Fluorescence Imaging of Neural Activity, Neurochemical Dynamics, and Drug-Specific Receptor Conformation with Genetically Encoded Sensors.

Recent advances in fluorescence imaging permit large-scale recording of neural activity and dynamics of neurochemical release with unprecedented resolution in behaving animals. Calcium imaging with highly optimized genetically encoded indicators provides a mesoscopic view of neural activity from genetically defined populations at cellular and subcellular resolutions. Rigorously improved voltage sensors and microscopy allow for robust spike imaging of populational neurons in various brain regions. In addition, recent protein engineering efforts in the past few years have led to the development of sensors for neurotransmitters and neuromodulators. Here, we discuss the development and applications of these genetically encoded fluorescent indicators in reporting neural activity in response to various behaviors in different biological systems as well as in drug discovery. We also report a simple model to guide sensor selection and optimization.

Superresolution imaging of antibiotic-induced structural disruption of bacteria enabled by photochromic glycomicelles.

Bacterial evolution, particularly in hospital settings, is leading to an increase in multidrug resistance. Understanding the basis for this resistance is critical as it can drive discovery of new antibiotics while allowing the clinical use of known antibiotics to be optimized. Here, we report a photoactive chemical probe for superresolution microscopy that allows for the in situ probing of antibiotic-induced structural disruption of bacteria. Conjugation between a spiropyran (SP) and galactose via click chemistry produces an amphiphilic photochromic glycoprobe, which self-assembles into glycomicelles in water. The hydrophobic inner core of the glycomicelles allows encapsulation of antibiotics. Photoirradiation then serves to convert the SP to the corresponding merocyanine (MR) form. This results in micellar disassembly allowing for release of the antibiotic in an on-demand fashion. The glycomicelles of this study adhere selectively to the surface of a Gram-negative bacterium through multivalent sugar-lectin interaction. Antibiotic release from the glycomicelles then induces membrane collapse. This dynamic process can be imaged in situ by superresolution spectroscopy owing to the "fluorescence blinking" of the SP/MR photochromic pair. This research provides a high-precision imaging tool that may be used to visualize how antibiotics disrupt the structural integrity of bacteria in real time.

Pressure-enhanced sensing of tissue oxygenation via endogenous porphyrin: Implications for dynamic visualization of cancer in surgery.

Fluorescence guidance is routinely used in surgery to enhance perfusion contrast in multiple types of diseases. Pressure-enhanced sensing of tissue oxygenation (PRESTO) via fluorescence is a technique extensively analyzed here, that uses an FDA-approved human precursor molecule, 5-aminolevulinic acid (ALA), to stimulate a unique delayed fluorescence signal that is representative of tissue hypoxia. The ALA precontrast agent is metabolized in most tissues into a red fluorescent molecule, protoporphyrin IX (PpIX), which has both prompt fluorescence, indicative of the concentration, and a delayed fluorescence, that is amplified in low tissue oxygen situations. Applied pressure from palpation induces transient capillary stasis and a resulting transient PRESTO contrast, dominant when there is near hypoxia. This study examined the kinetics and behavior of this effect in both normal and tumor tissues, with a prolonged high PRESTO contrast (contrast to background of 7.3) across 5 tumor models, due to sluggish capillaries and inhibited vasodynamics. This tissue function imaging approach is a fundamentally unique tool for real-time palpation-induced tissue response in vivo, relevant for chronic hypoxia, such as vascular diseases or oncologic surgery.

ExoJ - a Fiji/ImageJ2 plugin for automated spatiotemporal detection and analysis of exocytosis.

