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Perineuronal nets (PNNs), a specialized form of extra cellular matrix (ECM), surround numerous neurons in the CNS and allow synaptic connectivity through holes in its structure. We hypothesize that PNNs serve as gatekeepers that guard and protect synaptic territory and thus may stabilize an engram circuit. We present high-resolution and 3D EM images of PNN-engulfed neurons in mice brains, showing that synapses occupy the PNN holes and that invasion of other cellular components is rare. PNN constituents in mice brains are long-lived and can be eroded faster in an enriched environment, while synaptic proteins have a high turnover rate. Preventing PNN erosion by using pharmacological inhibition of PNN-modifying proteases or matrix metalloproteases 9 (MMP9) knockout mice allowed normal fear memory acquisition but diminished long-term memory stabilization, supporting the above hypothesis. [ABSTRACT FROM AUTHOR]

The technique of colour EM that was recently developed enabled localisation of specific macromolecules/proteins of interest by the targeted deposition of diaminobenzidine (DAB) conjugated to lanthanide chelates. By acquiring lanthanide elemental maps by energy‐filtered transmission electron microscopy (EFTEM) and overlaying them in pseudo‐colour over the conventional greyscale TEM image, a colour EM image is generated. This provides a powerful tool for visualising subcellular component/s, by the ability to clearly distinguish them from the general staining of the endogenous cellular material. Previously, the lanthanide elemental maps were acquired at the high‐loss M4,5 edge (excitation of 3d electrons), where the characteristic signal is extremely low and required considerably long exposures. In this paper, we explore the possibility of acquiring the elemental maps of lanthanides at their N4,5 edge (excitation of 4d electrons), which occurring at a much lower energy‐loss regime, thereby contains significantly greater total characteristic signal owing to the higher inelastic scattering cross‐sections at the N4,5 edge. Acquiring EFTEM lanthanide elemental maps at the N4,5 edge instead of the M4,5 edge, provides ∼4× increase in signal‐to‐noise and ∼2× increase in resolution. However, the interpretation of the lanthanide maps acquired at the N4,5 edge by the traditional 3‐window method, is complicated due to the broad shape of the edge profile and the lower signal‐above‐background ratio. Most of these problems can be circumvented by the acquisition of elemental maps with the more sophisticated technique of EFTEM Spectrum Imaging (EFTEM SI). Here, we also report the chemical synthesis of novel second‐generation DAB lanthanide metal chelate conjugates that contain 2 lanthanide ions per DAB molecule in comparison with 0.5 lanthanide ion per DAB in the first generation. Thereby, fourfold more Ln3+ per oxidised DAB would be deposited providing significant amplification of signal. This paper applies the colour EM technique at the intermediate‐loss energy‐loss regime to three different cellular targets, namely using mitochondrial matrix‐directed APEX2, histone H2B‐Nucleosome and EdU‐DNA. All the examples shown in the paper are single colour EM images only. [ABSTRACT FROM AUTHOR]

Using mRNA sequencing and de novo transcriptome assembly, we identified, cloned, and characterized 9 previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from A. victoria green fluorescent protein (avGFP). Among these FPs are the brightest green fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including 2 that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing. Aequorea victoria, the jellyfish that yielded GFP and transformed the study of living cells, held more secrets than first suspected. This study reveals that A. victoria and its Australian cousin encode a diverse set of unusual fluorescent proteins, including the brightest one ever described. [ABSTRACT FROM AUTHOR]

Purpose: A bottleneck in developing cell therapies for cancer is assaying cell biodistribution, persistence, and survival in vivo. Ex vivo cell labeling using perfluorocarbon (PFC) nanoemulsions, paired with 19F MRI detection, is a non‐invasive approach for cell product detection in vivo. Lymphocytes are small and weakly phagocytic limiting PFC labeling levels and MRI sensitivity. To boost labeling, we designed PFC nanoemulsion imaging probes displaying a cell‐penetrating peptide, namely the transactivating transcription sequence (TAT) of the human immunodeficiency virus. We report optimized synthesis schemes for preparing TAT co‐surfactant to complement the common surfactants used in PFC nanoemulsion preparations. Methods: We performed ex vivo labeling of primary human chimeric antigen receptor (CAR) T cells with nanoemulsion. Intracellular labeling was validated using electron microscopy and confocal imaging. To detect signal enhancement in vivo, labeled CAR T cells were intra‐tumorally injected into mice bearing flank glioma tumors. Results: By incorporating TAT into the nanoemulsion, a labeling efficiency of ~1012 fluorine atoms per CAR T cell was achieved that is a >8‐fold increase compared to nanoemulsion without TAT while retaining high cell viability (~84%). Flow cytometry phenotypic assays show that CAR T cells are unaltered after labeling with TAT nanoemulsion, and in vitro tumor cell killing assays display intact cytotoxic function. The 19F MRI signal detected from TAT‐labeled CAR T cells was 8 times higher than cells labeled with PFC without TAT. Conclusion: The peptide‐PFC nanoemulsion synthesis scheme presented can significantly enhance cell labeling and imaging sensitivity and is generalizable for other targeted imaging probes. [ABSTRACT FROM AUTHOR]

