Nucleic Acid-Based Nanodevices in Biological Imaging.

The nanoscale engineering of nucleic acids has led to exciting molecular technologies for high-end biological imaging. The predictable base pairing, high programmability, and superior new chemical and biological methods used to access nucleic acids with diverse lengths and in high purity, coupled with computational tools for their design, have allowed the creation of a stunning diversity of nucleic acid-based nanodevices. Given their biological origin, such synthetic devices have a tremendous capacity to interface with the biological world, and this capacity lies at the heart of several nucleic acid-based technologies that are finding applications in biological systems. We discuss these diverse applications and emphasize the advantage, in terms of physicochemical properties, that the nucleic acid scaffold brings to these contexts. As our ability to engineer this versatile scaffold increases, its applications in structural, cellular, and organismal biology are clearly poised to massively expand.

Design and synthesis of a small molecular NIR-II chemiluminescence probe for in vivo-activated H2S imaging.

Chemiluminescence (CL) with the elimination of excitation light and minimal autofluorescence interference has been wieldy applied in biosensing and bioimaging. However, the traditional emission of CL probes was mainly in the range of 400 to 650 nm, leading to undesired resolution and penetration in a biological object. Therefore, it was urgent to develop CL molecules in the near-infrared window [NIR, including NIR-I (650 to 900 nm) and near-infrared-II (900 to 1,700 nm)], coupled with unique advantages of long-time imaging, sensitive response, and high resolution at depths of millimeters. However, no NIR-II CL unimolecular probe has been reported until now. Herein, we developed an H2S-activated NIR-II CL probe [chemiluminiscence donor 950, (CD-950)] by covalently connecting two Schaap's dioxetane donors with high chemical energy to a NIR-II fluorophore acceptor candidate via intramolecular CL resonance energy transfer strategy, thereby achieving high efficiency of 95%. CD-950 exhibited superior capacity including long-duration imaging (~60 min), deeper tissue penetration (~10 mm), and specific H2S response under physiological conditions. More importantly, CD-950 showed detection capability for metformin-induced hepatotoxicity with 2.5-fold higher signal-to-background ratios than that of NIR-II fluorescence mode. The unimolecular NIR-II CL probe holds great potential for the evaluation of drug-induced side effects by tracking its metabolites in vivo, further facilitating the rational design of novel NIR-II CL-based detection platforms.

Hourglass, a compass navigating global and regional heterogeneity of pancreatic cancer†.

Advances in the digital pathology field have facilitated the characterization of histology samples for both clinical and preclinical research. However, uncovering subtle correlations between bioimaging, clinical and molecular parameters requires extensive statistical analysis. As a user-friendly software, Hourglass, simplifies multiparametric dataset analysis through intuitive data visualization and statistical tools. Systemic analysis of interleukin-6 (IL-6)/pStat3 signaling pathway through Hourglass revealed differences in regional immune cell composition within tumors. Moreover, these regional disparities were partially mediated by sex. Overall, Hourglass simplifies information extraction from complex datasets, resolving overlooked regional and global spatial tumor differences. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

An aggregation-induced emission-based ratiometric fluorescent chemosensor for Hg(II) and its application in Caenorhabditis elegans imaging.

A chromone-based ratiometric fluorescent probe L2 was developed for the selective detection of Hg(II) in a semi-aqueous solution based on aggregation-induced emission (AIE) and chelation-enhanced fluorescence (CHEF) effect. The probe L2 fluoresced significantly at 498 nm in its aggregated state, and when chelated with Hg(II), the soluble state fluoresced 1-fold higher. In addition, Job's plot reveals that the probe forms a 1:1 stoichiometry complex with Hg(II) with an association constant of 9.10 × 103M-1 estimated by the BH plot. The probe L2 detects Hg(II) down to 22.47 nM without interference from other interfering ions. The FTIR, ESI mass, and DFT-based computational studies investigated the binding mechanism of probe L2 with Hg(II). Taking advantage of its AIE characteristics, the probe L2 was successfully applied for bio-capability analysis in Caenorhabditis elegans (a nematode worm) imaging of Hg(II) in a living model.

Bodipy-based quinoline derivative as a highly Hg2+-selective fluorescent chemosensor and its potential applications.