Exocytosis is a dynamic physiological process that enables the release of biomolecules to the surrounding environment via the fusion of membrane compartments to the plasma membrane. Understanding its mechanisms is crucial, as defects can compromise essential biological functions. The development of pH-sensitive optical reporters alongside fluorescence microscopy enables the assessment of individual vesicle exocytosis events at the cellular level. Manual annotation represents, however, a time-consuming task that is prone to selection biases and human operational errors. Here, we introduce ExoJ, an automated plugin based on Fiji/ImageJ2 software. ExoJ identifies user-defined genuine populations of exocytosis events, recording quantitative features including intensity, apparent size and duration. We designed ExoJ to be fully user-configurable, making it suitable for studying distinct forms of vesicle exocytosis regardless of the imaging quality. Our plugin demonstrates its capabilities by showcasing distinct exocytic dynamics among tetraspanins and vesicular SNARE protein reporters. Assessment of performance on synthetic data shows that ExoJ is a robust tool that is capable of correctly identifying exocytosis events independently of signal-to-noise ratio conditions. We propose ExoJ as a standard solution for future comparative and quantitative studies of exocytosis.

The scaffolding protein IQGAP1 enhances EGFR signaling by promoting oligomerization and preventing degradation.

IQGAP1 is a large, multi-domain scaffold that connects and modulates different signaling networks including the one initiated by epidermal growth factor (EGF). In this study, we have used live cell fluorescence imaging methods along with other biochemical techniques to follow the mechanisms used by IQGAP1 to enhance EGF signaling. We show that IQGAP1 enhances EGF signaling by promoting the oligomerization of its receptor, EGFR, upon EGF addition along with concurrent IQGAP oligomerization. Using cellular markers, we find that IQGAP1 promotes the plasma membrane localization of EGFR and promotes association to one of its phosphoinositide lipid pathway ligands, PI(3,4,5)P3. Additionally, we find that binding of EGFR to IQGAP1 protects EGFR from lysosomal degradation. Taken together, our results show that IQGAP1 enhances EGF-mediated pathway progression through mechanisms that augment simple scaffolding activities.

Fluorescent tagging of endogenous IRS2 with an auxin-dependent degron to assess dynamic intracellular localization and function.

Insulin Receptor Substrate 2 (IRS2) is a signaling adaptor protein for the insulin (IR) and Insulin-like Growth Factor-1 (IGF-1R) receptors. In breast cancer, IRS2 contributes to both the initiation of primary tumor growth and the establishment of secondary metastases through regulation of cancer stem cell (CSC) function and invasion. However, how IRS2 mediates its diverse functions is not well understood. We used CRISPR/Cas9-mediated gene editing to modify endogenous IRS2 to study the expression, localization, and function of this adaptor protein. A cassette containing an auxin-inducible degradation (AID) sequence, 3x-FLAG tag, and mNeon-green was introduced at the N-terminus of the IRS2 protein to provide rapid and reversible control of IRS2 protein degradation and analysis of endogenous IRS2 expression and localization. Live fluorescence imaging of these cells revealed that IRS2 shuttles between the cytoplasm and nucleus in response to growth regulatory signals in a PI3K-dependent manner. Inhibition of nuclear export or deletion of a putative nuclear export sequence in the C-terminal tail promotes nuclear retention of IRS2, implicating nuclear export in the mechanism by which IRS2 intracellular localization is regulated. Moreover, the acute induction of IRS2 degradation reduces tumor cell invasion, demonstrating the potential for therapeutic targeting of this adaptor protein. Our data highlight the value of our model of endogenously tagged IRS2 as a tool to study IRS2 localization and function.

Localization of the tubby domain, a PI(4,5)P2 biosensor, to E-Syt3-rich endoplasmic reticulum-plasma membrane junctions.

The phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] acts as a signaling lipid at the plasma membrane (PM) with pleiotropic regulatory actions on multiple cellular processes. Signaling specificity might result from spatiotemporal compartmentalization of the lipid and from combinatorial binding of PI(4,5)P2 effector proteins to additional membrane components. Here, we analyzed the spatial distribution of tubbyCT, a paradigmatic PI(4,5)P2-binding domain, in live mammalian cells by total internal reflection fluorescence (TIRF) microscopy and molecular dynamics simulations. We found that unlike other well-characterized PI(4,5)P2 recognition domains, tubbyCT segregates into distinct domains within the PM. TubbyCT enrichment occurred at contact sites between PM and endoplasmic reticulum (ER) (i.e. at ER-PM junctions) as shown by colocalization with ER-PM markers. Localization to these sites was mediated in a combinatorial manner by binding to PI(4,5)P2 and by interaction with a cytosolic domain of extended synaptotagmin 3 (E-Syt3), but not other E-Syt isoforms. Selective localization to these structures suggests that tubbyCT is a novel selective reporter for a ER-PM junctional pool of PI(4,5)P2. Finally, we found that association with ER-PM junctions is a conserved feature of tubby-like proteins (TULPs), suggesting an as-yet-unknown function of TULPs.

