Cortex-wide transcranial localization microscopy with fluorescently labeled red blood cells.

Large-scale imaging of brain activity with high spatio-temporal resolution is crucial for advancing our understanding of brain function. The existing neuroimaging techniques are largely limited by restricted field of view, slow imaging speed, or otherwise do not have the adequate spatial resolution to capture brain activities on a capillary and cellular level. To address these limitations, we introduce fluorescence localization microscopy aided with sparsely-labeled red blood cells for cortex-wide morphological and functional cerebral angiography with 4.9 µm spatial resolution and 1 s temporal resolution. When combined with fluorescence calcium imaging, the proposed method enables extended recordings of stimulus-evoked neuro-vascular changes in the murine brain while providing simultaneous multiparametric readings of intracellular neuronal activity, blood flow velocity/direction/volume, and vessel diameter. Owing to its simplicity and versatility, the proposed approach will become an invaluable tool for deciphering the regulation of cortical microcirculation and neurovascular coupling in health and disease.

Amplified Assembly of G-Quadruplex-Decorated DNA Network Nanostructure toward AIE Signaling-Based Sensitive Biosensing.

Aggregation-induced emission (AIE) has offered a promising approach for developing low-background fluorescent methods; however, its applications often suffer from complex probe synthesis and poor biocompatibility. Herein, a novel AIE biosensing method for kanamycin antibiotic assays was developed by utilizing a DNA network nanostructure assembled from an aptamer recognition reaction to capture a large number of tetraphenylethylene fluorogen-labeled signal DNA (DTPE) probes. Due to the excellent hydrophilicity of the oligonucleotides, DTPE exhibited excellent water solubility without obvious background signal emission. Based on an ingenious nucleotide design, an abundance of G-quadruplex blocks neighboring the captured DTPE were formed on the DNA nanostructure. Because of the greatly restricted free motion of DTPE by this unique nanostructure, a strong AIE fluorescence signal response was produced to construct the signal transduction strategy. Together with target recycling and rolling circle amplification-based cascade nucleic acid amplification, this method exhibited a wide linear range from 75 fg mL-1 to 1 ng mL-1 and a detection limit down to 24 fg mL-1. The excellent analytical performance and effective manipulation improvement of the method over previous approaches determine its promising potential for various applications.

Fluorescent Protein-Based Sensors for Detecting Essential Metal Ions across the Tree of Life.

Genetically encoded fluorescent metal ion sensors are powerful tools for elucidating metal dynamics in living systems. Over the last 25 years since the first examples of genetically encoded fluorescent protein-based calcium indicators, this toolbox of probes has expanded to include other essential and non-essential metal ions. Collectively, these tools have illuminated fundamental aspects of metal homeostasis and trafficking that are crucial to fields ranging from neurobiology to human nutrition. Despite these advances, much of the application of metal ion sensors remains limited to mammalian cells and tissues and a limited number of essential metals. Applications beyond mammalian systems and in vivo applications in living organisms have primarily used genetically encoded calcium ion sensors. The aim of this Perspective is to provide, with the support of historical and recent literature, an updated and critical view of the design and use of fluorescent protein-based sensors for detecting essential metal ions in various organisms. We highlight the historical progress and achievements with calcium sensors and discuss more recent advances and opportunities for the detection of other essential metal ions. We also discuss outstanding challenges in the field and directions for future studies, including detecting a wider variety of metal ions, developing and implementing a broader spectral range of sensors for multiplexing experiments, and applying sensors to a wider range of single- and multi-species biological systems.

Ratiometric determination of etomidate based on an albumin-based indicator displacement assay (IDA).

The first fluorescent sensor based on the indicator displacement assay (IDA) for on-site determination of etomidate.

Force-controlled release of small molecules with a rotaxane actuator.

Force-controlled release of small molecules offers great promise for the delivery of drugs and the release of healing or reporting agents in a medical or materials context1-3. In polymer mechanochemistry, polymers are used as actuators to stretch mechanosensitive molecules (mechanophores)4. This technique has enabled the release of molecular cargo by rearrangement, as a direct5,6 or indirect7-10 consequence of bond scission in a mechanophore, or by dissociation of cage11, supramolecular12 or metal complexes13,14, and even by 'flex activation'15,16. However, the systems described so far are limited in the diversity and/or quantity of the molecules released per stretching event1,2. This is due to the difficulty in iteratively activating scissile mechanophores, as the actuating polymers will dissociate after the first activation. Physical encapsulation strategies can be used to deliver a larger cargo load, but these are often subject to non-specific (that is, non-mechanical) release3. Here we show that a rotaxane (an interlocked molecule in which a macrocycle is trapped on a stoppered axle) acts as an efficient actuator to trigger the release of cargo molecules appended to its axle. The release of up to five cargo molecules per rotaxane actuator was demonstrated in solution, by ultrasonication, and in bulk, by compression, achieving a release efficiency of up to 71% and 30%, respectively, which places this rotaxane device among the most efficient release systems achieved so far1. We also demonstrate the release of three representative functional molecules (a drug, a fluorescent tag and an organocatalyst), and we anticipate that a large variety of cargo molecules could be released with this device. This rotaxane actuator provides a versatile platform for various force-controlled release applications.

