ACS Publications. Most Trusted. Most Cited. Most Read
My Activity

Figure 1Loading Img

pHlameleons: A Family of FRET-Based Protein Sensors for Quantitative pH Imaging

View Author Information
Laboratory for Molecular and Cellular Systems, Department of Neuro- and Sensory Physiology, University Medicine Göttingen, Humboldtalee 23, 37073 Göttingen, Germany
†F.S.W. is member of, and financed by, the “Molecular Microscopy” section and the Excellence Cluster 171 “Microscopy on the Nanometer Scale” of the DFG-funded (German Research Council) Center for Molecular Physiology of the Brain (CMPB). Additional financing from the German Federal Ministry for Education and Research (BMBF) for the project “FLI-Cam” in the Biophotonik III program is acknowledged. M.G. is funded by the Fritz Thyssen Foundation.
* To whom correspondence should be addressed. Phone: +44-1223-334193. Fax: +44-1223-334796. E-mail: [email protected]
‡Present address: Laser Analytics Group, Department of Chemical Engineering and Biotechnology, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, U.K.
§Present address: Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, 04103 Leipzig, Germany.
Cite this: Biochemistry 2008, 47, 49, 13115–13126
Publication Date (Web):November 13, 2008
Copyright © 2008 American Chemical Society

    Article Views





    Read OnlinePDF (883 KB)
    Supporting Info (1)»


    Abstract Image

    Intracellular pH is an important indicator for cellular metabolism and pathogenesis. pH sensing in living cells has been achieved using a number of synthetic organic dyes and genetically expressible sensor proteins, even allowing the specific targeting of intracellular organelles. Ideally, a class of genetically encodeable sensors need to cover relevant cellular pH ranges. We present a FRET-based pH sensor platform, based on the pH modulation of YFP acceptor fluorophores in a fusion construct with ECFP. The concurrent loss of the overlap integral upon acidification results in a proportionally reduced FRET coupling. The readout of FRET over the sensitized YFP fluorescence lifetime yields a highly sensitive and robust pH measurement that is self-calibrated. The principle is demonstrated in the existing high-efficiency FRET fusion Cy11.5, and tunability of the platform design is demonstrated by genetic alteration of the pH sensitivity of the acceptor moiety.

    Supporting Information

    Jump To

    Morphological changes typical for necrosis induced by N-dodecyl (C12) imidazole (Figure 1). This material is available free of charge via the Internet at

    Terms & Conditions

    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system:

    Cited By

    This article is cited by 67 publications.

