@article{pmid41162396,

title = {A red fluorescent genetically encoded biosensor for in vivo imaging of extracellular L-lactate dynamics},

author = {Yuki Kamijo and Philipp Mächler and Natalie Ness and Cong Quang Vu and Tsukasa Kusakizako and Jamsad Mannuthodikayil and Zaneta Ku and Marc Boisvert and Ekaterina Grebenik and Ikumi Miyazaki and Rina Hashizume and Haruaki Sato and Rui Liu and Yukiko Hori and Taisuke Tomita and Tetsuro Katayama and Akihiro Furube and Gabriela Caraveo and Marie-Eve Paquet and Mikhail Drobizhev and Osamu Nureki and Satoshi Arai and Marco Brancaccio and Robert E Campbell and David Kleinfeld and Yusuke Nasu},

doi = {10.1038/s41467-025-64484-x},

issn = {2041-1723},

year  = {2025},

date = {2025-10-01},

journal = {Nat Commun},

volume = {16},

number = {1},

pages = {9531},

abstract = {L-Lactate is increasingly recognized as an intercellular energy currency in mammals, but mysteries remain regarding the spatial and temporal dynamics of its release and uptake between cells via the extracellular environment. Here we introduce R-eLACCO2.1, a red fluorescent extracellular L-lactate biosensor that is superior to previously reported green fluorescent biosensors in in vivo sensitivity to increases in extracellular L-lactate and spectral orthogonality. R-eLACCO2.1 exhibits excellent fluorescence response in cultured cells, mouse brain slices, and live mice. R-eLACCO2.1 also serves as an effective fluorescence lifetime-based biosensor. Using R-eLACCO2.1, we monitor whisker stimulation and locomotion-induced changes in endogenous extracellular L-lactate in the somatosensory cortex of awake mice. To highlight the potential insights gained from in vivo measurements with R-eLACCO2.1, we perform dual-color imaging from the somatosensory cortex of actively locomoting mice. This enables us to simultaneously observe the neural activity, reported by a green fluorescent GCaMP calcium ion biosensor, and extracellular L-lactate. As the high-performance tool in the suite of extracellular L-lactate biosensors, R-eLACCO2.1 is ideally suited to delimit the emerging roles of L-lactate in mammalian metabolism.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40888292,

title = {Bipartite Genetically Encoded Biosensors to Sense Calcium Ion Dynamics at Membrane-Membrane Contact Sites},

author = {Issei Yamaguchi and Lucia Barazzuol and Giulia Dematteis and Wenchao Zhu and Yurong Wen and Mikhail Drobizhev and Dmitry Lim and Robert E Campbell and Tito Calì and Yusuke Nasu},

doi = {10.1021/acs.analchem.5c03831},

issn = {1520-6882},

year  = {2025},

date = {2025-09-01},

journal = {Anal Chem},

volume = {97},

number = {36},

pages = {19848--19861},

abstract = {Self-complementing bipartite fluorescent proteins (FPs) are useful tools for the detection of protein-protein proximity and for localizing fluorophores to membrane-membrane contact sites. Here, we report versions of circularly permuted green FP (GFP), red FP (RFP), and mNeonGreen (NG), which are split into a large fragment composed of nine β-strands and a small fragment composed of two β-strands. In each case, the large and small fragments can associate in live cells to form the complete 11-stranded FP β-barrel. We further converted each of these three self-complementing FPs into bipartite calcium ion (Ca) biosensors. We demonstrate that appropriately targeted versions of these split FPs, and split FP-based biosensors, can be functionally assembled at membrane-membrane contact sites. We employ the bipartite NG-based Ca biosensor for visualization of pharmacologically induced Ca release at mitochondria-endoplasmic reticulum contact sites (MERCs).},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40631294,

title = {High-performance genetically-encoded green and red fluorescent biosensors for pyruvate},

author = {Shosei Imai and Saaya Hario and Carl Suerte and Issei Yamaguchi and Kenryo Sakoi and Adam Thuen and Brekken Meznarich and Mikhail Drobizhev and Takuya Terai and Kei Takahashi-Yamashiro and Robert E Campbell},

doi = {10.1101/2025.04.17.649293},

issn = {2692-8205},

year  = {2025},

date = {2025-09-01},

journal = {bioRxiv},

abstract = {Pyruvate is the end-product of glycolysis and a central metabolite involved in many biochemical pathways. However, a lack of high-performance (i.e., Δ/  > 10) single fluorescent protein (FP)-based biosensors has hindered efforts to investigate the physiological role of pyruvate. Here, we present the GreenPy1 and ApplePy1 series, which are green FP (GFP)-based and red FP (RFP)-based pyruvate biosensors, respectively. Both series exhibit large fluorescence intensity change (Δ/  ~ 20 to >40) and a range of affinities (10s of μM to several mM). We demonstrate the utility of these pyruvate biosensors for multicolor imaging of metabolite concentration changes in mammalian cells.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40195305,

