform+extracellular+stimulus+triggers+distinct+cAMP+signals+in+different+compartments+of+a+simple+cell.">A uniform extracellular stimulus triggers distinct cAMP signals in different compartments of a simple cell.</a> Proceedings of the National Academy of Sciences of the United States of America. 98, 13049-13054 (2001).</li><li>Sayner, S. L., Alexeyev, M., Dessauer, C. W., Stevens, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+adenylyl+cyclase+reveals+the+significance+of+cAMP+compartmentation+on+pulmonary+microvascular+endothelial+cell+barrier.">Soluble adenylyl cyclase reveals the significance of cAMP compartmentation on pulmonary microvascular endothelial cell barrier.</a> Circulation Research. 98, 675-681 (2006).</li><li>Rich, T. C., Tse, T. E., Rohan, J. G., Schaack, J., Karpen, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+assessment+of+local+phosphodiesterase+activity+using+tailored+cyclic+nucleotide-gated+channels+as+cAMP+sensors.">In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors.</a> The Journal of General Physiology. 118, 63-78 (2001).</li><li>Blackman, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDE4D+and+PDE4B+function+in+distinct+subcellular+compartments+in+mouse+embryonic+fibroblasts.">PDE4D and PDE4B function in distinct subcellular compartments in mouse embryonic fibroblasts.</a> Journal of Biological Chemistry. 286, 12590-12601 (2011).</li><li>Sayner, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paradoxical+cAMP-induced+lung+endothelial+hyperpermeability+revealed+by+Pseudomonas+aeruginosa+ExoY.">Paradoxical cAMP-induced lung endothelial hyperpermeability revealed by Pseudomonas aeruginosa ExoY.</a> Circulation Research. 95, 196-203 (2004).</li><li>Klarenbeek, J., Jalink, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+cAMP+with+an+EPAC-based+FRET+sensor+in+single+living+cells.">Detecting cAMP with an EPAC-based FRET sensor in single living cells.</a> Methods in Molecular Biology. 1071, 49-58 (2014).</li><li>Surdo, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET+biosensor+uncovers+cAMP+nano-domains+at+&#946;-adrenergic+targets+that+dictate+precise+tuning+of+cardiac+contractility.">FRET biosensor uncovers cAMP nano-domains at &#946;-adrenergic targets that dictate precise tuning of cardiac contractility.</a> Nature Communications. 8, 15031 (2017).</li><li>Ponsioen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+cAMP-induced+Epac+activation+by+fluorescence+resonance+energy+transfer:+Epac+as+a+novel+cAMP+indicator.">Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator.</a> EMBO Reports. 5, 1176-1180 (2004).</li><li>Vogel, S. S., Thaler, C., Koushik, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fanciful+FRET.">Fanciful FRET.</a> Science's STKE. 2006, (2006).</li><li>Clegg, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+History+of+FRET:+From+conception+through+the+labors+of+birth.">The History of FRET: From conception through the labors of birth.</a> Reviews in Fluorescence. 3, (2006).</li><li>Giepmans, B. N. G., Adams, S. R., Ellisman, M. H., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluorescent+toolbox+for+assessing+protein+location+and+function.">The fluorescent toolbox for assessing protein location and function.</a> Science. 312, 217-224 (2006).</li><li>Manzella-Lapeira, J., Brzostowski, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+protein-protein+interactions+by+F&#246;rster+resonance+energy+transfer+(FRET)+microscopy+in+live+cells.">Imaging protein-protein interactions by F&#246;rster resonance energy transfer (FRET) microscopy in live cells.</a> Current Protocols in Protein Science. 93, 58 (2018).</li><li>Cooper, D. M. F., Mons, N., Karpen, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenylyl+cyclases+and+the+interaction+between+calcium+and+cAMP+signalling.">Adenylyl cyclases and the interaction between calcium and cAMP signalling.</a> Nature. 374, 421-424 (1995).</li><li>Sassone-Corsi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+gene+expression+to+cAMP+signalling:+role+of+CREB+and+CREM.">Coupling gene expression to cAMP signalling: role of CREB and CREM.</a> The International Journal of Biochemistry &amp; Cell Biology. 30, 27-38 (1998).</li><li>Rebhun, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+nucleotides,+calcium,+and+cell+division.">Cyclic nucleotides, calcium, and cell division.</a> International Review of Cytology. 49, 1-54 (1977).</li><li>Klarenbeek, J., Goedhart, J., Van Batenburg, A., Groenewald, D., Jalink, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fourth-generation+Epac-based+FRET+sensors+for+cAMP+feature+exceptional+brightness,+photostability+and+dynamic+range:+characterization+of+dedicated+sensors+for+FLIM,+for+ratiometry+and+with+high+affinity.">Fourth-generation Epac-based FRET sensors for cAMP feature exceptional brightness, photostability and dynamic range: characterization of dedicated sensors for FLIM, for ratiometry and with high affinity.</a> PLOS ONE. 10, 0122513 (2015).</li><li>Leavesley, S. J., Rich, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET:+signals+hidden+within+the+noise.">FRET: signals hidden within the noise.</a> Cytometry Part A. 85, 918-920 (2014).</li><li>Rich, T. C., Webb, K. J., Leavesley, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+we+decipher+the+information+content+contained+within+cyclic+nucleotide+signals.">Can we decipher the information content contained within cyclic nucleotide signals.