Nuclear morphogenesis: forming a heterogeneous nucleus during embryogenesis.

An embryo experiences increasingly complex spatial and temporal patterns of gene expression as it matures, guiding the morphogenesis of its body. Using super-resolution fluorescence microscopy in Drosophila melanogaster embryos, we observed that the nuclear distributions of transcription factors and histone modifications undergo a similar transformation of increasing heterogeneity. This spatial partitioning of the nucleus could lead to distinct local regulatory environments in space and time that are tuned for specific genes. Accordingly, transcription sites driven by different cis-regulatory regions each had their own temporally and spatially varying local histone environments, which could facilitate the finer spatial and temporal regulation of genes to consistently differentiate cells into organs and tissues. Thus, 'nuclear morphogenesis' may be a microscopic counterpart of the macroscopic process that shapes the animal body.

High-precision tumor resection down to few-cell level guided by NIR-IIb molecular fluorescence imaging.

In vivo fluorescence/luminescence imaging in the near-infrared-IIb (NIR-IIb, 1,500 to 1,700 nm) window under <1,000 nm excitation can afford subcentimeter imaging depth without any tissue autofluorescence, promising high-precision intraoperative navigation in the clinic. Here, we developed a compact imager for concurrent visible photographic and NIR-II (1,000 to 3,000 nm) fluorescence imaging for preclinical image-guided surgery. Biocompatible erbium-based rare-earth nanoparticles (ErNPs) with bright down-conversion luminescence in the NIR-IIb window were conjugated to TRC105 antibody for molecular imaging of CD105 angiogenesis markers in 4T1 murine breast tumors. Under a ∼940 ± 38 nm light-emitting diode (LED) excitation, NIR-IIb imaging of 1,500- to 1,700-nm emission afforded noninvasive tumor­to­normal tissue (T/NT) signal ratios of ∼40 before surgery and an ultrahigh intraoperative tumor-to-muscle (T/M) ratio of ∼300, resolving tumor margin unambiguously without interfering background signal from surrounding healthy tissues. High-resolution imaging resolved small numbers of residual cancer cells during surgery, allowing thorough and nonexcessive tumor removal at the few-cell level. NIR-IIb molecular imaging afforded 10-times-higher and 100-times-higher T/NT and T/M ratios, respectively, than imaging with IRDye800CW-TRC105 in the ∼900- to 1,300-nm range. The vastly improved resolution of tumor margin and diminished background open a paradigm of molecular imaging-guided surgery.

Programmed Remodeling of the Tumor Milieu to Enhance NK Cell Immunotherapy Combined with Chemotherapy for Pancreatic Cancer.

Natural killer (NK) cell-based adoptive immunotherapy has demonstrated encouraging therapeutic effects in clinical trials for hematological cancers. However, the effectiveness of treatment for solid tumors remains a challenge due to insufficient recruitment and infiltration of NK cells into tumor tissues. Herein, a programmed nanoremodeler (DAS@P/H/pp) is designed to remodel dense physical stromal barriers and for dysregulation of the chemokine of the tumor environment to enhance the recruitment and infiltration of NK cells in tumors. The DAS@P/H/pp is triggered by the acidic tumor environment, resulting in charge reversal and subsequent hyaluronidase (HAase) release. HAase effectively degrades the extracellular matrix, promoting the delivery of immunoregulatory molecules and chemotherapy drugs into deep tumor tissues. In mouse models of pancreatic cancer, this nanomediated strategy for the programmed remodeling of the tumor microenvironment significantly boosts the recruitment of NK92 cells and their tumor cell-killing capabilities under the supervision of multiplexed near-infrared-II fluorescence.

X-ray Activated Nanoprodrug for Visualization of Cortical Microvascular Alterations and NIR-II Image-Guided Chemo-Radiotherapy of Glioblastoma.