A highly efficient sensor has been successfully developed using quinoline-based BODIPY compounds (8-quinoline-4,4-difluoro-4-boro-3a, 4a-diazaindacene (C1) and 7-hydroxy-8-quinoline-4,4-difluoro-4-boro-3a, 4a-diazindacene (C2) to detect Hg2+ ions. The sensor C1 exhibits remarkable selectivity in detecting Hg2+ with a limit of detection 3.06 × 10-8 mol/L. The developed chemical sensors have shown stability, cost-effectiveness, ease of preparation, and remarkable selectivity towards Hg2+ ions compared to other commonly occurring metal ions. The total recovery of the sensor C1 can be achieved by using a 0.1 mol/L solution of KI. The proposed sensor C1 has been applied to determine Hg2+ in tap and distilled water, yielding excellent results. In addition, the binding mode of C1-Hg2+ and C2-Hg2+ complexes was a 1:1 ratio confirmed by mass spectra, Job's plot, and DFT study. Moreover, the sensor C1 successfully applied for the biological studies results in negligible cytotoxicity, which demonstrates it can be used to determine Hg2+ in HT22 cells.

A fluorescence turn "on-off" imaging probe for sequential detection of Al3+ and L-Cysteine in HeLa cells.

At this "Aluminum Age", exposure to aluminum (metallic or ionic form) is inevitable and inestimable. The presence of aluminum in biological systems is evident but more often aluminum toxicity is less understood. Therefore, the presence of biologically reactive aluminum needs to be identified and quantified. Alongside metals, L-cysteine, an essential amino acid, plays a pivotal role in the homeostasis of cellular oxidative and reductive stress. However, excess (<7g) could be lethal and can lead to death. Thus, in-situ selective detection of aluminum and L-cysteine is of larger interest. Here we report a fluorogenic probe (R) for the sequential selective detection and quantification of Al3+ and L-cysteine in a semi-aqueous medium (3:7; water: DMSO). The probe (R) was synthesized by a one-step acid-mediated condensation reaction between pyridine-3,4-diamine and 2-hydroxy-1-napthaldehyde. The synthesized probe was characterized using 1H and 13C NMR, and HR-Mass spectroscopic techniques. The probe (R) is non-emissive in nature, but on recognition of Al3+, the probe R showed "turn-on" emission (bright yellow colour) showing two emission maxima (522 nm and 547 nm), and no naked eye observable color change. Other competing cations do not show any noticeable fluorescence outcome. The R + Al3+ ensemble can specifically detect L-cysteine among all the essential amino acids by showing a fluorescence "turn-off" response. The sensing mechanism of Al3+ is obeying the chelation-enhanced fluorescence (CHEF) effect. The binding constant of R + Al3+ is 0.3 × 104 M-1. The limit of detection (LoD) for Al3+ and L-cysteine are 2.02 × 10-7 M and 0.5 × 10-5 M respectively. The probe (R) can show maximum efficiency within the pH range (7.0-10.0). The probe is found non-toxic (>80 % cell viability with 15 µM concentration) and employed for the in-vitro fluorescence imaging in the HeLa cell.

In-Brain Multiphoton Imaging of Vaterite Cargoes Loaded with Carbon Dots.

Biocompatible fluorescent agents are key contributors to the theranostic paradigm by enabling real-time in vivo imaging. This study explores the optical properties of phenylenediamine carbon dots (CDs) and demonstrates their potential for fluorescence imaging in cells and brain blood vessels. The nonlinear absorption cross-section of the CDs was measured and achieved values near 50 Goeppert-Mayer (GM) units with efficient excitation in the 775-895 nm spectral range. Mesoporous vaterite nanoparticles were loaded with CDs to examine the possibility of a biocompatible imaging platform. Efficient one- and two-photon imaging of the CD-vaterite composites uptaken by diverse cells was demonstrated. For an in vivo scenario, CD-vaterite composites were injected into the bloodstream of a mouse, and their flow was monitored within the blood vessels of the brain through a cranial window. These results show the potential of the platform for high-brightness biocompatible imaging with the potential for both sensing and simultaneous drug delivery.

Deep learning for bioimage analysis in developmental biology.