</a> The Journal of General Physiology. 143, 17-27 (2014).</li><li>Annamdevula, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+imaging+of+FRET-based+sensors+reveals+sustained+cAMP+gradients+in+three+spatial+dimensions.">Spectral imaging of FRET-based sensors reveals sustained cAMP gradients in three spatial dimensions.</a> Cytometry Part A. 93 (10), 1029-1038 (2018).</li><li>Leavesley, S. J., Britain, A. L., Cichon, L. K., Nikolaev, V. O., Rich, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+FRET+using+spectral+techniques.">Assessing FRET using spectral techniques.</a> Cytometry Part A. 83, 898-912 (2013).</li><li>Leavesley, S. J., Rich, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+limitations+of+FRET+measurements.">Overcoming limitations of FRET measurements.</a> Cytometry Part A. 89, 325-327 (2016).</li><li>Fink, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+Earcths+Resources+from+Space.">Monitoring Earcths Resources from Space.</a> Technology Review. 75, 32-41 (1973).</li><li>Goetz, A. F. H., Vane, G., Solomon, J. E., Rock, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Spectrometry+for+Earth+Remote+Sensing.">Imaging Spectrometry for Earth Remote Sensing.</a> Science. 228, 1147-1153 (1985).</li><li>B&#252;cherl, C. A., Bader, A., Westphal, A. H., Laptenok, S. P., Borst, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET-FLIM+applications+in+plant+systems.">FRET-FLIM applications in plant systems.</a> Protoplasma. 251, 383-394 (2014).</li><li>Chen, Y., Mauldin, J. P., Day, R. N., Periasamy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+spectral+FRET+imaging+microscopy+for+monitoring+nuclear+protein+interactions.">Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions.</a> Journal of Microscopy. 228, 139-152 (2007).</li><li>Zimmermann, T., Rietdorf, J., Girod, A., Georget, V., Pepperkok, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+imaging+and+linear+un-mixing+enables+improved+FRET+efficiency+with+a+novel+GFP2-YFP+FRET+pair.">Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair.</a> FEBS Letters. 531, 245-249 (2002).</li><li>Griswold, J. R., Annamdevula, N., Deal, J., Rich, T., Leavesley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+FRET+Efficiency+using+Excitation-Scanning+Hyperspectral+Imaging.">Estimating FRET Efficiency using Excitation-Scanning Hyperspectral Imaging.</a> Biophysical Journal. 112, 586 (2017).</li><li>Favreau, P. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitation-scanning+hyperspectral+imaging+microscope.">Excitation-scanning hyperspectral imaging microscope.</a> Journal of Biomedical Optics. 19, 046010 (2014).</li><li>King, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+characteristics+of+lung+macro-+and+microvascular+endothelial+cell+phenotypes.">Structural and functional characteristics of lung macro- and microvascular endothelial cell phenotypes.</a> Microvascular Research. 67, 139-151 (2004).</li><li>Thaler, C., Koushik, S. V., Blank, P. S., Vogel, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+multiphoton+spectral+imaging+and+its+use+for+measuring+resonance+energy+transfer.">Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer.</a> Biophysical Journal. 89, 2736-2749 (2005).</li><li>Agarwal, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalized+cAMP+signaling+associated+with+lipid+raft+and+non-raft+membrane+domains+in+adult+ventricular+myocytes.">Compartmentalized cAMP signaling associated with lipid raft and non-raft membrane domains in adult ventricular myocytes.</a> Frontiers in Pharmacology. 9, 332 (2018).</li><li>Johnstone, T. B., Agarwal, S. R., Harvey, R. D., Ostrom, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cAMP+signaling+compartmentation:+Adenylyl+cyclases+as+anchors+of+dynamic+signaling+complexes.">cAMP signaling compartmentation: Adenylyl cyclases as anchors of dynamic signaling complexes.</a> Mol Pharmacol. , (2017).</li><li>Zhang, J., Li, H., Chai, L., Zhang, L., Qu, J., Chen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+FRET+measurement+using+emission-spectral+unmixing+with+independent+excitation+crosstalk+correction.">Quantitative FRET measurement using emission-spectral unmixing with independent excitation crosstalk correction.</a> Journal of Microscopy. 257, 104-116 (2015).</li><li>Zhang, J., Lin, F., Chai, L., Wei, L., Chen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IIem-spFRET:+improved+Iem-spFRET+method+for+robust+FRET+measurement.">IIem-spFRET: improved Iem-spFRET method for robust FRET measurement.</a> Journal of Biomedical Optics. 21, 105003 (2016).</li><li>Levy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SpRET:+highly+sensitive+and+reliable+spectral+measurement+of+absolute+FRET+efficiency.">SpRET: highly sensitive and reliable spectral measurement of absolute FRET efficiency.</a> Microscopy and Microanalysis. 17, 176-190 (2011).</li><li>West, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperspectral+Measurements+Allow+Separation+of+FRET+Signals+from+Non-Uniform+Background+Fluorescence.">Hyperspectral Measurements Allow Separation of FRET Signals from Non-Uniform Background Fluorescence.</a> Biophysical Journal. 112, 453 (2017).</li><li>Annamdevula, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+approach+for+characterizing+and+comparing+hyperspectral+microscopy+systems.">An approach for characterizing and comparing hyperspectral microscopy systems.</a> Sensors. 13, 9267-9293 (2013).</li></ol>

