作者感谢Kees Jalink博士（荷兰癌症研究所和荷兰阿姆斯特丹范利乌文霍克高级显微镜中心）为我们提供了H188 cAMP FRET生物传感器和肯尼·特里恩（南阿拉巴马大学工程学院）的技术帮助，以减少运行我们自定义开发的编程脚本所花的时间。
作者要感谢资金来源：美国心脏协会（16PRE27130004），国家科学基金会：（1725937） NIH， S100D020149， S10RR027535， R01HL058506， P01HL066299）.）

<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>

Leavesley博士和Rich博士披露了一家初创公司SpectraCyte的财务利益，该公司的成立是为了将光谱成像技术商业化。但是，本协议中描述的所有程序都是使用与 SpectraCyte 有限责任公司无关的商用产品进行的。

FRET生物传感器的研制使单细胞环核苷酸信号的测量和可视化，对13、22、37、38等亚细胞信号事件的可视化大有可为。然而，使用FRET生物传感器存在一些局限性，包括许多荧光蛋白基FRET记者的低信号到噪声特性，以及FRET记者的弱透射或表达效率（这在某些细胞系中可能特别具有挑战性，如PMVECs）23、24...

环腺苷单磷酸盐（cAMP）是参与关键细胞和生理过程的第二个信使，包括细胞分裂、钙的涌入、基因转录和信号转导。越来越多的证据表明，细胞中存在cAMP隔间，通过这些隔间可以达到1、2、3、4、5、6、7的信号特异性。直到最近，CAMP分隔断是根据不同的G耦合受体激动剂8，9，10，11诱导的独特的生理或细胞效应推断的。最近，基于FRET的荧光成像探测器为直接测量和观察12、13、14细胞中的CAMP信号提供了新的方法。
Fürster共振能量转移（FRET）是一种物理现象，当分子接近15、16时，能量转移以非辐射的方式发生在供体和接受分子之间。随着基于FRET的荧光指标的发展，这种物理现象已用于生物应用，研究蛋白质-蛋白质相互作用17，蛋白质共定位18，Ca+2信号19，基因表达20，细胞分裂21和环核苷酸信号。基于FRET的cAMP指标通常包括一个CAMP结合域，一个捐赠者氟和一个接受荧光22。例如，H188 cAMP 传感器12，22用于此方法包括从 Epac 获得的 cAMP 结合域，夹在绿松石（捐赠者）和金星（接受者）荧光之间。在基础条件下（未绑定），绿松石和金星处于一个方向，因此FRET发生在萤管之间。将 cAMP 与绑定域结合后，会发生构象变化，使绿松石和金星分开，导致 FRET 减少。
基于 FRET 的成像方法为研究细胞内的 cAMP 信号提供了一个很有前途的工具。然而，目前的基于FRET的微观成像技术往往只部分成功达到足够的信号强度，以测量具有亚细胞空间清晰度的FRET。这是由于几个因素，包括许多FRET记者的信号强度有限，准确量化FRET效率变化所需的高精度，以及存在混淆因素，如细胞自发光23，24。结果往往是受弱 SNR 困扰的 FRET 图像，使得 FRET 的子细胞变化的可视化变得非常困难。此外，空间定位 CAMP 信号的调查几乎只在两个空间维度中执行，轴向 CAMP 分布很少被视为25。这可能是因为低 SNR 妨碍了在三个空间维度中测量和可视化 cAMP 梯度的能力。为了克服使用低SNR的FRET传感器的限制，我们采用了高光谱成像和分析方法，在单个细胞25、26、27中测量FRET。
美国宇航局开发了超光谱成像方法，以区分卫星图像28、29中存在的地球物体。此后，这些技术被翻译成荧光显微镜场30，几个商业共聚焦显微镜系统提供光谱探测器。在传统的（非光谱）荧光成像中，样品使用带通滤镜或激光线激发，并且使用第二个带通滤波器收集发射，通常选择以匹配荧光的峰值发射波长。相比之下，高光谱成像方法寻求以特定波长间隔对荧光发射26、31、32或激发33、34的完整光谱剖面进行采样。在我们之前的研究中，我们发现，与传统的基于过滤器的FRET成像技术相比，高光谱成像和分析方法可以改善细胞中FRET信号的量化。在这里，我们提出了一种执行 4 维（x、y、z 和 +）高光谱 FRET 成像和分析的方法，以测量和可视化三个空间维度的 cAMP 分布。这些方法使单细胞25中激动剂诱导的CAMP空间梯度可视化。有趣的是，根据激动剂的不同，CAMP梯度在细胞中可能很明显。此处提供的方法利用不均匀背景和细胞自发光的光谱解密，以提高 FRET 测量的准确性。虽然这种方法在使用 cAMP FRET 生物传感器的肺微血管内皮细胞 （PMVECs） 中得到了证明，但该方法可以很容易地修改，用于替代 FRET 记者或替代细胞系。