title = {Far-red fluorescent genetically encoded calcium ion indicators},

author = {Rochelin Dalangin and Bill Z Jia and Yitong Qi and Abhi Aggarwal and Kenryo Sakoi and Mikhail Drobizhev and Rosana S Molina and Ronak Patel and Ahmed S Abdelfattah and Jihong Zheng and Daniel Reep and Jeremy P Hasseman and  and Yufeng Zhao and Jiahui Wu and Kaspar Podgorski and Alison G Tebo and Eric R Schreiter and Thomas E Hughes and Takuya Terai and Marie-Eve Paquet and Sean G Megason and Adam E Cohen and Yi Shen and Robert E Campbell},

doi = {10.1038/s41467-025-58485-z},

issn = {2041-1723},

year  = {2025},

date = {2025-04-01},

journal = {Nat Commun},

volume = {16},

number = {1},

pages = {3318},

abstract = {Genetically encoded calcium ion (Ca) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca dynamics and neuronal activities with single-cell resolution. Here we report the design and development of two far-red fluorescent GECIs, FR-GECO1a and FR-GECO1c, based on the monomeric far-red fluorescent proteins mKelly1 and mKelly2. FR-GECOs have excitation and emission maxima at ~596 nm and ~644 nm, respectively, display large responses to Ca in vitro (ΔF/F = 6 for FR-GECO1a, 18 for FR-GECO1c), are bright under both one-photon and two-photon illumination, and have high affinities (apparent K = 29 nM for FR-GECO1a, 83 nM for FR-GECO1c) for Ca. FR-GECOs offer sensitive and fast detection of single action potentials in neurons, and enable in vivo all-optical manipulation and measurement of cellular activities in combination with optogenetic actuators.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40188278,

title = {Synthesis and application of a photocaged-L-lactate for studying the biological roles of L-lactate},

author = {Ikumi Miyazaki and Kelvin K Tsao and Yuki Kamijo and Yusuke Nasu and Takuya Terai and Robert E Campbell},

doi = {10.1038/s42004-025-01495-1},

issn = {2399-3669},

year  = {2025},

date = {2025-04-01},

journal = {Commun Chem},

volume = {8},

number = {1},

pages = {104},

abstract = {L-Lactate, once considered a metabolic waste product of glycolysis, is now recognized as a vitally important metabolite and signaling molecule in multiple biological pathways. However, exploring L-lactate's emerging intra- and extra-cellular roles is hindered by a lack of tools to perturb L-lactate concentration intracellularly and extracellularly. Photocaged compounds are a powerful way to introduce bioactive molecules with spatiotemporal precision using illumination. Here, we report the development of a photocaged derivative of L-lactate, 4-methoxy-7-nitroindolinyl-L-lactate (MNI-L-lac), that releases L-lactate upon illumination. We validated MNI-L-lac in cell culture by demonstrating that the photorelease of L-lactate elicits a response from genetically encoded extra- and intracellular L-lactate biosensors (eLACCO1, eLACCO2.1, R-iLACCO1.2). To demonstrate the utility of MNI-L-lac, we employed the photorelease of L-lactate to activate G protein-coupled receptor 81 (GPR81), as revealed by the inhibition of adenylyl cyclase activity and concomitant decrease of cAMP. These results indicate that MNI-L-lac may be useful for perturbing the concentration of endogenous L-lactate in order to investigate L-lactate's roles in metabolic and signaling pathways.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39811063,

title = {Multimodal fluorescence-optoacoustic  imaging of the near-infrared calcium ion indicator NIR-GECO2G},

author = {Sarah F Shaykevich and Justin P Little and Yong Qian and Marie-Eve Paquet and Robert E Campbell and Daniel Razansky and Shy Shoham},