FRET biyosensörlerinin gelişimi, döngüsel nükleotid sinyallerinin tek hücrelerde ölçülmesini ve görselleştirilmesini sağladı ve hücre altı sinyal olaylarını görselleştirmek için büyük bir vaat var13,22,37,38. Bununla birlikte, FRET biyosensörlerinin kullanımı, birçok floresan protein bazlı FRET muhabirinin düşük sinyal-gürültü özellikleri ve FRET muhabirlerinin...

Siklik adenozin monofosfat (cAMP), hücre bölünmesi, kalsiyum akışı, gen transkripsiyonu ve sinyal transdüksiyonu dahil olmak üzere önemli hücresel ve fizyolojik süreçlerde yer alan ikinci bir habercidir. Büyüyen bir kanıt gövdesi, hücrede sinyal özgüllüğünün elde edildiği cAMP bölmelerinin varlığını göstermektedir1, 2,3,4,5,6,7. Yakın zamana kadar, cAMP bölmeleme, farklı G bağlantılı reseptör agonistleri 8 ,9, 10,11tarafından indüklenen farklı fizyolojik veya hücresel etkilere dayanarakçıkarılmıştır. Daha yakın zamanda, FRET tabanlı floresan görüntüleme probları,12, 13,14hücresinde cAMP sinyallerinin doğrudan ölçümü ve gözlemlenmesi için yeni yaklaşımlar sağlamıştır.
Förster rezonans enerji transferi (FRET), donör ve alıcı moleküller arasında, moleküller yakın mesafedeyken radyatif olmayan bir şekilde enerji transferinin gerçekleştiği fiziksel bir olgudur15,16. FRET tabanlı floresan göstergelerin geliştirilmesiyle, bu fiziksel fenomen biyolojik uygulamalarda protein-protein etkileşimlerini incelemek için kullanılmıştır17, protein ko-lokalizasyonu18, Ca+2 sinyal19, gen ekspresyasyonu20, hücre bölünmesi21 ve döngüsel nükleotid sinyali. FRET tabanlı cAMP göstergeleri tipik olarak bir cAMP bağlama etki alanı, bir donör floroforu ve bir kabul edici florofor22'denoluşur. Örneğin, bu metodolojide kullanılan H188 cAMP sensör12,22, Turkuaz (donör) ve Venüs (kabul eden) floroforlar arasında sıkışmış, Epac'tan elde edilen bir cAMP bağlama alanından oluşur. Bazal koşullarda (ilişkisiz), Turkuaz ve Venüs, FRET'in floroforlar arasında meydana geldiği bir yönelimdedir. cAMP'nin bağlama alanına bağlanması üzerine, Turkuaz ve Venüs'ün birbirinden uzaklaşması ve FRET'te bir azalmaya neden olması için uygun bir değişiklik meydana gelir.
FRET tabanlı görüntüleme yaklaşımları, bir hücre içindeki cAMP sinyallerini araştırmak ve görselleştirmek için umut verici bir araç sunar. Bununla birlikte, mevcut FRET tabanlı mikroskobik görüntüleme teknikleri genellikle FRET'i hücre altı mekansal netlik ile ölçmek için yeterli sinyal gücü elde etmede kısmen başarılıdır. Bunun nedeni, birçok FRET muhabirinin sınırlı sinyal gücü, FRET verimliliğindeki değişiklikleri doğru bir şekilde ölçmek için gereken yüksek hassasiyet seviyesi ve hücresel otofluoresans23,24gibi şaşırtıcı faktörlerin varlığı da dahil olmak üzere çeşitli faktörlerden kaynaklanmaktadır. Sonuç genellikle zayıf SNR ile boğuşan ve FRET'teki hücre altı değişikliklerin görselleştirilmesini çok zor hale getiren bir FRET görüntüsüdür. Ek olarak, mekansal olarak lokalize cAMP sinyallerinin araştırılması neredeyse sadece iki mekansal boyutta gerçekleştirilmiştir ve eksenel cAMP dağılımı nadiren25olarak kabul edilmiştir. Bunun nedeni, düşük SNR'nin cAMP degradelerini üç uzamsal boyutta ölçme ve görselleştirme yeteneğini engellemesidir. Düşük SNR'li FRET sensörlerini kullanmanın sınırlamalarını aşmak için, FRET'i tek hücrelerde ölçmek için hiperspektral görüntüleme ve analiz yaklaşımları uyguladık25,26,27.
Hiperspektral görüntüleme yaklaşımları NASA tarafından uydu görüntülerinde bulunan karasal nesneleri ayırt etmek için geliştirilmiştir28,29. Bu teknikler o zamandan beri floresan mikroskopi alanı30spektral dedektörler sunan çeşitli ticari konfokal mikroskop sistemleri ile çevrilmiştir. Geleneksel (spektral olmayan) floresan görüntülemede, örnek bir bant geçiş filtresi veya lazer çizgisi kullanılarak heyecanlanır ve emisyon, genellikle floroforların tepe emisyon dalga boylarına uyacak şekilde seçilen ikinci bir bant geçiş filtresi kullanılarak toplanır. Bunun aksine, hiperspektral görüntüleme yaklaşımları, floresanemisyonu 26,31,32veya heyecan33 ,34'üntam bir spektral profilini belirli dalga boyu aralıklarında örneklemeyi amaçlamaktadır. Önceki çalışmalarımızda, hiperspektral görüntüleme ve analiz yaklaşımlarının, geleneksel filtre tabanlı FRET görüntüleme tekniklerine kıyasla hücrelerdeki FRET sinyallerinin daha iyi nicelleştirilmesini sunabileceğini gösterdik26. Burada, cAMP dağılımlarını üç mekansal boyutta ölçmek ve görselleştirmek için 4 boyutlu (x, y, z ve φ) hiperspektral FRET görüntüleme ve analizi yapmak için bir metodoloji sunuyoruz. Bu yaklaşımlar, agonist kaynaklı cAMP uzamsal gradyanlarının tek hücrelerde görselleştirilmesine izin verdi25. İlginçtir ki, agoniste bağlı olarak, hücrelerde cAMP gradyanları belirgin olabilir. Burada sunulan metodoloji, FRET ölçümlerinin doğruluğunu artırmak için tekdüze olmayan arka plan ve hücresel otofluoresans spektral karıştırmayı kullanır. Bu metodoloji pulmoner mikrovasküler endotel hücrelerinde (PMVEC) cAMP FRET biyosensörü kullanılarak gösterilmiş olsa da, metodoloji alternatif FRET muhabirleri veya alternatif hücre hatları ile kullanılmak üzere kolayca değiştirilebilir.