    1. Reshma Bano, Mohd Mohsin, Mohammad Zeyaullah, Mohammad Suhail Khan. Real-Time Monitoring of Selenium in Living Cells by Fluorescence Resonance Energy Transfer-Based Genetically Encoded Ratiometric Nanosensors. ACS Omega 2023, 8 (9) , 8625-8633.
    2. Andreas Steinegger, Otto S. Wolfbeis, Sergey M. Borisov. Optical Sensing and Imaging of pH Values: Spectroscopies, Materials, and Applications. Chemical Reviews 2020, 120 (22) , 12357-12489.
    3. Pedro J. Pacheco-Liñán, Iván Bravo, María L. Nueda, José Albaladejo, Andrés Garzón-Ruiz. Functionalized CdSe/ZnS Quantum Dots for Intracellular pH Measurements by Fluorescence Lifetime Imaging Microscopy. ACS Sensors 2020, 5 (7) , 2106-2117.
    4. Joe Chin-Hun Kuo, Marc C. Goudge, Ann E. Metzloff, Ling-Ting Huang, Marshall J. Colville, Sangwoo Park, Warren R. Zipfel, Matthew J. Paszek. Litmus-Body: A Molecularly Targeted Sensor for Cell-Surface pH Measurements. ACS Sensors 2020, 5 (6) , 1555-1566.
    5. Emily P. Haynes, Megha Rajendran, Chace K. Henning, Abhipri Mishra, Angeline M. Lyon, Mathew Tantama. Quantifying Acute Fuel and Respiration Dependent pH Homeostasis in Live Cells Using the mCherryTYG Mutant as a Fluorescence Lifetime Sensor. Analytical Chemistry 2019, 91 (13) , 8466-8475.
    6. Sandra Burgstaller, Helmut Bischof, Thomas Gensch, Sarah Stryeck, Benjamin Gottschalk, Jeta Ramadani-Muja, Emrah Eroglu, Rene Rost, Sabine Balfanz, Arnd Baumann, Markus Waldeck-Weiermair, Jesse C. Hay, Tobias Madl, Wolfgang F. Graier, Roland Malli. pH-Lemon, a Fluorescent Protein-Based pH Reporter for Acidic Compartments. ACS Sensors 2019, 4 (4) , 883-891.
    7. Lydia A. Perkins, Qi Yan, Brigitte F. Schmidt, Dmytro Kolodieznyi, Saumya Saurabh, Mads Breum Larsen, Simon C. Watkins, Laura Kremer, and Marcel P. Bruchez . Genetically Targeted Ratiometric and Activated pH Indicator Complexes (TRApHIC) for Receptor Trafficking. Biochemistry 2018, 57 (5) , 861-871.
    8. Jingfang Shangguan, Dinggeng He, Xiaoxiao He, Kemin Wang, Fengzhou Xu, Jinquan Liu, Jinlu Tang, Xue Yang, and Jin Huang . Label-Free Carbon-Dots-Based Ratiometric Fluorescence pH Nanoprobes for Intracellular pH Sensing. Analytical Chemistry 2016, 88 (15) , 7837-7843.
    9. Luyuan Zhang, Fang Xu, Zhixing Chen, Xinxin Zhu, and Wei Min . Bioluminescence Assisted Switching and Fluorescence Imaging (BASFI). The Journal of Physical Chemistry Letters 2013, 4 (22) , 3897-3902.
    10. Angel Orte, Jose M. Alvarez-Pez, and Maria J. Ruedas-Rama . Fluorescence Lifetime Imaging Microscopy for the Detection of Intracellular pH with Quantum Dot Nanosensors. ACS Nano 2013, 7 (7) , 6387-6395.
    11. Mathew Tantama, Yin Pun Hung, and Gary Yellen . Imaging Intracellular pH in Live Cells with a Genetically Encoded Red Fluorescent Protein Sensor. Journal of the American Chemical Society 2011, 133 (26) , 10034-10037.
    12. Binila K Korah, Aiswarya Murali, Bony K John, Neenamol John, Beena Mathew. Carbon dots as a sustainable nanoplatform. Biomass Conversion and Biorefinery 2023, 126
    13. Mark C. Leake, Steven D. Quinn. A guide to small fluorescent probes for single-molecule biophysics. Chemical Physics Reviews 2023, 4 (1)
    14. Sergi Padilla‐Parra. Time‐resolved single virus tracking and spectral imaging to understand HIV‐1 entry and fusion. Biology of the Cell 2023, 115 (3)
    15. Dipak Kumar Rana, Subhash Chandra Bhattacharya. Implication toward a simple strategy to generate pH tunable FRET-based biosensing. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 2022, 282 , 121687.
    16. Lennard Karsten, Lukas Goett-Zink, Julian Schmitz, Raimund Hoffrogge, Alexander Grünberger, Tilman Kottke, Kristian M. Müller. Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates. Biosensors 2022, 12 (5) , 271.