The permeability of the highly selective blood-brain barrier (BBB) to anticancer drugs and the difficulties in defining deep tumor boundaries often reduce the effectiveness of glioma treatment. Thus, exploring the combination of multiple treatment modalities under the guidance of second-generation near-infrared (NIR-II) window fluorescence (FL) imaging is considered a strategic approach in glioma theranostics. Herein, a hybrid X-ray-activated nanoprodrug was developed to precisely visualize the structural features of glioma microvasculature and delineate the boundary of glioma for synergistic chemo-radiotherapy. The nanoprodrug comprised down-converted nanoparticle (DCNP) coated with X-ray sensitive poly(Se-Se/DOX-co-acrylic acid) and targeted Angiopep-2 peptide (DCNP@P(Se-DOX)@ANG). Because of its ultrasmall size and the presence of DOX, the nanoprodrug could easily cross BBB to precisely monitor and localize glioblastoma via intracranial NIR-II FL imaging and synergistically administer antiglioblastoma chemo-radiotherapy through specific X-ray-induced DOX release and radiosensitization. This study provides a novel and effective strategy for glioblastoma imaging and chemo-radiotherapy.

Heteroions Radii Matching Produced Intensely Luminescent Bismuth-Ag2S Nanocrystals for through-Skull NIR-II Imaging of Orthotopic Glioma.

Heteroions doped Ag2S nanocrystals (NCs) exhibiting enhanced near-infrared-II emission (NIR-II) hold great promise for glioma diagnosis. Nevertheless, current doped Ag2S NCs paradoxically improved properties via toxic dopants, and the blood-brain barrier (BBB) constitutes another challenge for orthotopic glioma imaging. Thus, it is urgent to develop biofriendly high-bright Ag2S NCs with active BBB-penetration for glioma-targeted imaging. Herein, bismuth (Bi) was screened to obtain Bi-Ag2S NCs with high absolute PLQY (∼13.3%) for its matched ionic-radius (1.03 Å) with Ag+. The Bi-Ag2S NCs exhibited a higher luminance and deeper penetration (5-6 mm) than clinical indocyanine green. Upon conjugation with lactoferrin, the NCs acquired BBB-crossing and glioma-targeting abilities. Time-dependent NIR-II-imaging demonstrated their effective accumulation in glioma with skull/scalp intact after intravenous injection. Moreover, the toxic-metal-free NCs exhibited negligible toxicity and great biocompatibility. The success of leveraging the ion-radii comparison may unlock the full potential of doped-Ag2S NCs in bioimaging and inspire the development of various doped NIR-II NCs.

Designable Nanoadaptor for Enhanced Recognition of Natural Killer Cell to Tumor via Bio-orthogonal Click Reaction.

Highly efficient recognition of cancer cells by immune cells is important for successful therapeutic-cell-based cancer immunotherapy. Herein, we present a facile NIR-II nanoadaptor [hyaluronic acid (HA)/dibenzocyclooctyne (DBCO)-Au:Ag2Te quantum dots (QDs)] for enhancing the tumor recognition and binding ability of natural killer (NK) cells via a bio-orthogonal click reaction in vivo. The Nanoadaptor possesses superior tumor-targeting capacity, facilitating the accumulation of the chemical receptor DBCO at the tumor sites. Subsequently, the enrichment of DBCO on tumor cell surfaces provides multivalent recognition sites for capturing pretreated azide engineered NK92 cells (NK92-N3) through an efficient click reaction, thereby significantly enhancing the therapeutical efficiency. The dynamic process of nanoadaptor-mediated recognition of NK cells to tumor cells could be vividly observed using multiplexed NIR-II fluorescence imaging in a mouse model of lung cancer. Such a nanoadaptor strategy can be extended to other therapeutic cellular systems and holds promise for future clinical applications.

Injectable Fluorescent Neural Interfaces for Cell-Specific Stimulating and Imaging.