<table>
	<tbody>
		<tr>
			<td>Attofluor Cell Chamber</td>
			<td>Invitrogen</td>
			<td>A7816</td>
			<td>Attofluor contains steel cell chambers and a rubber O-ring. Cell chamber holds the coverslip and O-ring provides a lock in mechanism to hold the buffer in cell chamber with out leakage</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Fisher Scientific</td>
			<td>BP231-100</td>
			<td>Solvent used to prepare stock solution forskolin.</td>
		</tr>
		<tr>
			<td>DRAQ5 Fluoroscent Probe Solution</td>
			<td>Thermo Scientific</td>
			<td>62252</td>
			<td>Nuclear label</td>
		</tr>
		<tr>
			<td>Dulbecco Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td>Contains nutrients and growth factors for the cells to grow and divide in the culture dishes.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td>F6178</td>
			<td>Growth factor suppliment that is added to culture medium, DMEM</td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma</td>
			<td>F3917</td>
			<td>Adenyly cyclase activator.</td>
		</tr>
		<tr>
			<td>H188 Cyclic AMP FRET biosensor</td>
			<td>Netherland Cancer Institute, Dr. K. Jalink</td>
			<td>Gift</td>
			<td>Plasmid encoding Turquoise (donor fluorophore), Venus (acceptor fluorophore), and binding domain obtained from Epac.</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>image.net</td>
			<td>Free download</td>
			<td>Another image processing platform used to extact spectral information and image processing.</td>
		</tr>
		<tr>
			<td>Integrating Sphere</td>
			<td>Ocean Optics</td>
			<td>FOIS-1</td>
			<td>Used to measure illumination intensity of the laser line at different laser intensities (?).</td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein, Natural</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
			<td>Coverslips are coated with laminin and this helps in cell attachment, growth and motility of the cell.</td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Kit</td>
			<td>Invitrogen</td>
			<td>L3000-015</td>
			<td>Transfection reagent used to transfect cells with H188 FRET biosensor</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td>R2019a</td>
			<td>Image processing operations (linear unmixing and FRET efficiency calculations) are performed by writing custom programs in MATLAB programming environment</td>
		</tr>
		<tr>
			<td>Nikon A1R confocal microscope</td>
			<td>Nikon Instruments</td>
			<td>Nikon A1R</td>
			<td>Spectral image acquisition is performed using confocal microscope.</td>
		</tr>
		<tr>
			<td>Nikon Elements Software</td>
			<td>Nikon Instruments</td>
			<td>Software dongle</td>
			<td>used to export and handle nd2 image files (multidimensional image files) that are aquired using Nikon A1R</td>
		</tr>
		<tr>
			<td>NIST-Traceable Calibration Lamp</td>
			<td>Ocean Optics</td>
			<td>LS-1-CAL-INT</td>
			<td>A lamp with a known spectrum for use as a standard</td>
		</tr>
		<tr>
			<td>PBS pH 7.4 (1X)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td>coomonly used buffer suring cell culture</td>
		</tr>
		<tr>
			<td>Pulmonary Microvascular Endothelial Cells (PMVECs)</td>
			<td>In house - Cell culture core, Univeristy of South Alabama</td>
			<td>Isolated from Rat pulmonary microvasculature</td>
			<td>PMVECs form inner lining of a blood vessel.</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/ml)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>antibiotics are added to culture medum to prevent contamination of the cells.</td>
		</tr>
		<tr>
			<td>Pre-Cleaned Gold Seal Micro Slides</td>
			<td>Clay Adams</td>
			<td>3010</td>
			<td>Microscope slides used for cell fixation</td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mounting Media</td>
			<td>Invitrogen</td>
			<td>P36961</td>
			<td>If samples are fixed using antifade mountant, then the later protects fluoroscent dyes and chromophores from fading.</td>
		</tr>
		<tr>
			<td>Spectrometer</td>
			<td>Ocean Optics</td>
			<td>QE65000</td>
			<td>Used to measure spectral response of the light source (?)</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>Digests the protein-protein bond between the cell and cell matrix and helps to disscociate and lift the cells during cell plating.</td>
		</tr>
		<tr>
			<td>Tyrodes Buffer</td>
			<td>Made in-house</td>
			<td>Made in-house</td>
			<td>Tyrodes buffer is used to make working solutions and to maintain cells in aqueous solution during image acquisition.</td>
		</tr>
		<tr>
			<td>6 Well Cell Culture Plate</td>
			<td>Corning</td>
			<td>3506</td>
			<td>Laminin coated coverslips are placed in 6-well culture dish (one coverlisps/well). Cells along with medium is added into each well.</td>
		</tr>
		<tr>
			<td>25 mm Round Microscope Cover Slips</td>
			<td>Fisher Scientific</td>
			<td>12545102</td>
			<td>Cells were grown on round glass coverslips</td>
		</tr>
		<tr>
			<td>60X Ojective</td>
			<td>Nikon Instruments</td>
			<td>Plan Apo VC 60X/1.2 WI &#8734;/0.15-0.18 WD 0.27</td>
			<td>water immersion and commonly used objective for cells</td>
		</tr>
	</tbody>
</table>