<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

循环AMP是第二个信使，参与广泛的细胞和生理活动。一些研究表明，cAMP 信号是分隔的，隔间化有助于 cAMP 信号通路中的信号特异性。基于Fürster共振能量转移（FRET）的生物传感器的发展，进一步增强了测量和可视化细胞中CAMP信号的能力。但是，这些测量通常局限于两个空间维度，这可能导致对数据的误解。迄今为止，由于使用本身信号与噪声比 （SNR） 较低的 FRET 传感器的技术限制，在三个空间维度（x、y 和 z）中对 CAMP 信号的测量非常有限。此外，由于光谱相声、信号强度有限和自发光等一系列因素，传统的基于滤波器的成像方法通常对精确测量局部子细胞区域的 CAMP 信号无效。为了克服这些限制，并允许基于FRET的生物传感器与多个荧光素一起使用，我们开发了高光谱FRET成像和分析方法，为计算FRET效率提供光谱特异性，并能够将FRET信号与混淆的自发光和/或来自其他荧光标签的信号隔离开来。在这里，我们介绍了实施高光谱FRET成像的方法，以及需要构建一个适当的光谱库，既不采样不足，也不采样过采，以执行光谱未混合。虽然我们提出了测量肺微血管内皮细胞（PMVECs）中三维CAMP分布的方法，但这种方法可用于研究CAMP在一系列细胞类型的空间分布。

本协议描述了使用高光谱 FRET 成像和分析方法测量活细胞中三个空间维度的 cAMP 梯度。生成这些结果涉及几个关键步骤，在分析和量化数据时需要仔细注意这些步骤。这些关键步骤包括构建适当的光谱库、背景光谱未混合、识别细胞边框的阈值以及 FRET 效率计算。 图1 说明了测量活细胞中FRET效率和cAMP水平所涉及的所有步骤的示意图流。如果执行得当，这些成像和分析步骤?...

Watch this Scientific Journal Video about Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and ÃŽÂ» 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

由于基于 Fürster 共振能量转移 （FRET） 传感器的固有的低信号与噪声比 （SNR），CAMP 信号的测量一直具有挑战性，尤其是在三个空间维度方面。在这里，我们描述了一个高光谱FRET成像和分析方法，允许测量三个空间尺寸的CAMP分布。

使用 4 维（x、y、z 和 +）高光谱 FRET 成像和分析测量活细胞中的三维 cAMP 分布

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 ...

measurement-3-dimensional-camp-distributions-living-cells-using-4

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

3.8K 次观看.  University of South Alabama. 由于基于 Fürster 共振能量转移 （FRET） 传感器的固有的低信号与噪声比 （SNR），CAMP 信号的测量一直具有挑战性，尤其是在三个空间维度方面。在这里，我们描述了一个高光谱FRET成像和分析方法，允许测量三个空间尺寸的CAMP分布。

视频: Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and λ Hyperspectral FRET Imaging and Analysis

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

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

N -连接糖链的糖蛋白在哺乳动物细胞中的脉冲大通分析

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

科研

生物学

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

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell sources due to their limitless self-renewal capabilities and their potential to differentiate into multiple cell types. The pluripotent state of hPSCs is typically assessed by techniques such as qPCR, immunocytochemistry, and by other in vitro and in vivo differentiation strategies into multiple cell types. Among these, immunocytochemical techniques have been developed for routine characterization of the undifferentiated state of hPSCs based on analysis of candidate intracellular and cell-surface biomarkers. Given the fact that hPSCs grow as colonies, problems arise in quantifying the expression of these markers at the individual cell level on a routine basis. Flow cytometry analyses serve to address this issue but require cell numbers and use of reagents that are not normally conducive for routine quality control assessment of hPSC cultures. Thus, the development of practical and reproducible means of creating monolayer cell samples with preserved integrity for marker evaluation has many advantages in stem cell research. This greatly benefits immunocytochemical analysis because individual cells from the monolayer can be easily observed and quantified for the expression of specific markers. Towards this goal, a self-made cytospin apparatus was constructed and optimized for use with immunocytochemical staining. Two cell-surface markers (SSEA3/SSEA4) expression were analyzed in a variant BG01 stem cell line for the purpose of this protocol.

Suspension immunocytochemical staining of human pluripotent stem cells (hPSCs) for cell-surface markers (SSEA-3/SSEA-4) was achieved based on use of a self-made cytospin apparatus to create a monolayer of cells for observation and quantification.

人多能干细胞的免疫组织化学分析，使用自制Cytospin器械

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

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

影像生活细胞的形状变化果蝇胚胎

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

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

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

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

同时进行pH测量的内吞和胞车厢中使用共焦显微镜活细胞

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

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

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

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

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

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

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) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

荧光成像与一纳米精度（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

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

基于FRET钙内波动培养平滑肌细胞使用的肌质网的测量共焦成像

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

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 employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET成像的三维水凝胶

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 ...