Building on current explorations in chronic optical neural interfaces, it is essential to address the risk of photothermal damage in traditional optogenetics. By focusing on calcium fluorescence for imaging rather than stimulation, injectable fluorescent neural interfaces significantly minimize photothermal damage and improve the accuracy of neuronal imaging. Key advancements including the use of injectable microelectronics for targeted electrical stimulation and their integration with cell-specific genetically encoded calcium indicators have been discussed. These injectable electronics that allow for post-treatment retrieval offer a minimally invasive solution, enhancing both usability and reliability. Furthermore, the integration of genetically encoded fluorescent calcium indicators with injectable bioelectronics enables precise neuronal recording and imaging of individual neurons. This shift not only minimizes risks such as photothermal conversion but also boosts safety, specificity, and effectiveness of neural imaging. Embracing these advancements represents a significant leap forward in biomedical engineering and neuroscience, paving the way for advanced brain-machine interfaces.

Harnessing CRISPR/Cas12a Activity and DNA-Based Ultrabright FluoroCube for In Situ Imaging of Metabolically Labeled Cell Membrane Glycoproteins.

Fluorescence imaging of cell membrane glycoproteins based on metabolic labeling faces challenges including the sensitivity and spatial specificity and the use of a high concentration of unnatural sugars. To overcome these limitations, we developed a method for in situ imaging of cell membrane glycoproteins by operating Cas12a activity, and employing the ultrabright DNA nanostructure, FluoroCube (FC), as a signal reporter. Following Cas12a activation, we observed stable and intense fluorescence signals within 15 min. The combination of bright FC and Cas12a's amplification capability allows for effective imaging with only 5 µM of unnatural sugars and a brief 24-h incubation. Computational modeling demonstrates that Cas12a specifically cleaves FC in the 11-17 nm range of the glycosylation site, enabling spatially precise imaging. This approach successfully enabled fluorescence imaging of glycoproteins across various cell lines and the detection of changes in glycoprotein levels induced by drugs.

Molecular Engineering of Bright NIR-I/NIR-II Nanofluorophores for High-Resolution Bioimaging and Tumor Detection in Vivo.

A comprehensive approach for the construction of NIR-I/NIR-II nanofluorophores with exceptional brightness and excellent chemo- and photostability has been developed. This study first confirmed that the amphiphilic molecules with stronger hydrophobic moieties and weaker hydrophilic moieties are superior candidates for constructing brighter nanofluorophores, which are attributed to its higher efficiency in suppressing the intramolecular charge transfer/aggregation-caused fluorescence quenching of donor-acceptor-donor type fluorophores. The prepared nanofluorophore demonstrates a fluorescence quantum yield exceeding 4.5% in aqueous solution and exhibits a strong NIR-II tail emission up to 1300 nm. The superior performance of the nanofluorophore enabled the achievement of high-resolution whole-body vessel imaging and brain vessel imaging, as well as high-contrast fluorescence imaging of the lymphatic system in vivo. Furthermore, their potential for highly sensitive fluorescence detection of tiny tumors in vivo has been successfully confirmed, thus supporting their future applications in precise fluorescence imaging-guided surgery in the early stages of cancer.

Temperature-Responsive "Nano-to-Micro" Transformed Polymersomes for Enhanced Ultrasound/Fluorescence Dual Imaging-Guided Tumor Phototherapy.

The perfect integration of microbubbles for efficient ultrasound imaging and nanocarriers for intelligent tumor-targeting delivery remains a challenge in precise tumor theranostics. Herein, we exquisitely fabricated laser-activated and targeted polymersomes (abbreviated as FIP-NPs) for simultaneously encapsulating the photosensitizer indocyanine green (ICG) and the phase change agent perfluorohexane (PFH). The formulated FIP-NPs were nanosize and effectively accumulated into tumors as observed by ICG fluorescence imaging. When the temperature rose above 56 °C, the encapsulated PFH transformed from liquid to gas and the FIP-NPs underwent balloon-like enlargement without structure destruction. Impressively, the enlarged FIP-NPs fused with adjacent polymersomes to form even larger microparticles. This temperature-responsive "nano-to-micro" transformation and fusion process was clearly demonstrated, and FIP-NPs showed greatly improved ultrasound signals. More importantly, FIP-NPs achieved dramatic antitumor efficacy through ICG-mediated phototherapy. Taken together, the novel polymersomes achieved excellent ultrasound/fluorescence dual imaging-guided tumor phototherapy, providing an optimistic candidate for the application of tumor theranostics.