measurement of 3-dimensional camp distributions in living cells using 4-dimensional (x, y, z, and &lambda;) hyperspectral fret imaging and analysis

Siklik AMP, çok çeşitli hücresel ve fizyolojik aktivitelerde yer alan ikinci bir habercidir. Çeşitli çalışmalar, cAMP sinyallerinin bölümlere ayırdığını ve bölümlere ayırmanın cAMP sinyal yolu içindeki sinyal özgüllüğüne katkıda bulunduğunu göstermektedir. Fӧrster rezonans enerji transferi (FRET) bazlı biyosensörlerin geliştirilmesi, hücrelerdeki cAMP sinyallerini ölçme ve görselleştirme yeteneğini daha da ileriye taşıdı. Bununla birlikte, bu ölçümler genellikle verilerin yanlış yorumlanmasına neden olabilecek iki uzamsal boyutla sınırlıdır. Bugüne kadar, doğası gereği düşük sinyal-gürültü oranı (SNR) sergileyen FRET sensörlerinin kullanılmasındaki teknik sınırlamalar nedeniyle, üç uzamsal boyutta (x, y ve z) cAMP sinyallerinin çok sınırlı ölçümleri olmuştur. Buna ek olarak, geleneksel filtre tabanlı görüntüleme yaklaşımları, spektral çapraz konuşma, sınırlı sinyal gücü ve otofluoresans gibi çeşitli faktörler nedeniyle yerelleştirilmiş hücre altı bölgelerdeki cAMP sinyallerinin doğru ölçümü için genellikle etkisizdir. Bu sınırlamaların üstesinden gelmek ve FRET tabanlı biyosensörlerin birden fazla floresanla kullanılmasına izin vermek için, FRET verimliliklerini hesaplamak için spektral özgüllük ve FRET sinyallerini ek floresan etiketlerden gelen şaşırtıcı otoflüoresan ve/veya sinyallerden spektral olarak ayırma yeteneği sağlayan hiperspektral FRET görüntüleme ve analiz yaklaşımları geliştirdik. Burada, hiperspektral FRET görüntülemenin uygulanması için metodolojinin yanı sıra spektral karıştırma yapmak için ne az örneklenmiş ne de aşırı örneklenmiş uygun bir spektral kütüphane inşa etme ihtiyacını sunuyoruz. Pulmoner mikrovasküler endotel hücrelerinde (PMVEC) üç boyutlu cAMP dağılımlarının ölçümü için bu metodolojiyi sunarken, bu metodoloji cAMP'nin çeşitli hücre tiplerindeki mekansal dağılımlarını incelemek için kullanılabilir.

