45 research outputs found
GAF-CaMP3–sfGFP, An Enhanced Version of the Near-Infrared Genetically Encoded Positive Phytochrome-Based Calcium Indicator for the Visualization of Neuronal Activity
No full textThe first generation of near-infrared, genetically encoded calcium indicators (NIR-GECIs) was developed from bacterial phytochrome-based fluorescent proteins that utilize biliverdin (BV) as the chromophore moiety. However, NIR-GECIs have some main drawbacks such as either an inverted response to calcium ions (in the case of NIR-GECO1) or a limited dynamic range and a lack of data about their application in neurons (in the case of GAF-CaMP2–superfolder green fluorescent protein (sfGFP)). Here, we developed an enhanced version of the GAF-CaMP2–sfGFP indicator, named GAF-CaMP3–sfGFP. The GAF-CaMP3–sfGFP demonstrated spectral characteristics, molecular brightness, and a calcium affinity similar to the respective characteristics for its progenitor, but a 2.9-fold larger DF/F response to calcium ions. As compared to GAF-CaMP2–sfGFP, in cultured HeLa cells, GAF-CaMP3–sfGFP had similar brightness but a 1.9-fold larger DF/F response to the elevation of calcium ions levels. Finally, we successfully utilized the GAF-CaMP3–sfGFP for the monitoring of the spontaneous and stimulated activity of neuronal cultures and compared its performance with the R-GECO1 indicator using two-color confocal imaging. In the cultured neurons, GAF-CaMP3–sfGFP showed a linear DF/F response in the range of 0–20 APs and in this range demonstrated a 1.4-fold larger DF/F response but a 1.3- and 2.4-fold slower rise and decay kinetics, respectively, as compared to the same parameters for the R-GECO1 indicator
A sensitive soma-localized red fluorescent calcium indicator for in vivo imaging of neuronal populations at single-cell resolution.
No full textRecent advancements in genetically encoded calcium indicators, particularly those based on green fluorescent proteins, have optimized their performance for monitoring neuronal activities in a variety of model organisms. However, progress in developing red-shifted GECIs, despite their advantages over green indicators, has been slower, resulting in fewer options for end users. In this study, we explored topological inversion and soma-targeting strategies, which are complementary to conventional mutagenesis, to re-engineer a red genetically encoded calcium indicator, FRCaMP, for enhanced in vivo performance. The resulting sensors, FRCaMPi and soma-targeted FRCaMPi (SomaFRCaMPi), exhibit up to 2-fold higher dynamic range and peak ΔF/F0 per single AP compared to widely used jRGECO1a in neurons both in culture and in vivo. Compared to jRGECO1a and FRCaMPi, SomaFRCaMPi reduces erroneous correlation of neuronal activity in the brains of mice and zebrafish by two- to 4-fold due to diminished neuropil contamination without compromising the signal-to-noise ratio. Under wide-field imaging in primary somatosensory and visual cortices in mice with high labeling density (80-90%), SomaFRCaMPi exhibits up to 40% higher SNR and decreased artifactual correlation across neurons. Altogether, SomaFRCaMPi improves the accuracy and scale of neuronal activity imaging at single-neuron resolution in densely labeled brain tissues due to a 2-3-fold enhanced automated neuronal segmentation, 50% higher fraction of responsive cells, up to 2-fold higher SNR compared to jRGECO1a. Our findings highlight the potential of SomaFRCaMPi, comparable to the most sensitive soma-targeted GCaMP, for precise spatial recording of neuronal populations using popular imaging modalities in model organisms such as zebrafish and mice
NeMeHg, genetically encoded indicator for mercury ions based on mNeonGreen green fluorescent protein and merP protein from Shigella flexneri
No full textThe detection of mercury ions is an important task in both environmental monitoring and cell biology research. However, existing genetically encoded sensors for mercury ions have certain limitations, such as negative fluorescence response, narrow dynamic range, or the need for cofactor supplementation. To address these limitations, we have developed novel sensors by fusing a circularly permutated version of the mNeonGreen green fluorescent protein with the merP mercury-binding protein from Gram-negative bacteria Shigella flexneri. The developed NeMeHg and iNeMeHg sensors responded to mercury ions with positive and negative fluorescence changes, respectively. We characterized their properties in vitro. Using the developed biosensors, we were able to successfully visualize changes in mercury ion concentration in mammalian cultured cells
Advances in Engineering and Application of Optogenetic Indicators for Neuroscience
No full textOur ability to investigate the brain is limited by available technologies that can record biological processes in vivo with suitable spatiotemporal resolution. Advances in optogenetics now enable optical recording and perturbation of central physiological processes within the intact brains of model organisms. By monitoring key signaling molecules noninvasively, we can better appreciate how information is processed and integrated within intact circuits. In this review, we describe recent efforts engineering genetically-encoded fluorescence indicators to monitor neuronal activity. We summarize recent advances of sensors for calcium, potassium, voltage, and select neurotransmitters, focusing on their molecular design, properties, and current limitations. We also highlight impressive applications of these sensors in neuroscience research. We adopt the view that advances in sensor engineering will yield enduring insights on systems neuroscience. Neuroscientists are eager to adopt suitable tools for imaging neural activity in vivo, making this a golden age for engineering optogenetic indicators. Keywords: optogenetic tools; neuroscience; calcium sensor; voltage sensor; neurotransmitter
