@article {47527, title = {A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo.}, journal = {Nature Biotechnology}, volume = {34}, year = {2016}, month = {2016 May 30}, pages = {760-7}, abstract = {

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals owing to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright, engineered, orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.

}, issn = {1546-1696}, doi = {10.1038/nbt.3550}, author = {Chu, Jun and Oh, Younghee and Sens, Alex and Ataie, Niloufar and Dana, Hod and Macklin, John J and Laviv, Tal and Welf, Erik S and Dean, Kevin M and Zhang, Feijie and Kim, Benjamin B and Tang, Clement Tran and Hu, Michelle and Baird, Michelle A and Davidson, Michael W and Kay, Mark A and Fiolka, Reto and Yasuda, Ryohei and Kim, Douglas S and Ng, Ho-Leung and Lin, Michael Z} } @article {5163, title = {Imaging neural activity using Thy1-GCaMP transgenic mice.}, journal = {Neuron}, volume = {76}, year = {2012}, month = {2012 Oct 18}, pages = {297-308}, abstract = {

The ability to chronically monitor neuronal activity in the living brain is essential for understanding the organization and function of the nervous system. The genetically encoded green fluorescent protein based calcium sensor GCaMP provides a powerful tool for detecting calcium transients in neuronal somata, processes, and synapses that are triggered by neuronal activities. Here we report the generation and characterization of transgenic mice that express improved GCaMPs in various neuronal subpopulations under the control of the Thy1 promoter. In vitro and in vivo studies show that calcium transients induced by spontaneous and stimulus-evoked neuronal activities can be readily detected at the level of individual cells and synapses in acute brain slices, as well as in awake behaving animals. These GCaMP transgenic mice allow investigation of activity patterns in defined neuronal populations in the living brain, and will greatly facilitate dissecting complex structural and functional relationships of neural networks.

}, issn = {1097-4199}, doi = {10.1016/j.neuron.2012.07.011}, author = {Chen, Qian and Cichon, Joseph and Wang, Wenting and Qiu, LI and Lee, Seok-Jin R. and Campbell, Nolan R. and DeStefino, Nicholas and Fu, Zhanyan and Yasuda, Ryohei and Looger, Loren L and Arenkiel, Benjamin R. and Gan, Wenbiao and Feng, Guoping} } @article {4254, title = {A genetically encoded fluorescent sensor of ERK activity.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {105}, year = {2008}, month = {2008 Dec 9}, pages = {19264-9}, abstract = {

The activity of the ERK has complex spatial and temporal dynamics that are important for the specificity of downstream effects. However, current biochemical techniques do not allow for the measurement of ERK signaling with fine spatiotemporal resolution. We developed a genetically encoded, FRET-based sensor of ERK activity (the extracellular signal-regulated kinase activity reporter, EKAR), optimized for signal-to-noise ratio and fluorescence lifetime imaging. EKAR selectively and reversibly reported ERK activation in HEK293 cells after epidermal growth factor stimulation. EKAR signals were correlated with ERK phosphorylation, required ERK activity, and did not report the activities of JNK or p38. EKAR reported ERK activation in the dendrites and nucleus of hippocampal pyramidal neurons in brain slices after theta-burst stimuli or trains of back-propagating action potentials. EKAR therefore permits the measurement of spatiotemporal ERK signaling dynamics in living cells, including in neuronal compartments in intact tissues.

}, keywords = {Action Potentials, Animals, Artifacts, Bacterial Proteins, Cell Line, Dendrites, Endothelial Cells, Enzyme Activation, Extracellular Signal-Regulated MAP Kinases, Fluorescence Resonance Energy Transfer, Fluorescent Dyes, Genes, Green Fluorescent Proteins, Hippocampus, Humans, Kidney, Luminescent Proteins, MAP Kinase Signaling System, Organ Culture Techniques, Patch-Clamp Techniques, Pyramidal Cells, Rats, Reporter}, issn = {1091-6490}, doi = {10.1073/pnas.0804598105}, author = {Harvey, Christopher D and Ehrhardt, Anka G and Cellurale, Cristina and Zhong, Haining and Yasuda, Ryohei and Davis, Roger J and Svoboda, Karel} } @article {5213, title = {The spread of Ras activity triggered by activation of a single dendritic spine.}, journal = {Science}, volume = {321}, year = {2008}, month = {2008 Jul 4}, pages = {136-40}, abstract = {

In neurons, individual dendritic spines isolate N-methyl-d-aspartate (NMDA) receptor-mediated calcium ion (Ca2+) accumulations from the dendrite and other spines. However, the extent to which spines compartmentalize signaling events downstream of Ca2+ influx is not known. We combined two-photon fluorescence lifetime imaging with two-photon glutamate uncaging to image the activity of the small guanosine triphosphatase Ras after NMDA receptor activation at individual spines. Induction of long-term potentiation (LTP) triggered robust Ca2+-dependent Ras activation in single spines that decayed in approximately 5 minutes. Ras activity spread over approximately 10 micrometers of dendrite and invaded neighboring spines by diffusion. The spread of Ras-dependent signaling was necessary for the local regulation of the threshold for LTP induction. Thus, Ca2+-dependent synaptic signals can spread to couple multiple synapses on short stretches of dendrite.

}, keywords = {Animals, Calcium, Cell Membrane, Dendritic Spines, Diffusion, Fluorescence Resonance Energy Transfer, Glutamic Acid, GTPase-Activating Proteins, Guanine Nucleotide Exchange Factors, Hippocampus, Long-Term Potentiation, N-Methyl-D-Aspartate, Pyramidal Cells, ras Proteins, Rats, Receptors, Recombinant Fusion Proteins, Signal Transduction, Synapses, Transfection}, issn = {1095-9203}, doi = {10.1126/science.1159675}, author = {Harvey, Christopher D and Yasuda, Ryohei and Zhong, Haining and Svoboda, Karel} }