Multiparameter Sensing of Oxygen and pH at Biological Interfaces via Hyperspectral Imaging of Luminescent Sensor Nanoparticles.

Chemical dynamics in biological samples are seldom stand-alone processes but represent the outcome of complicated cascades of interlinked reaction chains. In order to understand these processes and how they correlate, it is important to monitor several parameters simultaneously at high spatial and temporal resolution. Hyperspectral imaging is a promising tool for this, as it provides broad-range spectral information in each pixel, enabling the use of multiple luminescent indicator dyes, while simultaneously providing information on sample structures and optical properties. In this study, we first characterized pH- and O2-sensitive indicator dyes incorporated in different polymer matrices as optical sensor nanoparticles to provide a library for (hyperspectral) chemical imaging. We then demonstrate the successful combination of a pH-sensitive indicator dye (HPTS(DHA)3), an O2-sensitive indicator dye (PtTPTBPF), and two reference dyes (perylene and TFPP), incorporated in polymer nanoparticles for multiparameter chemical imaging of complex natural samples such as green algal biofilms (Chlorella sorokiniana) and seagrass leaves (Zostera marina) with high background fluorescence. We discuss the system-specific challenges and limitations of our approach and further optimization possibilities. Our study illustrates how multiparameter chemical imaging with hyperspectral read-out can now be applied on natural samples, enabling the alignment of several chemical parameters to sample structures.

Click Chemistry with Cell-Permeable Fluorophores Expands the Choice of Bioorthogonal Markers for Two-Color Live-Cell STED Nanoscopy.

STED nanoscopy allows for the direct observation of dynamic processes in living cells and tissues with diffraction-unlimited resolution. Although fluorescent proteins can be used for STED imaging, these labels are often outperformed in photostability by organic fluorescent dyes. This feature is especially crucial for time-lapse imaging. Unlike fluorescent proteins, organic fluorophores cannot be genetically fused to a target protein but require different labeling strategies. To achieve simultaneous imaging of more than one protein in the interior of the cell with organic fluorophores, bioorthogonal labeling techniques and cell-permeable dyes are needed. In addition, the fluorophores should preferentially emit in the red spectral range to reduce the potential phototoxic effects that can be induced by the STED light, which further restricts the choice of suitable markers. In this work, we selected five different cell-permeable organic dyes that fulfill all of the above requirements and applied them for SPIEDAC click labeling inside living cells. By combining click-chemistry-based protein labeling with other orthogonal and highly specific labeling methods, we demonstrate two-color STED imaging of different target structures in living specimens using different dye pairs. The excellent photostability of the dyes enables STED imaging for up to 60 frames, allowing the observation of dynamic processes in living cells over extended time periods at super-resolution.

Dual-functional porphyrinic zirconium-based metal-organic framework for the fluorescent sensing of histidine enantiomers and Hg2.

A novel fluorescence sensor based on a porphyrinic zirconium-based metal-organic framework, L-cysteine-modified PCN-222 (L-Cys/PCN-222), was developed to selectively recognize histidine enantiomers and sensitively detect Hg2+. The dual-functional sensor was successfully prepared via the solvent-assisted ligand incorporation method and characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), 1H nuclear magnetic resonance (1H NMR) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD), X-ray photoelectron spectroscopy (XPS), and nitrogen adsorption-desorption analyses. L-Cys/PCN-222 not only showed a higher quenching response for L-histidine than that for D-histidine with a fast fluorescent response rate of <40 s but also exhibited low detection limits for L- and D-histidine (2.48 µmol L-1 and 3.85 µmol L-1, respectively). Moreover, L-Cys/PCN-222 was employed as a fluorescent and visual sensor for the highly sensitive detection of Hg2+ in the linear range of 10-500 µmol L-1, and the detection limit was calculated to be 2.79 µmol L-1 in surface water. The specific and selective recognition of chiral compounds and metal ions by our probe make it suitable for real field applications.