Polyfunctional Arylamine Based Nanofiltration Membranes with Enhanced Aggressive Organic Solvents Resistance.

Organic solvent nanofiltration (OSN) membranes with high separation performance and excellent stability in aggressive organic solvents are urgently desired for chemical separation. Herein, we utilized a polyfunctional arylamine tetra-(4-aminophenyl) ethylene (TAPE) to prepare a highly cross-linked polyamide membrane with a low molecular weight cut-off (MWCO) of 312 Da. Owing to its propeller-like conformation, TAPE formed micropores within the polyamide membrane and provided fast solvent transport channels. Importantly, the rigid conjugated skeleton and high connectivity between micropores effectively prevented the expansion of the polyamide matrix in aggressive organic solvents. The membrane maintained high separation performance even immersed in N,N-dimethylformamide for 90 days. Based on the aggregation-induced emission (AIE) effect of TAPE, the formation of polyamide membrane can be visually monitored by fluorescence imaging technology, which achieved visual guidance for membrane fabrication. This work provides a vital foundation for utilizing polyfunctional monomers in the interfacial polymerization reaction to prepare high-performance OSN membranes.

Ultrabright NIR-II Nanoprobe for Image-Guided Accurate Resection of Tiny Metastatic Lesions.

Fluorescence imaging is a vital way to delineate the tumor boundaries. Here, we achieve a NIR-II aggregation-induced emission luminogen (AIEgen) with a fluorescence quantum yield (QY) of 12.6% in water through straightforward alkyl side chain modification. After loading of NIR-II AIEgen into polystyrene (PS) nanospheres, the thermal deactivation pathway is extremely limited, thereby concentrating absorption excitation on fluorescence emission. The fluorescence intensity is further enhanced by 5.4 times, the QY increases to 21.1%, and the NIR-II imaging signal is accordingly enhanced by 8.7 times, surpassing conventional DSPE-PEG carriers. The NIR-II@PS nanoprobe showcases superior resolution and tissue penetration depth compared to indocyanine green (ICG) and short-range near-infrared AIEgens. In vivo investigations underscore its tumor-to-normal tissue ratio (3.9) at 24 h post intravenous injection, enabling complete resection of ≤1 mm metastases under NIR-II bioimaging guidance. Additionally, the PS carrier-nanoparticles exhibit low toxicity in vivo, laying a promising foundation for the future design of medical nanomaterials.

Quantifying dynamic pro-inflammatory gene expression and heterogeneity in single macrophage cells.

Macrophages must respond appropriately to pathogens and other pro-inflammatory stimuli in order to perform their roles in fighting infection. One way in which inflammatory stimuli can vary is in their dynamics-that is, the amplitude and duration of stimulus experienced by the cell. In this study, we performed long-term live cell imaging in a microfluidic device to investigate how the pro-inflammatory genes IRF1, CXCL10, and CXCL9 respond to dynamic interferon-gamma (IFNγ) stimulation. We found that IRF1 responds to low concentration or short duration IFNγ stimulation, whereas CXCL10 and CXCL9 require longer or higherconcentration stimulation to be expressed. We also investigated the heterogeneity in the expression of each gene and found that CXCL10 and CXCL9 have substantial cell-to-cell variability. In particular, the expression of CXCL10 appears to be largely stochastic with a subpopulation of nonresponding cells across all the stimulation conditions tested. We developed both deterministic and stochastic models for the expression of each gene. Our modeling analysis revealed that the heterogeneity in CXCL10 can be attributed to a slow chromatin-opening step that is on a similar timescale to that of adaptation of the upstream signal. In this way, CXCL10 expression in individual cells can remain stochastic in response to each pulse of repeated stimulation, which we also validated by experiments. Together, we conclude that pro-inflammatory genes in the same signaling pathway can respond to dynamic IFNγ stimulus with very different response features and that upstream signal adaptation can contribute to shaping heterogeneous gene expression.