A luciferase prosubstrate and a red bioluminescent calcium indicator to image neuronal activity

Genetically encoded fluorescent indicators have been broadly used to monitor neuronal activity in live animals, but invasive surgical procedures are required. This study presents a functional bioluminescence imaging (fBLI) method for recording the activity of neuronal ensembles in the brain in awake mice. We developed a luciferase prosubstrate activatable in vivo by nonspecific esterase to enhance the brain delivery of the luciferin. We further engineered a bright, bioluminescent indicator with robust responsiveness to calcium ions (Ca2+) and appreciable emission above 600 nm. Integration of these advantageous components enabled the imaging of Ca2+ dynamics in awake mice minimally invasively with excellent signal- to-background and subsecond temporal resolution. This study thus establishes a new paradigm for studying brain functions in health and disease.

Fluorescent Indicators for Live-Cell and in Vitro Detection of Inorganic Cadmium Dynamics

Cadmium contamination is a severe threat to the environment and food safety. Thus, there is an urgent need to develop highly sensitive and selective cadmium detection tools. The engineered fluorescent indicator is a powerful tool for the rapid detection of inorganic cadmium in the environment. In this study, the development of yellow fluorescent indicators of cadmium chloride by inserting a fluorescent protein at different positions of the high cadmium-specific repressor and optimizing the flexible linker between the connection points is reported. These indicators provide a fast, sensitive, specific, high dynamic range, and real-time readout of cadmium ion dynamics in solution. Under optimal conditions, the fluorescent indicators N0C0/N1C1 showed a linear response to cadmium concentration within the range from 10/30 to 50/100 nM and with a detection limit of 10/33 nM. Escherichia coli cells containing the indicator were used to further study the response of cadmium ion concentration in living cells. E. coli N1C1 could respond to different concentrations of cadmium ions. This study provides a rapid and straightforward method for cadmium ion detection in vitro and the potential for biological imaging.

Fluorescent and bioluminescent calcium indicators with tuneable colors and affinities

We introduce a family of bright, rhodamine-based calcium indicators with tuneable affinities and colors. The indicators can be specifically localized to different cellular compartments and are compatible with both fluorescence and bioluminescence readouts through conjugation to HaloTag fusion proteins. Importantly, their increase in fluorescence upon localization enables no-wash live-cell imaging, which greatly facilitates their use in biological assays. Applications as fluorescent indicators in rat hippocampal neurons include the detection of single action potentials and of calcium fluxes in the endoplasmic reticulum (ER). Applications as bioluminescent indicators include the recording of the pharmacological modulation of nuclear calcium in high-throughput-compatible assays. The versatility and remarkable ease of use of these indicators make them powerful tools for bioimaging and bioassays.

Stop the beat to see the rhythm: excitation-contraction uncoupling in cardiac research

Optical mapping is an imaging technique that is extensively used in cardiovascular research, wherein parameter-sensitive fluorescent indicators are used to study the electrophysiology and excitation-contraction coupling of cardiac tissues. Despite the many benefits of optical mapping, eliminating motion artifacts within the optical signals is a major challenge, as myocardial contraction interferes with the faithful acquisition of action potentials and intracellular calcium transients. As such, excitation-contraction uncoupling agents are frequently used to reduce signal distortion by suppressing contraction. Compared to other uncoupling agents, blebbistatin is the most frequently used as it offers increased potency with minimal direct effects on cardiac electrophysiology. Nevertheless, blebbistatin may exert secondary effects on electrical activity, metabolism, and coronary flow, and the incorrect administration of blebbistatin to cardiac tissue can prove detrimental, resulting in erroneous interpretation of optical mapping results. In this "Getting It Right" perspective, we briefly review the literature regarding the use of blebbistatin in cardiac optical mapping experiments, highlight potential secondary effects of blebbistatin on cardiac electrical activity and metabolic demand, and conclude with the consensus of the authors on best practices for effectively using blebbistatin in optical mapping studies of cardiac tissue.