Construction of a novel flavonol fluorescent probe for copper (II) ion detection and its application in actual samples.

Copper is an essential trace element in the human body, and its level is directly related to many diseases. While the source of copper in human body is mainly intake from food, then the detection of copper ions (Cu2+) in food becomes crucial. Here, we synthesized a novel probe (E)-3-hydroxy-2-styryl-4H-benzo[h]chromen-4-one (NSHF) and explored the binding ability of NSHF for Cu2+ using nuclear magnetic resonance hydrogen spectroscopy (1H NMR), high-resolution mass spectrometry (HRMS), Job's plot method and density functional theory (DFT). NSHF shows the advantages of fast response time, good selectivity and high sensitivity for Cu2+. The fluorescence intensity ratio (F/F0) of NSHF shows a good linear relationship with the concentration of Cu2+ and the detection limit is 0.061 µM. NSHF was successfully applied to the detection of Cu2+ in real samples. In addition, a simple and convenient Cu2+ detection platform was constructed by combining NSHF with a smartphone and a UV lamp, which can realize the rapid detection of Cu2+. This work provides an effective tool for the real-time detection of Cu2+.

A combined experimental and theoretical approach for doxycycline sensing using simple fluorescent probe with distinct fluorescence change in wide range of interferences.

Overuse of doxycycline (DOXY) can cause serious problems to human health, environment and food quality. So, it is essential to develop a new sensing methodology that is both sensitive and selective for the quantitative detection of DOXY. In our current research, we synthesized a simple fluorescent probe 4,4'-bis(benzyloxy)-1,1'-biphenyl (BBP) for the highly selective detection of doxycycline by through fluorescence spectroscopy. The probe BBP displayed ultra-sensitivity towards doxycycline due to Forster resonance energy transfer (FRET). Fluorescence spectroscopy, density functional theory (DFT), 1H NMR titration, UV-Vis, and Job's plot were used to confirm the sensing mechanism. The charge transfer between the probe and analyte was further examined qualitatively by electron density differences (EDD) and quantitively by natural bond orbital (NBO) analyses. Whereas the non-covalent nature of probe BBP towards DOXY was verified by theoretical non-covalent interaction (NCI) analysis as along with Bader's quantum theory of atoms in molecules (QTAIM) analysis. Furthermore, probe BBP was also practically employed for the detection of doxycycline in fish samples, pharmaceutical wastewater and blood samples.

Optical recordings of organellar membrane potentials and the components of membrane conductance in lysosomes.

The eukaryotic cell is highly compartmentalized with organelles. Owing to their function in transporting metabolites, metabolic intermediates and byproducts of metabolic activity, organelles are important players in the orchestration of cellular function. Recent advances in optical methods for interrogating the different aspects of organellar activity promise to revolutionize our ability to dissect cellular processes with unprecedented detail. The transport activity of organelles is usually coupled to the transport of charged species; therefore, it is not only associated with the metabolic landscape but also entangled with membrane potentials. In this context, the targeted expression of fluorescent probes for interrogating organellar membrane potential (Ψorg) emerges as a powerful approach, offering less-invasive conditions and technical simplicity to interrogate cellular signalling and metabolism. Different research groups have made remarkable progress in adapting a variety of optical methods for measuring and monitoring Ψorg. These approaches include using potentiometric dyes, genetically encoded voltage indicators, hybrid fluorescence resonance energy transfer sensors and photoinduced electron transfer systems. These studies have provided consistent values for the resting potential of single-membrane organelles, such as lysosomes, the Golgi and the endoplasmic reticulum. We can foresee the use of dynamic measurements of Ψorg to study fundamental problems in organellar physiology that are linked to serious cellular disorders. Here, we present an overview of the available techniques, a survey of the resting membrane potential of internal membranes and, finally, an open-source mathematical model useful to interpret and interrogate membrane-bound structures of small volume by using the lysosome as an example.

Luminescence-Based Techniques for KRAS Thermal Stability Monitoring.

Various biochemical methods have been introduced to detect and characterize KRAS activity and interactions, from which the vast majority is based on luminescence detection in its varying forms. Among these methods, thermal stability assays, using luminophore-conjugated proteins or external environment sensing dyes, are widely used. In this chapter, we describe methods enabling KRAS stability monitoring in vitro, with an emphasis on ligand-induced stability. This chapter focuses mainly on luminescence-based techniques utilizing external dye molecules and fluorescence detection.

Direct fluoride monitoring using a fluorogenic RNA-based biosensor.