[super 1]H NMR spectroscopy is used for determining the solution structure of the complex formed between the peptide and the fluorophore ReAsH moiety. The biarsenical-tetracysteine motif is used for genetic labeling of proteins with small molecules and the studies have shown that the backbone structure of the peptide is well defined, with a hairpinline turn formed by the central CPGCC segment.

The kinetic and dissociation constants for FIAsH, a membrane-permanent fluorescein derivative with two As(III) substituents, binding to model tetracysteine peptides are investigated. Affinities in vitro and detection limits in living cells suggest that the preferred peptide conformation is a hairpin instead of alpha-helix.

Fluorescence is enhanced by cloning and transcribing malachite green (MG) aptamer sequences in tandem, revealing concatenate aptamers result in increased levels of fluorescence without quenching one another. The specificity of MG and Sulforhodamine B aptamers for different sets of dyes suggests that it could be possible t discriminate multiple RNAs simultaneously.

Summary Electron microscopy (EM) remains the primary method for imaging cellular and tissue ultrastructure, although simultaneous localization of multiple specific molecules continues to be a challenge for EM. We present a method for obtaining multicolor EM views of multiple subcellular components. The method uses sequential, localized deposition of different lanthanides by photosensitizers, small-molecule probes, or peroxidases. Detailed view of biological structures is created by overlaying conventional electron micrographs with pseudocolor lanthanide elemental maps derived from distinctive electron energy-loss spectra of each lanthanide deposit via energy-filtered transmission electron microscopy. This results in multicolor EM images analogous to multicolor fluorescence but with the benefit of the full spatial resolution of EM. We illustrate the power of this methodology by visualizing hippocampal astrocytes to show that processes from two astrocytes can share a single synapse. We also show that polyarginine-based cell-penetrating peptides enter the cell via endocytosis, and that newly synthesized PKMζ in cultured neurons preferentially localize to the postsynaptic membrane. [ABSTRACT FROM AUTHOR]

In Echtzeit und ratiometrisch kann die Thrombin ‐ Aktivierung in vivo mithilfe aktivierbarer zellgängiger Peptide (RACPPs) verfolgt werden. RACPPs verbinden die 1) Ermittlung des Verhältnisses zweier Emissionen, 2) In ‐ vivo ‐ Bildgebung in Säugern im fernen Rot ‐ bis Infrarotbereich und 3) spaltungsabhängige räumliche Lokalisierung. Das beste RACPP nutzt Norleucin(Nle) ‐ TPRSFL als Linker, was die Empfindlichkeit für Thrombin um das 90 ‐ Fache erhöht (siehe Bild). [ABSTRACT FROM AUTHOR]

Intracellular Ca2+ regulates numerous proteins and cellular functions and can vary substantially over submicron and submillisecond scales, so precisely localized fast detection is desirable. We have created a ∼1-kDa biarsenical Ca2+ indicator, called Calcium Green FlAsH (CaGF, 1), to probe [Ca2+] surrounding genetically targeted proteins. CaGF attached to a tetracysteine motif becomes ten-fold more fluorescent upon binding Ca2+, with a Kd of ∼100 μM, <1-ms kinetics and good Mg2+ rejection. In HeLa cells expressing tetracysteine-tagged connexin 43, CaGF labels gap junctions and reports Ca2+ waves after injury. Total internal reflection microscopy of tetracysteine-tagged, CaGF-labeled α1C L-type calcium channels shows fast-rising depolarization-evoked Ca2+ transients, whose lateral nonuniformity suggests that the probability of channel opening varies greatly over micron dimensions. With moderate Ca2+ buffering, these transients decay surprisingly slowly, probably because most of the CaGF signal comes from closed channels feeling Ca2+ from a tiny minority of clustered open channels. With high Ca2+ buffering, CaGF signals decay as rapidly as the calcium currents, as expected for submicron Ca2+ domains immediately surrounding active channels. Thus CaGF can report highly localized, rapid [Ca2+] dynamics. [ABSTRACT FROM AUTHOR]