Bu protokol, canlı hücrelerde üç mekansal boyutta cAMP gradyanlarını ölçmek için hiperspektral FRET görüntüleme ve analiz yaklaşımlarının kullanımını açıklamaktadır. Verileri analiz ederken ve ölçerken dikkatli olunmesi gereken bu sonuçların oluşturulmasında birkaç önemli adım vardır. Bu önemli adımlar arasında uygun bir spektral kütüphanenin inşası, arka plan spektral karıştırması, hücre kenarlıklarını tanımlamak için eşik oluşturma ve FRET verimlilik hesaplamaları yer...

Watch this Scientific Journal Video about Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and Î» Hyperspectral FRET Imaging and Analysis at JoVE.com

Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and &lambda; Hyperspectral FRET Imaging and Analysis

Fӧrster rezonans enerji transferi (FRET) tabanlı sensörlerin doğal düşük sinyal-gürültü oranı (SNR) nedeniyle, cAMP sinyallerinin ölçümü, özellikle üç uzamsal boyutta zor olmuştur. Burada, cAMP dağılımının üç mekansal boyutta ölçülemesine izin veren hiperspektral FRET görüntüleme ve analiz metodolojisini açıklıyoruz.

4 Boyutlu (x, y, z ve φ) Hiperspektral FRET Görüntüleme ve Analizi Kullanılarak Canlı Hücrelerde 3 Boyutlu cAMP Dağılımlarının Ölçümü

Cyclic AMP is a second messenger that is involved in a wide range of cellular and physiological activities. Several studies suggest that cAMP signals are ...

This protocol is significant as it provides a method for measuring and visualizing FRET or cyclic AMP signals in three spatial dimensions in individual cells. Cyclic AMP distributions may occur axially in addition to laterally and it is important to acquire FRET cyclic AMP image data in three dimensions to sample these distributions. On the day of imaging, warm Tyrodes buffer to 37 degrees Celsius in a water bath and mount a cover slip seated with the transfected cells of interest into a cell chamber.Secure the top of the chamber with a mounting gasket to prevent leaking and use a delicate task wiper to remove any excess medium or adherent cells from the bottom of the cover slip. Add 800 microliters of working buffer and four microliters of five millimolar nuclear label to the cell chamber and gently rock the chamber for five to 10 seconds. Then cover the cell chamber with aluminum foil for a 10 minute incubation at room temperature.To image the cells, select the 60X water immersion objective on a confocal microscope equipped with a spectral detector and add a drop of water to the objective. Place the loaded cell chamber onto the microscope stage and tune the filter knob to select the appropriate filter set. Select the fluorescence widefield mode and use the eyepieces to select a field of view containing cells expressing the FRET cyclic AMP sensor.Open the imaging software, select the confocal mode, and unlock the laser interlock button. Open the A1 settings menu. Check the boxes corresponding to the 405 and 561 nanometer laser lines and set these spectral detections to SD, the resolution to 10, and the channels to 32.Select the start and end wavelength values to set the wavelength range. Open the A1 Settings menu to select the binning skip icon. Select the number 15 box and click OK.Set the laser intensities to eight percent for the 405 nanometer laser and to two percent for the 561 nanometer laser.Set the detector gain to 149 and the pinhole radius to 2.4 Airy disk units. Set the scan speed to 25 spectral frames per second, and select the unidirectional scan direction. Set the count to four and the scan size to 1024 by 1024.To define the z-stack acquisition parameters, click View, Acquisition Control, and ND Acquisition, and enter the file destination and name in the pop window to save the ND file. Click the z-series box and click Live in the A1 Plus Settings window to open a live viewing window. Adjust the focus knob to focus on the cells while looking in the preview window.Adjust the focus knob on the microscope to select the top of the cell and click Top