Deep learning has transformed the way large and complex image datasets can be processed, reshaping what is possible in bioimage analysis. As the complexity and size of bioimage data continues to grow, this new analysis paradigm is becoming increasingly ubiquitous. In this Review, we begin by introducing the concepts needed for beginners to understand deep learning. We then review how deep learning has impacted bioimage analysis and explore the open-source resources available to integrate it into a research project. Finally, we discuss the future of deep learning applied to cell and developmental biology. We analyze how state-of-the-art methodologies have the potential to transform our understanding of biological systems through new image-based analysis and modelling that integrate multimodal inputs in space and time.

Multimodal Therapeutic Platforms Based on Self-Assembled Metallacycles/Metallacages for Cancer Radiochemotherapy.

Discrete organometallic complexes with defined structures are proceeding rapidly in combating malignant tumors due to their multipronged treatment modalities. Many innovative superiorities, such as high antitumor activity, extremely low systemic toxicity, active targeting ability, and enhanced cellular uptake, make them more competent for clinical applications than individual precursors. In particular, coordination-induced regulation of luminescence and photophysical properties of organic light-emitting ligands has demonstrated significant potential in the timely evaluation of therapeutic efficacy by bioimaging and enabled synergistic photodynamic therapy (PDT) or photothermal therapy (PTT). This review highlights instructive examples of multimodal radiochemotherapy platforms for cancer ablation based on self-assembled metallacycles/metallacages, which would be classified by functions in a progressive manner. Finally, the essential demands and some plausible prospects in this field for cancer therapy are also presented.

Biocompatible Silica-Coated Europium-Doped CsPbBr3 Nanoparticles with Luminescence in Water for Zebrafish Bioimaging.

Cesium lead halide (CsPbX3, X = Br, Cl, and I) nanocrystals (NCs) are widely concerned and applied in many fields due to the excellent photoelectric performance. However, the toxicity of Pb and the loss of luminescence in water limit its application in vivo. A stable perovskite nanomaterial with good bioimaging properties is developed by incorporating europium (Eu) in CsPbX3 NCs followed with the surface coating of silica (SiO2) shell (CsPbX3:Eu@SiO2). Through the surface coating of SiO2, the luminescence stability of CsPbBr3 in water is improved and the leakage of Pb2+ is significantly reduced. In particular, Eu doping inhibits the photoluminescence quantum yield reduction of CsPbBr3 caused by SiO2 coating, and further reduces the release of Pb2+. CsPbBr3:Eu@SiO2 nanoparticles (NPs) show efficient luminescence in water and good biocompatibility to achieve cell imaging. More importantly, CsPb(ClBr)3:Eu@SiO2 NPs are obtained by adjusting the halogen components, and green light and blue light are realized in zebrafish imaging, showing good imaging effect and biosafety. The work provides a strategy for advanced perovskite nanomaterials toward biological practical application.

Smart Stimuli-Responsive Spherical Nucleic Acids: Cutting-Edge Platforms for Biosensing, Bioimaging, and Therapeutics.

Spherical nucleic acids (SNAs) with exceptional colloidal stability, multiple modularity, and programmability are excellent candidates to address common molecular delivery-related issues. Based on this, the higher targeting accuracy and enhanced controllability of stimuli-responsive SNAs render them precise nanoplatforms with inestimable prospects for diverse biomedical applications. Therefore, tailored diagnosis and treatment with stimuli-responsive SNAs may be a robust strategy to break through the bottlenecks associated with traditional nanocarriers. Various stimuli-responsive SNAs are engineered through the incorporation of multifunctional modifications to meet biomedical demands with the development of nucleic acid functionalization. This review provides a comprehensive overview of prominent research in this area and recent advancements in the utilization of stimuli-responsive SNAs in biosensing, bioimaging, and therapeutics. For each aspect, SNA nanoplatforms that exhibit responsive behavior to both internal stimuli (including sequence, enzyme, redox reactions, and pH) and external stimuli (such as light and temperature) are highlighted. This review is expected to offer inspiration and guidance strategies for the rational design and development of stimuli-responsive SNAs in the field of biomedicine.

Nanoscale Metal-Organic Frameworks as a Photoluminescent Platform for Bioimaging and Biosensing Applications.