    17. Sandra Burgstaller, Helmut Bischof, Lucas Matt, Robert Lukowski. Assessing K+ ions and K+ channel functions in cancer cell metabolism using fluorescent biosensors. Free Radical Biology and Medicine 2022, 181 , 43-51.
    18. Heejung Kim, Jihye Seong. Fluorescent Protein-Based Autophagy Biosensors. Materials 2021, 14 (11) , 3019.
    19. T.P. Gerasimova, T.I. Burganov, S.A. Katsyuba, A.A. Kalinin, L.N. Islamova, G.M. Fazleeva, B.S. Ahmadeev, A.R. Mustafina, A. Monari, X. Assfeld, O.G. Sinyashin. Halochromic luminescent quinoxalinones as a basis for pH-sensing in organic and aqueous solutions. Dyes and Pigments 2021, 186 , 108958.
    20. Shaomei Xu, Xu He, Yibing Huang, Xin Liu, Lihe Zhao, Xinghua Wang, Ying Sun, Pinyi Ma, Daqian Song. Lysosome-targeted ratiometric fluorescent sensor for monitoring pH in living cells based on one-pot-synthesized carbon dots. Microchimica Acta 2020, 187 (8)
    21. Consuelo Ripoll, Mar Roldan, Rafael Contreras-Montoya, Juan J. Diaz-Mochon, Miguel Martin, Maria J. Ruedas-Rama, Angel Orte. Mitochondrial pH Nanosensors for Metabolic Profiling of Breast Cancer Cell Lines. International Journal of Molecular Sciences 2020, 21 (10) , 3731.
    22. Hamide Ehtesabi, Zahra Hallaji, Shima Najafi Nobar, Zeinab Bagheri. Carbon dots with pH-responsive fluorescence: a review on synthesis and cell biological applications. Microchimica Acta 2020, 187 (2)
    23. Sunaina Surana, David Villarroel‐Campos, Oscar M. Lazo, Edoardo Moretto, Andrew P. Tosolini, Elena R. Rhymes, Sandy Richter, James N. Sleigh, Giampietro Schiavo. The evolution of the axonal transport toolkit. Traffic 2020, 21 (1) , 13-33.
    24. Samira Bagheri, Amin TermehYousefi, Javad Mehrmashhadi. Carbon dot-based fluorometric optical sensors: an overview. Reviews in Inorganic Chemistry 2019, 39 (4) , 179-197.
    25. Maria R. Depaoli, Helmut Bischof, Emrah Eroglu, Sandra Burgstaller, Jeta Ramadani-Muja, Thomas Rauter, Maximilian Schinagl, Markus Waldeck-Weiermair, Jesse C. Hay, Wolfgang F. Graier, Roland Malli. Live cell imaging of signaling and metabolic activities. Pharmacology & Therapeutics 2019, 202 , 98-119.
    26. Qin Wang, Haitao Yang, Qiang Zhang, Hongguang Ge, Shengrui Zhang, Zhiyin Wang, Xiaohui Ji. Strong acid-assisted preparation of green-emissive carbon dots for fluorometric imaging of pH variation in living cells. Microchimica Acta 2019, 186 (7)
    27. Alejandro Arce‐Rodríguez, Daniel C. Volke, Sarina Bense, Susanne Häussler, Pablo I. Nikel. Non‐invasive, ratiometric determination of intracellular pH in Pseudomonas species using a novel genetically encoded indicator. Microbial Biotechnology 2019, 12 (4) , 799-813.
    28. Vladimir I. Martynov, Alexey A. Pakhomov, Igor E. Deyev, Alexander G. Petrenko. Genetically encoded fluorescent indicators for live cell pH imaging. Biochimica et Biophysica Acta (BBA) - General Subjects 2018, 1862 (12) , 2924-2939.
    29. Christian Rupprecht, Marcus Wingen, Janko Potzkei, Thomas Gensch, Karl-Erich Jaeger, Thomas Drepper. A novel FbFP-based biosensor toolbox for sensitive in vivo determination of intracellular pH. Journal of Biotechnology 2017, 258 , 25-32.
    30. James R.W. Conway, Sean C. Warren, Paul Timpson. Context-dependent intravital imaging of therapeutic response using intramolecular FRET biosensors. Methods 2017, 128 , 78-94.
    31. Haijun Yu, Chao Chen, Xiaodan Cao, Yueling Liu, Shengmin Zhou, Ping Wang. Ratiometric fluorescent pH nanoprobes based on in situ assembling of fluorescence resonance energy transfer between fluorescent proteins. Analytical and Bioanalytical Chemistry 2017, 409 (21) , 5073-5080.
    32. Fred S. Wouters. F örster Resonance Energy Transfer and Fluorescence Lifetime Imaging. 2017, 405-451.
    33. MAG de Castro, G Bunt, FS Wouters. Cathepsin B launches an apoptotic exit effort upon cell death-associated disruption of lysosomes. Cell Death Discovery 2016, 2 (1)