in the ND Acquisition window to set the current position as the top. Adjust the focus knob on the microscope to select the bottom of the cell and click Bottom to set the current position as the bottom. Enter one micrometer for the step size.Select Top Bottom for the z-scan direction and click run to acquire a z-stack. When the z-stack acquisition is complete, use a pipette to gently add the reagent of interest to the chamber without disturbing the cells. After 10 minutes, change the file name and acquire a second z-stack image.After imaging, create new folders with the same file names as the spectral z-stack images and open the spectral image file of interest. Click File, Import/Export, and Export ND Document. In the pop-up window, select the newly created folder, TIFF for the file type, Mono image for each channel, and keep bit depth.Click export to export the individual files to the folder and open the linear spectral unmixing software. Open the Linear Unmixing. m file and click Run.Browse and select the folder containing the exported TIFF file sequence and click OK to open the wavelength and z-slice window. Copy the file name of the first file without the z-slice and channel number from the folder and paste it into the first step of the Enter of the imagename box, enter the number of channels in the Enter the number of wavelength bands box, and the number of z-slices in the Enter the number of Z Slices box and click OK.Then in the pop-up window, browse and select the Wavelength. mat file and click Open.Browse and select the Library. mat file in the new pop-up window and click Open again to initiate the slice unmixing. To calculate the FRET efficiency, open the multiFRRCF.m programming script and enter the number of experimental trials to analyze in the How many folders to reslice box. Click OK.Browse to select the unmixed folders and click OK again. In the new pop-up window, set the scaling factor to 12.4, the threshold to 56, the X, Y, and Z frequency to five, five, and one respectively, and the smoothing algorithm to Gaussian.Then click Run to perform the FRET measurements and reslicing. To map the FRET efficiency to cyclic AMP levels, open the Mapping_FRET.Efficiency_to_cAMP_concentration. m file and click Run.Navigate to and select the first grayscale FRET image and click OK.Then open the FRET cyclic AMP images to inspect the distribution of the cyclic AMP signals in three dimensions. In these images, three-dimensional views of false colored raw hyperspectral image data acquired using confocal microscopy at baseline and 10 minutes after forskolin treatment can be observed. In this analysis, an acceptor fluorophore was completely photo destructed allowing spectral signatures of the donor and acceptor with one-to-one stoichiometry to be obtained.Non-transfected cells labeled with a nuclear dye were utilized to obtain the pure spectrum of the dye fluorophore. Combining the spectra of the donor, acceptor, and nuclear dye fluorophores allows the creation of a three component library. Here, the sources of three background spectral signatures can be observed.Although these signals are distributed non-uniformly within the sample and cannot simply be subtracted out, adding the spectral signatures of these signals to the spectral library and using linear unmixing to separate the signals provides an approach for removing these confounding signals from the donor and acceptor signals prior to calculating their FRET efficiencies. The six component spectral library can then be used to perform linear spectral unmixing for each slice in the axial image. Here, the changes in FRET efficiency and cyclic AMP levels in different XY plane slices can be observed allowing a comparison between the baseline conditions and the conditions 10 minutes after forskolin treatment.This technique could also be used to quantify the FRET efficiency of other reporters in three dimensions in both cells and tissues.