Fluorescent Analysis Reveals that Insulin Diminishes Superoxide Increase in Neurons Induced with Toxic Glutamate Treatment

Glutamate excitotoxicity is implicated in the pathogenesis of many disorders, including stroke, traumatic brain injury, and Alzheimer’s disease, for which central insulin resistance is a comorbid condition. Massive glutamate release primarily through ionotropic N-methyl-D-aspartate receptors (NMDARs) causes a sustained rise in [Ca2+]i, followed by mitochondrial depolarization and an increase in intracellular O2• (superoxide) production. Recently, we found that insulin protected neurons against excitotoxicity by diminishing the delayed calcium deregulation (DCD), However, a role of insulin in superoxide production in excitotoxicity still needs to be clarified. The present study is aimed to investigate the effects of insulin on glutamate-evoked superoxide generation and DCD using the fluorescent indicators dihydroethidium, MitoSOX Red, and Fura-FF in rats cultured cortical neurons. We found that insulin significantly diminished both the intracellular and mitochondrial superoxide production in neurons exposed to glutamate and there was a strong linear correlation between [Ca2+]i and intracellular superoxide. MK 801, an inhibitor of NMDAR-gated Ca2+ influx, completely abrogated the glutamate effects in both the presence and absence of insulin. In experiments on sister cultures, insulin diminishes neuronal death. Thus, collectively, data obtained suggest that insulin diminishes glutamate-induced superoxide production in neurons via fall of [Ca2+]i increased and thereby improves viability of neurons

One-Step Synthesis of Ribonucleases A–Ag Nanocomposites as Fluorescent Nanodrugs for in vivo Delivery

Proteins and enzymes with potential therapeutic function have been widely used in drug delivery. Meanwhile, nanocrystals (NCs) have showed their advantage as in vivo fluorescent indicators. Combining of these two kinds of materials can result in multifunctional composites. Here, a one-step synthesis of protein–NCs nanocomposites with near infrared (NIR) fluorescence and efficient therapeutic function was developed. Ag NCs were formed using ribonucleases A (RNase A) enzyme as a stabilizer. Meanwhile, the RNase A with long chain rapped around Ag NCs and formed RNase A–Ag nanocomposites. The nanocomposites remained both the NIR fluorescence of Ag NCs and antitumor activity of RNase A. The inhibition of the growth of tumor cells by the nanocomposites was confirmed. This study developed an efficient drug delivery system through combination of the protein and nanoparticles, which had potential clinical application in the future.

Azo-Dyes-Grafted Oligosaccharides—From Synthesis to Applications

Azobenzenes are photochromic molecules that possess a large range of applications. Their syntheses are usually simple and fast, and their purifications can be easy to perform. Oligosaccharide is also a wide family of biopolymer constituted of linear chain of saccharides. It can be extracted from biomass, as for cellulose, being the principal constituent of plant cell wall, or it can be enzymatically produced as for cyclodextrins, having properties not far from cellulose. Combining these two materials families can afford interesting applications such as controlled drug-release systems, photochromic liquid crystals, photoresponsive films or even fluorescent indicators. This review will compile the different syntheses of azo-dyes-grafted oligosaccharides, and will show their various applications.

FLIM Indicators for Quantitative Measurement of pH

Monitoring of intracellular pH changes in situ can provide valuable information about cellular metabolism and a deeper understanding of physiological processes. Most traditional fluorescent indicators are only capable of a relative assessment of changes in the studied parameter in the cell. We associate the possibility of measuring the absolute values that characterize the analyte with the detection of the indicator signal in the time domain, where its quantitative measure is the fluorescence lifetime (tau). In this project, we test promising pH-sensitive fluorophores with labile fluorescence lifetimes—EYFP-G65T and EGFP-Y145L/S205V—both as fluorescent cores for the previously described pH indicators and as independent pH indicators. Measurement of the fluorescence attenuation kinetics of four structures (EYFP-G65T, EGFP-Y145L/S205V, SypHer3s, and SypHer3s-G65T) over a wide pH range revealed areas where tau is linearly dependent on pH. The differences in the fluorescence excitation modes of these molecules makes it possible to use them in one experimental system to assess pH changes in a wide range, 4.0–9.0. We showed that under the conditions of traditional fluorescence microscopy (in the cytoplasm of HEK293 cells), the SypHer3s-G65T indicator shows a dynamic response range approximately 3 times wider than the original SypHer3s.