    34. M.G. Guillén, F. Gámez, T. Lopes-Costa, J. Cabanillas-González, J.M. Pedrosa. A fluorescence gas sensor based on Förster Resonance Energy Transfer between polyfluorene and bromocresol green assembled in thin films. Sensors and Actuators B: Chemical 2016, 236 , 136-143.
    35. Bryce Bajar, Emily Wang, Shu Zhang, Michael Lin, Jun Chu. A Guide to Fluorescent Protein FRET Pairs. Sensors 2016, 16 (9) , 1488.
    36. Dahdjim-Benoît Betolngar, Marie Erard, Hélène Pasquier, Yasmina Bousmah, Awa Diop-Sy, Elvire Guiot, Pierre Vincent, Fabienne Mérola. pH sensitivity of FRET reporters based on cyan and yellow fluorescent proteins. Analytical and Bioanalytical Chemistry 2015, 407 (14) , 4183-4193.
    37. Dana Meyen, Katsiaryna Tarbashevich, Torsten U Banisch, Carolina Wittwer, Michal Reichman-Fried, Benoît Maugis, Cecilia Grimaldi, Esther-Maria Messerschmidt, Erez Raz. Dynamic filopodia are required for chemokine-dependent intracellular polarization during guided cell migration in vivo. eLife 2015, 4
    38. Katsiaryna Tarbashevich, Michal Reichman-Fried, Cecilia Grimaldi, Erez Raz. Chemokine-Dependent pH Elevation at the Cell Front Sustains Polarity in Directionally Migrating Zebrafish Germ Cells. Current Biology 2015, 25 (8) , 1096-1103.
    39. Darpan Malhotra, Joseph R Casey. Intracellular p H Measurement. 2015, 1-7.
    40. Taichiro Tomida. Visualization of the spatial and temporal dynamics of MAPK signaling using fluorescence imaging techniques. The Journal of Physiological Sciences 2015, 65 (1) , 37-49.
    41. Robert J. Meier, Johann M. B. Simbürger, Tero Soukka, Michael Schäferling. A FRET based pH probe with a broad working range applicable to referenced ratiometric dual wavelength and luminescence lifetime read out. Chemical Communications 2015, 51 (28) , 6145-6148.
    42. Chi Chen, Pengfei Zhang, Li Zhang, Duyang Gao, Guanhui Gao, Yong Yang, Wenjun Li, Ping Gong, Lintao Cai. Long-decay near-infrared-emitting doped quantum dots for lifetime-based in vivo pH imaging. Chemical Communications 2015, 51 (56) , 11162-11165.
    43. Christoph Biskup, Thomas Gensch. Fluorescence lifetime imaging of ions in biological tissues. 2014, 497-534.
    44. Michal Stawarski, Izabela Rutkowska-Wlodarczyk, André Zeug, Monika Bijata, Hubert Madej, Leszek Kaczmarek, Jakub Wlodarczyk. Genetically encoded FRET-based biosensor for imaging MMP-9 activity. Biomaterials 2014, 35 (5) , 1402-1410.
    45. Maria J. Ruedas-Rama, Jose M. Alvarez-Pez, Luis Crovetto, Jose M. Paredes, Angel Orte. FLIM Strategies for Intracellular Sensing. 2014, 191-223.
    46. D. Aigner, R. I. Dmitriev, S. M. Borisov, D. B. Papkovsky, I. Klimant. pH-sensitive perylene bisimide probes for live cell fluorescence lifetime imaging. J. Mater. Chem. B 2014, 2 (39) , 6792-6801.
    47. Alice G. Byrne, Matthew M. Byrne, George Coker III, Kelly B. Gemmill, Christopher Spillmann, Igor Medintz, Seth L. Sloan, B. Wieb van der Meer. Data. 2013, 655-755.
    48. Fred S. Wouters. Förster Resonance Energy Transfer and Fluorescence Lifetime Imaging. 2013, 245-291.
    49. Marie Erard, Asma Fredj, Hélène Pasquier, Dahdjim-Benoît Beltolngar, Yasmina Bousmah, Valérie Derrien, Pierre Vincent, Fabienne Merola. Minimum set of mutations needed to optimize cyan fluorescent proteins for live cell imaging. Mol. BioSyst. 2013, 9 (2) , 258-267.
    50. Spencer C. Alford, Jiahui Wu, Yongxin Zhao, Robert E. Campbell, Thomas Knöpfel. Optogenetic reporters. Biology of the Cell 2013, 105 (1) , 14-29.
    51. Janko Potzkei, Martin Kunze, Thomas Drepper, Thomas Gensch, Karl-Erich Jaeger, Jochen Büchs. Real-time determination of intracellular oxygen in bacteria using a genetically encoded FRET-based biosensor. BMC Biology 2012, 10 (1)
    52. Maria J. Ruedas-Rama, Jamie D. Walters, Angel Orte, Elizabeth A.H. Hall. Fluorescent nanoparticles for intracellular sensing: A review. Analytica Chimica Acta 2012, 751 , 1-23.