Research

JoVE Journal

Biology

Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional (x, y, z, and &lambda;) Hyperspectral FRET Imaging and Analysis

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Memeli Hücrelerinde Glikoproteinler N-bağlı Şeker Zincirler Pulse-kovalamaca Analizi

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Biyoloji

Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus

Self-Made sitospin Aparatı kullanarak İnsan Pluripotent Kök Hücre İmmünositokimyasal Analizi

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell ...

Imaging Cell Shape Change in Living Drosophila Embryos

Imaging Cell Shape Change in Living Drosophila Embryos

Living Görüntüleme Hücre Şekli Değiştir Drosophila Embriyolar

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Saç Rejenerasyon sırasında epitelyal-mezenkimal Sinyal Rapid Genetik Analizi

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Konfokal mikroskopi kullanılarak Yaşayan hücreler içinde endositik ve Sitosolik Bölümleri eşzamanlı pH Ölçümü

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

Oranlı metrik Bimoleküler Fenerleri kullanma Yaşam Hücrelerde Tek Engineered RNA Transkriptlerinin Gerçek zamanlı Görüntüleme

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence Imaging with One-nanometer Accuracy FIONA

Bir nanometre Hassasiyet Floresans Görüntüleme (Fiona)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Kullanımı Kültür düz kas hücrelerinin sarkoplazmik retikulum içinde Kalsiyum Değişiminin Ölçülmesi FRET tabanlı Konfokal Görüntüleme

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

FRET Imaging in Three-dimensional Hydrogels

Üç boyutlu Hidrojeller içinde Görüntüleme FRET

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...