The tracking of nanomedicines in their concentration and location inside living systems has a pivotal effect on the understanding of the biological processes, early-stage diagnosis, and therapeutic monitoring of diseases. Nanoscale metal-organic frameworks (nano MOFs) possess high surface areas, definite structure, regulated optical properties, rich functionalized sites, and good biocompatibility that allow them to excel in a wide range of biomedical applications. Controllable syntheses and functionalization endow nano MOFs with better properties as imaging agents and sensing units for the diagnosis and treatment of diseases. This minireview summarizes the tunable synthesis strategies of nano MOFs with controllable size, shape, and regulated luminescent performance, and pinpoints their recent advanced applications as optical elements in bioimaging and biosensing. The current limitations and future development directions of nano MOF-contained materials in bioimaging and biosensing applications are also discussed, aiming to expand the biological applications of nano MOF-based nanomedicine and facilitate their production or clinical translation.

Lipid Droplets Specific Fluorophore for Demarcation of Normal and Diseased Tissues.

Using high-fidelity, permeable, lipophilic, and bright fluorophores for imaging lipid droplets (LDs) in tissues holds immense potential in diagnosing conditions such as diabetic or alcoholic fatty liver disease. In this work, we utilized linear and Λ-shaped polarity-sensitive fluorescent probes for imaging LDs in both cellular and tissue environments, specifically in rats with diabetic and alcoholic fatty liver disease. The fluorescent probes possess several key characteristics, including high permeability, lipophilicity, and brightness, which make them well-suited for efficient LD imaging. Notably, the probes exhibit a substantial Stokes shift, with 143 nm for DCS and 201 nm for DCN with selective targeting of the lipid droplets. Our experimental investigations successfully differentiated morphological variations between diseased and normal tissues in three distinct tissue types: liver, adipose, and small intestine. They could help provide pointers for improved detection and understanding of LD-related pathologies.

Tumor Acidity-Triggered Bioorthogonal Reactions for Biomedical Applications.

Cancer is one of the most serious threats to human health. Over the past few years, researchers have incrementally uncovered the pivotal role of tumor acidity in tumor formation, development and treatment through in-depth scientific research. In addition, bioorthogonal reactions have been widely used in tumor diagnosis and therapy, owing to their advantageous characteristics, including small ligand size, biocompatibility, fast reaction kinetics, and high chemospecificity. Consequently, bioorthogonal reactions triggered by tumor acidity have become an emerging strategy in biomedical applications. On this basis, we introduce the concept and major strategies of tumor acidity-triggered bioorthogonal reactions, and review their progress in biomedical applications, with a particular focus on their importance in disease diagnosis and treatment. Finally, clinical challenges and future trends are also outlooked.

Recent Advances in Electrochemiluminescence Visual Biosensing and Bioimaging.

Electrochemiluminescence (ECL) is one of the most powerful techniques that meet the needs of analysis and detection in a variety of scenarios, because of its highly analytical sensitivity and excellent spatiotemporal controllability. ECL combined with microscopy (ECLM) offers a promising approach for quantifying and mapping a wide range of analytes. To date, ECLM has been widely used to image biological entities and processes, such as cells, subcellular structures, proteins and membrane transport properties. In this review, we first introduced the mechanisms of several classic ECL systems, then highlighted the progress of visual biosensing and bioimaging by ECLM in the last decade. Finally, the characteristics of ECLM were summarized, as well as some of the current challenges. The future research interests and potential directions for the application of ECLM were also outlooked.

Exploiting the Photo-Physical Properties of Metal Halide Perovskite Nanocrystals for Bioimaging.

Perovskite nanomaterials have recently been exploited for bioimaging applications due to their unique photo-physical properties, including high absorbance, good photostability, narrow emissions, and nonlinear optical properties. These attributes outperform conventional fluorescent materials such as organic dyes and metal chalcogenide quantum dots and endow them with the potential to reshape a wide array of bioimaging modalities. Yet, their full potential necessitates a deep grasp of their structure-attribute relationship and strategies for enhancing water stability through surface engineering for meeting the stringent and unique requirements of each individual imaging modality. This review delves into this evolving frontier, highlighting how their distinctive photo-physical properties can be leveraged and optimized for various bioimaging modalities, including visible light imaging, near-infrared imaging, and super-resolution imaging.

Dual-Emissive Iridium(III) Complexes and Their Applications in Biological Sensing and Imaging.