doi = {10.1016/j.pacs.2024.100671},

issn = {2213-5979},

year  = {2025},

date = {2025-02-01},

journal = {Photoacoustics},

volume = {41},

pages = {100671},

abstract = {Measuring whole-brain distributed functional activity is an important unmet need in neuroscience, requiring high temporal resolution and cellular specificity across large volumes. Functional optoacoustic neuro-tomography (FONT) with genetically encoded calcium ion indicators is a promising approach towards this goal. However, it has not yet been applied in the near-infrared (NIR) range that provides deep penetration and low vascular background optimal for  neuroimaging. Here, we study the noninvasive multimodal fluorescence and optoacoustic imaging performance of state-of-the-art NIR calcium ion indicator NIR-GECO2G in the mouse brain. We observe robust  signals with widefield fluorescence, and for the first time, with FONT. We also show that in both modalities, the NIR-GECO2G signal improves more than twofold in the biliverdin-enriched   mouse line compared to wild type. Our findings demonstrate the potential of multimodal fluorescence and optoacoustic NIR imaging, opening new possibilities for whole-brain real-time functional imaging in rodents.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39678364,

title = {A chemigenetic indicator based on a synthetic chelator and a green fluorescent protein for imaging of intracellular sodium ions},

author = {Shiori Takeuchi and Shosei Imai and Takuya Terai and Robert E Campbell},

doi = {10.1039/d4cb00256c},

issn = {2633-0679},

year  = {2025},

date = {2025-02-01},

journal = {RSC Chem Biol},

volume = {6},

number = {2},

pages = {170--174},

abstract = {A chemigenetic indicator with an affinity suitable for imaging of intracellular sodium ions (Na) in mammalian cells was developed. The indicator, based on a chimera of green fluorescent protein (GFP) and HaloTag labeled with a synthetic crown ether chelator, was produced by a combination of rational design and directed evolution. In mammalian cells the indicator exhibited an approximately 100% increase in excitation ratio when the cells were treated with 20 mM Na and an ionophore.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39763884,

title = {PinkyCaMP a mScarlet-based calcium sensor with exceptional brightness, photostability, and multiplexing capabilities},

author = {Ryan Fink and Shosei Imai and Nala Gockel and German Lauer and Kim Renken and Jonas Wietek and Paul J Lamothe-Molina and Falko Fuhrmann and Manuel Mittag and Tim Ziebarth and Annika Canziani and Martin Kubitschke and Vivien Kistmacher and Anny Kretschmer and Eva Sebastian and Dietmar Schmitz and Takuya Terai and Jan Gründemann and Sami Hassan and Tommaso Patriarchi and Andreas Reiner and Martin Fuhrmann and Robert E Campbell and Olivia Andrea Masseck},

doi = {10.1101/2024.12.16.628673},

issn = {2692-8205},

year  = {2025},

date = {2025-01-01},

journal = {bioRxiv},

abstract = {Genetically encoded calcium (Ca) indicators (GECIs) are widely used for imaging neuronal activity, yet current limitations of existing red fluorescent GECIs have constrained their applicability. The inherently dim fluorescence and low signal-to-noise ratio of red-shifted GECIs have posed significant challenges. More critically, several red-fluorescent GECIs exhibit photoswitching when exposed to blue light, thereby limiting their applicability in all-optical experimental approaches. Here, we present the development of PinkyCaMP, the first mScarlet-based Ca sensor that outperforms current red fluorescent sensors in brightness, photostability, signal-to-noise ratio, and compatibility with optogenetics and neurotransmitter imaging. PinkyCaMP is well-tolerated by neurons, showing no toxicity or aggregation, both  and . All imaging approaches, including single-photon excitation methods such as fiber photometry, widefield imaging, miniscope imaging, as well as two-photon imaging in awake mice, are fully compatible with PinkyCaMP.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39601449,

title = {High-Performance Chemigenetic Potassium Ion Indicator},

author = {Dazhou Cheng and Zhenlin Ouyang and Xiaoyu He and Yusuke Nasu and Yurong Wen and Takuya Terai and Robert E Campbell},

doi = {10.1021/jacs.4c10917},

issn = {1520-5126},

year  = {2024},

date = {2024-12-01},

journal = {J Am Chem Soc},

volume = {146},

number = {51},

pages = {35117--35128},

abstract = {Potassium ion (K) is the most abundant metal ion in cells and plays an indispensable role in practically all biological systems. Although there have been reports of both synthetic and genetically encoded fluorescent K indicators, there remains a need for an indicator that is genetically targetable, has high specificity for K versus Na, and has a high fluorescent response in the red to far-red wavelength range. Here, we introduce a series of chemigenetic K indicators, designated as the HaloKbp1 series, based on the bacterial K-binding protein (Kbp) inserted into HaloTag7 self-labeled with environmentally sensitive rhodamine derivatives. This series of high-performance indicators features high brightness in the red to far-red region, large intensiometric fluorescence changes, and a range of  values. We demonstrate that they are suitable for the detection of physiologically relevant K concentration changes such as those that result from the Ca-dependent activation of the BK potassium channel.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}