Fluorinated compounds, whether naturally occurring or from anthropogenic origin, have been extensively exploited in the last century. Degradation of these compounds by physical or biochemical processes is expected to result in the release of fluoride. Several fluoride detection mechanisms have been previously described. However, most of these methods are not compatible with high- and ultrahigh-throughput screening technologies, lack the ability to real-time monitor the increase of fluoride concentration in solution, or rely on costly reagents (such as cell-free expression systems). Our group recently developed "FluorMango" as the first completely RNA-based and direct fluoride-specific fluorogenic biosensor. To do so, we merged and engineered the Mango-III light-up RNA aptamer and the fluoride-specific aptamer derived from a riboswitch, crcB. In this chapter, we explain how this RNA-based biosensor can be produced in large scale before providing examples of how it can be used to quantitatively detect (end-point measurement) or monitor in real-time fluoride release in complex biological systems by translating it into measurable fluorescent signal.

An efficient lipid droplet-targeted fluorescent probe for detection of intracellular viscosity.

Lipid droplet, an intracellular lipid reservoir, is vital for energy metabolism and signal transmission in cells. The viscosity directly affects the metabolism of lipid droplets, and the abnormal viscosity is associated with the occurrence and development of various diseases. Therefore, it is indispensable to develop techniques that can detect viscosity changes in intracellular lipid droplets. Based on twisted intramolecular charge transfer (TICT) mechanism, a novel small-molecule lipid droplet-targeted viscosity fluorescence probe PPF-1 was designed. The probe was easy to synthesize, it had a large Stokes shift, stable optical properties, and low bio-toxicity. Compared to being in methanol solution, the fluorescence intensity of PPF-1 in glycerol solution was increased 26.7-fold, and PPF-1 showed excellent ability to target lipid droplets. Thus, the probe PPF-1 could provide an effective means of detecting viscosity changes of lipid droplets and was of great value for physiological diagnosis of related diseases, pathological analysis, and medical research.

Novel activated NIR-II fluorescence/Ratio photoacoustic probe for dual-modality accurate imaging of palladium ions overload in mouse liver.

Palladium contaminants can pose risks to human health and the natural environment. Once Pd2+ enters the body, it can bind with DNA, proteins, and other macromolecules, disrupting cellular processes and causing serious harm to health. Therefore, it becomes critical to develop simple, highly selective and precise methods for detecting Pd2+in vivo. Here, we have successfully developed the first activated second near-infrared region fluorescence (NIR-II FL) and ratio photoacoustic (PA) probe NYR-1 for dual-modal accurate detection of Pd2+ levels. NYR-1 is capable of rapidly (< 60 s) and sensitively detection of Pd2+ in solution, providing switched on NIR-II FL920 and ratio PA808/PA720 dual-mode signal change. More notably, the probe NYR-1 was successfully used for non-invasive imaging of Pd2+ overload in mouse liver by NIR-II FL/Ratio PA dual-modality imaging technology for the first time. Thus, this work opens up a promising dual-modal detection method for the precise detection of Pd2+ in organisms and in the environment.

Imaging gastrointestinal damage due to acute mercury poisoning using a mitochondria-targeted dual near-infrared fluorescent probe.

Mercury (Hg) is one of the most widespread pollutants that pose serious threats to public health and the environment. People are inevitably exposed to Hg via different routes, such as respiration, dermal contact, drinking or diet. Hg poisoning could cause gingivitis, inflammation, vomiting and diarrhea, respiratory distress or even death. Especially during the developmental stage, there is considerable harm to the brain development of young children, causing serious symptoms such as intellectual disability and motor impairments, and delayed neural development. Therefore, it's of great significance to develop a specific, quick, practical and labor-saving assay for monitoring Hg2+. Herein, a mitochondria-targeted dual (excitation 700 nm and emission 728 nm) near-infrared (NIR) fluorescent probe JZ-1 was synthesized to detect Hg2+, which is a turn-on fluorescent probe designed based on the rhodamine fluorophore thiolactone, with advantages of swift response, great selectivity, and robust anti-interference capability. Cell fluorescence imaging results showed that JZ-1 could selectively target mitochondria in HeLa cells and monitor exogenous Hg2+. More importantly, JZ-1 has been successfully used to monitor gastrointestinal damage of acute mercury poisoning in a drug-induced mouse model, which provided a great method for sensing Hg species in living subjects, as well as for prenatal diagnosis.

Supermolecular Confined Silicon Phosphorescence Nanoprobes for Time-Resolved Hypoxic Imaging Analysis.