Phosphorescent iridium(III) complexes are widely recognized for their unique properties in the excited triplet state, making them crucial for various applications including biological sensing and imaging. Most of these complexes display single phosphorescence emission from the lowest-lying triplet state after undergoing highly efficient intersystem crossing (ISC) and ultrafast internal conversion (IC) processes. However, in cases where these excited-state processes are restricted, the less common phenomenon of dual emission has been observed. This dual emission phenomenon presents an opportunity for developing biological probes and imaging agents with multiple emission bands of different wavelengths. Compared to intensity-based biosensing, where the existence and concentration of an analyte are indicated by the brightness of the probe, the emission profile response involves modifications in emission color. This enables quantification by utilizing the intensity ratio of different wavelengths, which is self-calibrating and unaffected by the probe concentration and excitation laser power. Moreover, dual-emissive probes have the potential to demonstrate distinct responses to multiple analytes at separate wavelengths, providing orthogonal detection capabilities. In this concept, we focus on iridium(III) complexes displaying fluorescence-phosphorescence or phosphorescence-phosphorescence dual emission, along with their applications as biological probes for sensing and imaging.

Substitution Effects on Subcellular Organelle Localization in Live-cell Imaging.

A series of D-p-A indole-containing fluorescent probes were developed followed by an investigation of their photophysical properties and compounds' suitability for subcellular imaging in living cells. We demonstrate that the preference for mitochondrial localization was lost when morpholine was substituted, resulting in the accumulation of the molecule in the lysosomes. However, interestingly, the presence of a nitro group led to their localization within the lipid droplets despite the presence of the morpholine pendant. We also showcase the probes' sensitivity to pH, the influence of added chloroquine, and the temperature response on the changes in fluorescence intensity within lysosomes. The design of the probes with strong intramolecular charge transfer and substantial Stokes shift could facilitate extensive application in various cellular lysosomal models and contribute to a better understanding of the mechanisms involved in stimuli-responsive diseases.

High-angular-sensitivity X-ray phase-contrast microtomography of soft tissue through a two-directional beam-tracking synchrotron set-up.

Two-directional beam-tracking (2DBT) is a method for phase-contrast imaging and tomography that uses an intensity modulator to structure the X-ray beam into an array of independent circular beamlets that are resolved by a high-resolution detector. It features isotropic spatial resolution, provides two-dimensional phase sensitivity, and enables the three-dimensional reconstructions of the refractive index decrement, δ, and the attenuation coefficient, µ. In this work, the angular sensitivity and the spatial resolution of 2DBT images in a synchrotron-based implementation is reported. In its best configuration, angular sensitivities of ∼20 nrad and spatial resolution of at least 6.25 µm in phase-contrast images were obtained. Exemplar application to the three-dimensional imaging of soft tissue samples, including a mouse liver and a decellularized porcine dermis, is also demonstrated.

Bio-net dataset: AI-based diagnostic solutions using peripheral blood smear images.

Peripheral blood smear examination is one of the basic steps in the evaluation of different blood cells. It is a confirmatory step after an automated complete blood count analysis. Manual microscopy is time-consuming and requires professional laboratory expertise. Therefore, the turn-around time for peripheral smear in a health care center is approximately 3-4 hours. To avoid the traditional method of manual counting under the microscope a computerized automation of peripheral blood smear examination has been adopted, which is a challenging task in medical diagnostics. In recent times, deep learning techniques have overcome the challenges associated with human microscopic evaluation of peripheral smears and this has led to reduced cost and precise diagnosis. However, their application can be significantly improved by the availability of annotated datasets. This study presents a large customized annotated blood cell dataset (named the Bio-Net dataset from healthy individuals) and blood cell detection and counting in the peripheral blood smear images. A mini-version of the dataset for specialized WBC-based image processing tasks is also equipped to classify the healthy and mature WBCs in their respective classes. An object detection algorithm called You Only Look Once (YOLO) with a refashion disposition has been trained on the novel dataset to automatically detect and classify blood cells into RBCs, WBCs, and platelets and compare the results with other publicly available datasets to highlight the versatility. In short the introduction of the Bio-Net dataset and AI-powered detection and counting offers a significant potential for advancement in biomedical research for analyzing and understanding biological data.