Effects of Sputtered Platinum Counter Electrode and Integrated TiO2 Electrode with SWCNT on DSSC Performance
No full textabstract: Dye sensitized solar cells (DSSCs) are the third generation solar cells expected to outperform the first two generations of solar cells with their advantages of comparative higher efficiency and lower manufacturing costs. The manufacturing cost of Dye sensitized solar cells is one fifth of the conventional silicon solar cell. However, DSSCs have problems of low conversion efficiency, stability and reliability. Some effective approaches are required to improve their performance. This paper projects the work related to assessment and verification of the repeatability of the semi-automated fabrication process. Changes were introduced in to the fabrication process to enhance the efficiency and stability. The sealant step in the fabrication process was remodeled to a newer version with an improvement in efficiency from 11% to 11.8%. Sputtering was performed on counter electrode in 30 seconds intervals. Cells were fabricated to assess the performance & time dependent characteristics from EIS experiments. Series resistance increased three times in sputtered Pt electrode as compared to standard platinum electrode. This resulted in the degradation of conductive surface on glass electrode due to heavy bombardment of ions. The second phase of the project work relates to the incorporation of SWCNT on the TiO2 electrode and its effect on the cell efficiency. Different weight loadings (0.1 wt %, 0.2 wt%, 0.4 wt %) of SWCNTs were prepared and mixed with the commercial TiO2 paste and ethanol solvent. The TiO2-SWCNT layer was coated on the electrode using screen-printing technique. Both open circuit voltage and photocurrent were found to have measurable dependence on the TiO2 layer loading. Photo voltage ranged from ~0.73 V to ~0.43 V and photocurrent from ~8 to ~33 mA depending on weight percent loading. This behavior is due to aggregation of particles and most TiO2 aggregate particles are not connected to SWCNT. Transparency loss was observed leading to saturation in the photo current and limiting the light absorption within the TiO2 film.Dissertation/ThesisM.S.Tech Technology 201
Near-Infrared Genetically Encoded Positive Calcium Indicator Based on GAF-FP Bacterial Phytochrome
No full textA variety of genetically encoded calcium indicators are currently available for visualization of calcium dynamics in cultured cells and in vivo. Only one of them, called NIR-GECO1, exhibits fluorescence in the near-infrared region of the spectrum. NIR-GECO1 is engineered based on the near-infrared fluorescent protein mIFP derived from bacterial phytochromes. However, NIR-GECO1 has an inverted response to calcium ions and its excitation spectrum is not optimal for the commonly used 640 nm lasers. Using small near-infrared bacterial phytochrome GAF-FP and calmodulin/M13-peptide pair, we developed a near-infrared calcium indicator called GAF-CaMP2. In vitro, GAF-CaMP2 showed a positive response of 78% and high affinity (Kd of 466 nM) to the calcium ions. It had excitation and emission maxima at 642 and 674 nm, respectively. GAF-CaMP2 had a 2.0-fold lower brightness, 5.5-fold faster maturation and lower pH stability compared to GAF-FP in vitro. GAF-CaMP2 showed 2.9-fold higher photostability than smURFP protein. The GAF-CaMP2 fusion with sfGFP demonstrated a ratiometric response with a dynamic range of 169% when expressed in the cytosol of mammalian cells in culture. Finally, we successfully applied the ratiometric version of GAF-CaMP2 for the simultaneous visualization of calcium transients in three organelles of mammalian cells using four-color fluorescence microscopy
Advances in Engineering and Application of Optogenetic Indicators for Neuroscience
No full textOur ability to investigate the brain is limited by available technologies that can record biological processes in vivo with suitable spatiotemporal resolution. Advances in optogenetics now enable optical recording and perturbation of central physiological processes within the intact brains of model organisms. By monitoring key signaling molecules noninvasively, we can better appreciate how information is processed and integrated within intact circuits. In this review, we describe recent efforts engineering genetically-encoded fluorescence indicators to monitor neuronal activity. We summarize recent advances of sensors for calcium, potassium, voltage, and select neurotransmitters, focusing on their molecular design, properties, and current limitations. We also highlight impressive applications of these sensors in neuroscience research. We adopt the view that advances in sensor engineering will yield enduring insights on systems neuroscience. Neuroscientists are eager to adopt suitable tools for imaging neural activity in vivo, making this a golden age for engineering optogenetic indicators
ChemInform Abstract: Engineering of Bacterial Phytochromes for Near‐Infrared Imaging, Sensing, and Light‐Control in Mammals
No full textCorrection: Piatkevich et al. Advances in Engineering and Application of Optogenetic Indicators for Neuroscience. Appl. Sci. 2019, 9, 562
No full textWe, the authors, wish to make the following corrections to our paper [...
