Non-invasive observation of spatiotemporal activity of large neural populations distributed over entire brains is a longstanding goal of neuroscience.We developed a volumetric multispectral optoacoustic tomography pla...Non-invasive observation of spatiotemporal activity of large neural populations distributed over entire brains is a longstanding goal of neuroscience.We developed a volumetric multispectral optoacoustic tomography platform for imaging neural activation deep in scattering brains.It can record 100 volumetric frames per second across scalable fields of view ranging between 50 and 1000 mm^(3) with respective spatial resolution of 35–200μm.Experiments performed in immobilized and freely swimming larvae and in adult zebrafish brains expressing the genetically encoded calcium indicator GCaMP5G demonstrate,for the first time,the fundamental ability to directly track neural dynamics using optoacoustics while overcoming the longstanding penetration barrier of optical imaging in scattering brains.The newly developed platform thus offers unprecedented capabilities for functional whole-brain observations of fast calcium dynamics;in combination with optoacoustics'well-established capacity for resolving vascular hemodynamics,it could open new vistas in the study of neural activity and neurovascular coupling in health and disease.展开更多
GCaMP is one of the most widely used calcium indicators in neuronal imaging and calcium cell biology. The newly developed GCaMP6 shows superior brightness and ultrasensitivity to calcium concentration change. In this ...GCaMP is one of the most widely used calcium indicators in neuronal imaging and calcium cell biology. The newly developed GCaMP6 shows superior brightness and ultrasensitivity to calcium concentration change. In this study, we determined crystal structures of CaZ＋-bound GCaMP6 monomer and dimer and presented detailed structural analyses in comparison with its par- ent version GCaMP5G. Our analyses reveal the structural basis for the outperformance of this newly developed Ca2＋ indicator. Three substitution mutations and the resulting changes of local structure and interaction explain the ultrasensitivity and in- creased fluorescence intensity common to all three versions of GCaMP6. Each particular substitution in the three GCaMP6 is also structurally consistent with their differential sensitivity and intensity, maximizing the potential of using GCaMP6 in solving diverse problems in neuronal research and calcium signaling. Our studies shall also be beneficial to further structure-guided optimization of GCaMP and facilitate the design of novel calcium indicators.展开更多
Genetically encoded Ca^(2+) indicators(GECI)are important for the measurement of Ca^(2+) in vivo.GCaMP2,a widelyused GECI,has recently been iteratively improved.Among the improved variants,GCaMP3 exhibits significantl...Genetically encoded Ca^(2+) indicators(GECI)are important for the measurement of Ca^(2+) in vivo.GCaMP2,a widelyused GECI,has recently been iteratively improved.Among the improved variants,GCaMP3 exhibits significantly better fluorescent intensity.In this study,we developed a new GECI called GCaMPJ and determined the crystal structures of GCaMP3 and GCaMPJ.GCaMPJ has a 1.5-fold increase in fl uorescence and 1.3-fold increase in calcium affi nity over GCaMP3.Upon Ca^(2+) binding,GCaMP3 exhibits both monomeric and dimeric forms.The structural superposition of these two forms reveals the role of Arg-376 in improving monomer performance.However,GCaMPJ seldom forms dimers under conditions similar to GCaMP3.St ructural and mutagenesis studies on Tyr-380 confi rmed its importance in blocking the cpEGFPβ-barrel holes.Our study proposes an efficient tool for mapping Ca^(2+) signals in intact organs to facilitate the further improvement of GCaMP sensors.展开更多
基金support from the European Research Council ERC-2010-StG-260991(DR)and ERC-2012-StG_20111109(AL and GGW)the National Institute of Health R21-EY026382-01(DR and SS)+1 种基金the German-Israeli Foundation(GIF)for Scientific Research and Development 1142-46.10/2011(DR and SS)the Helmholtz Association of German Research Centers and the Technische Universität München(DR and GGW)。
文摘Non-invasive observation of spatiotemporal activity of large neural populations distributed over entire brains is a longstanding goal of neuroscience.We developed a volumetric multispectral optoacoustic tomography platform for imaging neural activation deep in scattering brains.It can record 100 volumetric frames per second across scalable fields of view ranging between 50 and 1000 mm^(3) with respective spatial resolution of 35–200μm.Experiments performed in immobilized and freely swimming larvae and in adult zebrafish brains expressing the genetically encoded calcium indicator GCaMP5G demonstrate,for the first time,the fundamental ability to directly track neural dynamics using optoacoustics while overcoming the longstanding penetration barrier of optical imaging in scattering brains.The newly developed platform thus offers unprecedented capabilities for functional whole-brain observations of fast calcium dynamics;in combination with optoacoustics'well-established capacity for resolving vascular hemodynamics,it could open new vistas in the study of neural activity and neurovascular coupling in health and disease.
基金supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute to Shao Fenggrant from the National Basic Research Program of China (2011CB910304 and 2011CB911103) to Wang DaChengNational Natural Science Foundation of China (31100535) to Ding JingJin
文摘GCaMP is one of the most widely used calcium indicators in neuronal imaging and calcium cell biology. The newly developed GCaMP6 shows superior brightness and ultrasensitivity to calcium concentration change. In this study, we determined crystal structures of CaZ＋-bound GCaMP6 monomer and dimer and presented detailed structural analyses in comparison with its par- ent version GCaMP5G. Our analyses reveal the structural basis for the outperformance of this newly developed Ca2＋ indicator. Three substitution mutations and the resulting changes of local structure and interaction explain the ultrasensitivity and in- creased fluorescence intensity common to all three versions of GCaMP6. Each particular substitution in the three GCaMP6 is also structurally consistent with their differential sensitivity and intensity, maximizing the potential of using GCaMP6 in solving diverse problems in neuronal research and calcium signaling. Our studies shall also be beneficial to further structure-guided optimization of GCaMP and facilitate the design of novel calcium indicators.
基金supported by grants from the National Basic Research Program of China(Nos.2011CB8091004 and 2009CB918701)。
文摘Genetically encoded Ca^(2+) indicators(GECI)are important for the measurement of Ca^(2+) in vivo.GCaMP2,a widelyused GECI,has recently been iteratively improved.Among the improved variants,GCaMP3 exhibits significantly better fluorescent intensity.In this study,we developed a new GECI called GCaMPJ and determined the crystal structures of GCaMP3 and GCaMPJ.GCaMPJ has a 1.5-fold increase in fl uorescence and 1.3-fold increase in calcium affi nity over GCaMP3.Upon Ca^(2+) binding,GCaMP3 exhibits both monomeric and dimeric forms.The structural superposition of these two forms reveals the role of Arg-376 in improving monomer performance.However,GCaMPJ seldom forms dimers under conditions similar to GCaMP3.St ructural and mutagenesis studies on Tyr-380 confi rmed its importance in blocking the cpEGFPβ-barrel holes.Our study proposes an efficient tool for mapping Ca^(2+) signals in intact organs to facilitate the further improvement of GCaMP sensors.