    53. Jithesh V. Veetil, Sha Jin, Kaiming Ye. Fluorescence Lifetime Imaging Microscopy of Intracellular Glucose Dynamics. Journal of Diabetes Science and Technology 2012, 6 (6) , 1276-1285.
    54. Sergi Padilla-Parra, Pedro M. Matos, Naoyuki Kondo, Mariana Marin, Nuno C. Santos, Gregory B. Melikyan. Quantitative imaging of endosome acidification and single retrovirus fusion with distinct pools of early endosomes. Proceedings of the National Academy of Sciences 2012, 109 (43) , 17627-17632.
    55. Elena Kardash, Jan Bandemer, Erez Raz. Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors. Nature Protocols 2011, 6 (12) , 1835-1846.
    56. Clara Bermejo, Farzad Haerizadeh, Hitomi Takanaga, Diane Chermak, Wolf B Frommer. Optical sensors for measuring dynamic changes of cytosolic metabolite levels in yeast. Nature Protocols 2011, 6 (11) , 1806-1817.
    57. Gilbert O. Fruhwirth, Luis P. Fernandes, Gregory Weitsman, Gargi Patel, Muireann Kelleher, Katherine Lawler, Adrian Brock, Simon P. Poland, Daniel R. Matthews, György Kéri, Paul R. Barber, Borivoj Vojnovic, Simon M. Ameer‐Beg, Anthony C. C. Coolen, Franca Fraternali, Tony Ng. How Förster Resonance Energy Transfer Imaging Improves the Understanding of Protein Interaction Networks in Cancer Biology. ChemPhysChem 2011, 12 (3) , 442-461.
    58. Alessandro Esposito, Arjen N. Bader, Simon C. Schlachter, Dave J. van den Heuvel, Gabriele S. Kaminski Schierle, Ashok R. Venkitaraman, Clemens F. Kaminski, Hans C. Gerritsen. Design and application of a confocal microscope for spectrally resolved anisotropy imaging. Optics Express 2011, 19 (3) , 2546.
    59. Fred S. Wouters. Imaging Molecular Physiology in Cells Using FRET-Based Fluorescent Nanosensors. 2011, 131-152.
    60. Jan Willem Borst, Antonie J W G Visser. Fluorescence lifetime imaging microscopy in life sciences. Measurement Science and Technology 2010, 21 (10) , 102002.
    61. Johannes T. Wessels, Kensuke Yamauchi, Robert M. Hoffman, Fred S. Wouters. Advances in cellular, subcellular, and nanoscale imaging in vitro and in vivo. Cytometry Part A 2010, 77A (7) , 667-676.
    62. Marleen Forkink, Jan A.M. Smeitink, Roland Brock, Peter H.G.M. Willems, Werner J.H. Koopman. Detection and manipulation of mitochondrial reactive oxygen species in mammalian cells. Biochimica et Biophysica Acta (BBA) - Bioenergetics 2010, 1797 (6-7) , 1034-1044.
    63. Fred Wouters, Gertrude Bunt. Molecular Resolution of Cellular Biochemistry and Physiology by FRET/FLIM. 2010, 12-1-12-26.
    64. Andreas Ibraheem, Robert E Campbell. Designs and applications of fluorescent protein-based biosensors. Current Opinion in Chemical Biology 2010, 14 (1) , 30-36.
    65. A. J. W. G. Visser, S. P. Laptenok, N. V. Visser, A. van Hoek, D. J. S. Birch, J.-C. Brochon, J. W. Borst. Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs. European Biophysics Journal 2010, 39 (2) , 241-253.
    66. Hélène A. Benink, Mark G. McDougall, Dieter H. Klaubert, Georgyi V. Los. Direct pH measurements by using subcellular targeting of 5(and 6-) carboxyseminaphthorhodafluor in mammalian cells. BioTechniques 2009, 47 (3) , 769-774.
    67. Alessandro Esposito, Simon Schlachter, Gabriele S. Kaminski Schierle, Alan D. Elder, Alberto Diaspro, Fred S. Wouters, Clemens F. Kaminski, Asparouh I. Iliev. Quantitative Fluorescence Microscopy Techniques. 2009, 117-142.

    Pair your accounts.

    Export articles to Mendeley

    Get article recommendations from ACS based on references in your Mendeley library.

    Pair your accounts.

    Export articles to Mendeley

    Get article recommendations from ACS based on references in your Mendeley library.

    You’ve supercharged your research process with ACS and Mendeley!

    STEP 1:
    Click to create an ACS ID

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Your Mendeley pairing has expired. Please reconnect