Room temperature phosphorescence (RTP) nanoprobes play crucial roles in hypoxia imaging due to their high signal-to-background ratio (SBR) in the time domain. However, synthesizing RTP probes in aqueous media with a small size and high quantum yield remains challenging for intracellular hypoxic imaging up to present. Herein, aqueous RTP nanoprobes consisting of naphthalene anhydride derivatives, cucurbit[7]uril (CB[7]), and organosilicon are reported via supermolecular confined methods. Benefiting from the noncovalent confinement of CB[7] and hydrolysis reactions of organosilicon, such small-sized RTP nanoprobes (5-10 nm) exhibit inherent tunable phosphorescence (from 400 to 680 nm) with microsecond second lifetimes (up to ∼158.7 µs) and high quantum yield (up to ∼30%). The as-prepared RTP nanoprobes illustrate excellent intracellular hypoxia responsibility in a broad range from ∼0.1 to 21% oxygen concentrations. Compared to traditional fluorescence mode, the SBR value (∼108.69) of microsecond-range time-resolved in vitro imaging is up to 2.26 times greater in severe hypoxia (<0.1% O2), offering opportunities for precision imaging analysis in a hypoxic environment.

Design and Screening of Fluorescent Probes Based upon Hemicyanine Dyes for Monitoring Mitochondrial Viscosity in Living Cells.

Viscosity, at the subcellular level, plays a crucial role as a physicochemical factor affecting microenvironment homeostasis. Abnormal changes in mitochondrial viscosity often lead to various diseases in the organism. Based on the twisted intramolecular charge transfer mechanism, four hemicyanine dye fluorescent probes (HT-SA, HT-SA-S, HT-Bzh, and HT-NA) were designed and synthesized for viscosity response. The single bond between the nitrogen-containing heterocycle and the carbon-carbon double in the structure of the probe bond served as the viscosity response site. Finally, the probe HT-Bzh was screened as the optimal mitochondrial viscosity probe according to its responsiveness, targeting, and interference resistance. The fluorescence intensity of the probe HT-Bzh increased 22-fold when the viscosity was increased from 13.75 to 811.2 cP. In summary, all four viscosity probes we have developed can be used in different applications depending on the external environment, providing a valuable reference for the design of potential tools to address viscosity monitoring in biological systems.

A ß-Galactosidase-Activated Fluorogenic Reporter for the Detection of Gastric Cancer In Vivo and in Urine.

Although gastric cancer (GC) is one of the most frequent malignant tumors in the digestive tract with high morbidity and mortality, it remains a diagnostic dilemma due to its reliance on invasive biopsy or insensitive assays. Herein, we report a fluorescent gastric cancer reporter (FGCR) with activatable near-infrared fluorescence (NIRF) signals and high renal-clearance efficiency for the detection of orthotopic GC in a murine model via real-time imaging and remote urinalysis. In the presence of gastric-tumor-associated ß-galactosidase (ß-Gal), FGCR can be fluorescently activated for in vivo NIRF imaging. Relying on its high renal-clearance efficiency (∼95% ID), it can be rapidly excreted through kidneys to urine for the ultrasensitive detection of tumors with a diameter down to ∼2.1 mm and for assessing the prognosis of oxaliplatin-based chemotherapy. This study not only provides a new approach for noninvasive auxiliary diagnosis and prognosis of GC but also provides guidelines for the development of fluorescence probes for cancer diagnosis.

Bridging biological and food monitoring: A colorimetric and fluorescent dual-mode sensor based on N-doped carbon dots for detection of pH and histamine.

Rapid and sensitive monitoring of pH and histamine is crucial for bridging biological and food systems and identifying corresponding abnormal situations. Herein, N-doped carbon dots (CDs) are fabricated by a hydrothermal method employing dipicolinic acid and o-phenylenediamine as precursors. The CDs exhibit colorimetric and fluorescent dual-mode responses to track pH and histamine variations in living cells and food freshness, respectively. The aggregation-induced emission enhancement and intramolecular charge transfer result in a decrease in absorbance and an increase in fluorescence, which become readily apparent as the pH changes from acidic to neutral. This property enables precise differentiation between normal and cancerous cells. Furthermore, given the intrinsic basicity of histamine, pH-responsive CDs are advantageous for additional colorimetric and fluorescent monitoring of histamine in food freshness, achieving linearities of 25-1000 µM and 30-1000 µM, respectively, which are broader than those of alternative nanoprobes. Interestingly, the smartphone-integrated sensing platform can portably and visually evaluate pH and histamine changes due to sensitive color changes. Therefore, the sensor not only establishes a dynamic connection between pH and histamine for the purposes of biological and food monitoring, but also presents a novel approach for developing a multifunctional biosensor that can accomplish environmental monitoring and biosensing simultaneously.