Destabilized near-infrared fluorescent nanobodies enable background-free targeting of GFP-based biosensors for imaging and manipulation.

Near-infrared (NIR) probes are highly sought after as fluorescent tags for multicolor cellular and in vivo imaging. Here we develop small NIR fluorescent nanobodies, termed NIR-Fb and NIR-Fb, enabling background-free visualization of various GFP-derived probes and biosensors. We also design a red-shifted variant, NIR-Fb, to simultaneously target several antigens within the NIR spectral range.

Technologies for large-scale mapping of functional neural circuits active during a user-defined time window.

The mapping of neural circuits activated during behavior down to individual neurons is crucial for decoding how the brain processes information. Technologies allowing activity-dependent labeling of neurons during user-defined restricted time windows are rapidly developing. Precise marking of the time window with light, in addition to chemicals, is now possible.

FRCaMP, a Red Fluorescent Genetically Encoded Calcium Indicator Based on Calmodulin from Schizosaccharomyces Pombe Fungus.

Red fluorescent genetically encoded calcium indicators (GECIs) have expanded the available pallet of colors used for the visualization of neuronal calcium activity in vivo. However, their calcium-binding domain is restricted by calmodulin from metazoans. In this study, we developed red GECI, called FRCaMP, using calmodulin (CaM) from fungus as a calcium binding domain.

FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity.

Genetically encoded calcium indicators (GECIs) have become a widespread tool for the visualization of neuronal activity. As compared to popular GCaMP GECIs, the FGCaMP indicator benefits from calmodulin and M13-peptide from the fungi and , which prevent its interaction with the intracellular environment. However, FGCaMP exhibits a two-phase fluorescence behavior with the variation of calcium ion concentration, has moderate sensitivity in neurons (as compared to the GCaMP6s indicator), and has not been fully characterized in vitro and in vivo.

Novel Genetically Encoded Bright Positive Calcium Indicator NCaMP7 Based on the mNeonGreen Fluorescent Protein.

Green fluorescent genetically encoded calcium indicators (GECIs) are the most popular tool for visualization of calcium dynamics in vivo. However, most of them are based on the EGFP protein and have similar molecular brightnesses. The NTnC indicator, which is composed of the mNeonGreen fluorescent protein with the insertion of troponin C, has higher brightness as compared to EGFP-based GECIs, but shows a limited inverted response with an ΔF/F of 1.

Click histochemistry for whole-mount staining of brain structures.

Labeling of the replicating DNA with synthetic thymidine analogs is commonly used for marking the dividing cells. However, until now this method has only been applied to histological sections. A growing number of current approaches for three-dimensional visualization of large tissue samples requires detection of dividing cells within whole organs.

Near-Infrared Genetically Encoded Positive Calcium Indicator Based on GAF-FP Bacterial Phytochrome.

A 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.

Slowly Reducible Genetically Encoded Green Fluorescent Indicator for In Vivo and Ex Vivo Visualization of Hydrogen Peroxide.

Hydrogen peroxide (HO) plays an important role in modulating cell signaling and homeostasis in live organisms. The HyPer family of genetically encoded indicators allows the visualization of HO dynamics in live cells within a limited field of view. The visualization of HO within a whole organism with a single cell resolution would benefit from a slowly reducible fluorescent indicator that integrates the HO concentration over desired time scales.

Suppressed neurogenesis without cognitive deficits: effects of fast neutron irradiation in mice.

This study assessed the effects of combined low-dose neutron and γ-ray irradiation on hippocampal neurogenesis and hippocampal-dependent memory. Neural progenitor cell division and survival were evaluated in brain sections and whole hippocampal preparations following head irradiation at a dose of 0.34 Gy for neutron radiation and 0.

The whither of bacteriophytochrome-based near-infrared fluorescent proteins: Insights from two-photon absorption spectroscopy.

We present one- and two-photon-absorption fluorescence spectroscopic analysis of biliverdin (BV) chromophore-based single-domain near-infrared fluorescent proteins (iRFPs). The results of these studies are used to estimate the internal electric fields acting on BV inside iRFPs and quantify the electric dipole properties of this chromophore, defining the red shift of excitation and emission spectra of BV-based iRFPs. The iRFP studied in this work is shown to fit well the global diagram of the red-shift tunability of currently available BV-based iRFPs as dictated by the quadratic Stark effect, suggesting the existence of the lower bound for the strongest red shifts attainable within this family of fluorescent proteins.

NTnC-like genetically encoded calcium indicator with a positive and enhanced response and fast kinetics.

The NTnC genetically encoded calcium indicator has an advantageous design because of its smaller size, GFP-like N- and C-terminal ends and two-fold reduced number of calcium binding sites compared with widely used indicators from the GCaMP family. However, NTnC has an inverted and modest calcium response and a low temporal resolution. By replacing the mNeonGreen fluorescent part in NTnC with EYFP, we engineered an NTnC-like indicator, referred to as YTnC, that had a positive and substantially improved calcium response and faster kinetics.

Green fluorescent genetically encoded calcium indicator based on calmodulin/M13-peptide from fungi.

Currently available genetically encoded calcium indicators (GECIs) utilize calmodulins (CaMs) or troponin C from metazoa such as mammals, birds, and teleosts, as calcium-binding domains. The amino acid sequences of the metazoan calcium-binding domains are highly conserved, which may limit the range of the GECI key parameters and cause undesired interactions with the intracellular environment in mammalian cells. Here we have used fungi, evolutionary distinct organisms, to derive CaM and its binding partner domains and design new GECI with improved properties.

A new design for a green calcium indicator with a smaller size and a reduced number of calcium-binding sites.

Genetically encoded calcium indicators (GECIs) are mainly represented by two- or one-fluorophore-based sensors. One type of two-fluorophore-based sensor, carrying Opsanus troponin C (TnC) as the Ca-binding moiety, has two binding sites for calcium ions, providing a linear response to calcium ions. One-fluorophore-based sensors have four Ca-binding sites but are better suited for in vivo experiments.