Motion artifacts continue to present a major challenge to single cell imaging in cardiothoracic organs such as the beating heart, blood vessels, or lung. In this study, we present a new water-immersion suctioning stabilizer that enables minimally invasive intravital fluorescence microscopy using water-based stick objectives. The stabilizer works by reducing major motion excursions and can be used in conjunction with both prospective or retrospective gating approaches. We show that the new approach offers cellular resolution in the beating murine heart without perturbing normal physiology. In addition, because this technique allows multiple areas to be easily probed, it offers the opportunity for wide area coverage at high resolution.
intravital microscopy; motion compensation; optical microscopy; in vivo imaging; cardiac imaging
The task of rapidly identifying patients infected with Mycobacterium tuberculosis (MTB) in resource-constrained environments remains a challenge. A sensitive and robust platform that does not require bacterial isolation or culture is critical in making informed diagnostic and therapeutic decisions. Here we introduce a platform for the detection of nucleic acids based on a magnetic barcoding strategy. PCR-amplified mycobacterial genes are sequence-specifically captured on microspheres, labeled by magnetic nanoprobes, and detected by nuclear magnetic resonance. All components are integrated into a single, small fluidic cartridge for streamlined on-chip operation. We use this platform to detect MTB and identify drug-resistance strains from mechanically processed sputum samples within 2.5 hours. The specificity of the assay is confirmed by a panel of clinically relevant non-MTB bacteria, and the clinical utility is demonstrated by the measurements in MTB-positive patient specimens. Combined with portable systems, the magnetic barcode assay holds promise to become a sensitive, high-throughput, and low-cost platform for point-of-care diagnostics.
New approaches that allow precise spatiotemporal control of gene expression in model organisms at the single cell level are necessary to better dissect the role of specific genes and cell populations in development, disease and therapy. Here, we describe a new optochemogenetic switch (OCG switch) to control CreER/loxP-mediated recombination via photoactivatable (“caged”) tamoxifen analogues in individual cells in cell culture, organoid culture and in vivo in adult mice. This approach opens opportunities to more fully exploit existing CreER transgenic mouse strains to achieve more precise temporal- and location-specific regulation of genetic events and gene expression.
Efficient methods to immobilize small molecules under continuous-flow microfluidic conditions would greatly improve label-free molecular interaction studies using biosensor technology. At present, small-molecule immobilization chemistries require special conditions and in many cases must be performed outside the detector and microfluidic system where real-time monitoring is not possible. Here, we have developed and optimized a method for on-chip bioorthogonal chemistry that enables rapid, reversible immobilization of small molecules with control over orientation and immobilization density, and apply this technique to surface plasmon resonance (SPR) studies. Immobilized small molecules reverse the orientation of canonical SPR interaction studies, and also enable a variety of new SPR applications including on-chip assembly and interaction studies of multicomponent structures such as functionalized nanoparticles, and measurement of bioorthogonal reaction rates. We use this approach to demonstrate that on-chip assembled functionalized nanoparticles show a preserved ability to interact with their target protein, and to measure rapid bioorthogonal reaction rates with k2 > 103 M−1 s−1. This method offers multiple benefits for microfluidic biological applications, including rapid screening of targeted nanoparticles with vastly decreased nanoparticle synthetic requirements, robust immobilization chemistry in the presence of serum, and a continuous flow technique that mimics biologic contexts better than current methods used to measure bioorthogonal reaction kinetics such as NMR or UV-vis spectroscopy (e.g., stopped flow kinetics). Taken together, this approach constitutes a flexible and powerful technique for evaluating a wide variety of reactions and intermolecular interactions for in vitro or in vivo applications.
A number of exendin derivatives have been developed to target glucagon-like peptide 1 (GLP-1) receptors on beta cells in vivo. Modifications of exendin analogues have been shown to have significant effects on pharmacokinetics and, as such, have been used to develop a variety of therapeutic compounds. Here, we show that an exendin-4, modified at position 12 with a cysteine conjugated to a tetrazine, can be labeled with 18F-trans-cyclooctene and converted into a PET imaging agent at high yields and with good selectivity. The agent accumulates in beta cells in vivo and has sufficiently high accumulation in mouse models of insulinomas to enable in vivo imaging.
18F-trans-cyclooctene; cancer; diabetes; exendin-4; insulinoma; positron emission tomography
Background and Objectives
The application of fluorescent molecular imaging to surgical oncology is a developing field with the potential to reduce morbidity and mortality. However, the detection thresholds and other requirements for successful intervention remain poorly understood. Here we modeled and experimentally validated depth and size of detection of tumor deposits, trade-offs in coverage and resolution of areas of interest, and required pharmacokinetics of probes based on differing levels of tumor target presentation.
Three orthotopic tumor models were imaged by widefield epifluorescence and confocal microscopes, and the experimental results were compared with pharmacokinetic models and light scattering simulations to determine detection thresholds.
Widefield epifluorescence imaging can provide sufficient contrast to visualize tumor margins and detect tumor deposits 3–5 mm deep based on labeled monoclonal antibodies at low objective magnification. At higher magnification, surface tumor deposits at cellular resolution are detectable at TBR ratios achieved with highly expressed antigens.
A widefield illumination system with the capability for macroscopic surveying and microscopic imaging provides the greatest utility for varying surgical goals. These results have implications for system and agent designs, which ultimately should aid complete resection in most surgical beds and provide real-time feedback to obtain clean margins.
antibody targeting; antigen expression; fluorescence; surgery
Magnetic relaxation switching (MRSw) assays that employ target-induced aggregation (or disaggregation) of magnetic nanoparticles (MNPs) can be used to detect a wide range of biomolecules. The precise working mechanisms, however, remain poorly understood, often leading to confounding interpretation. We herein present a systematic and comprehensive characterization of MRSw sensing. By using different types of MNPs with varying physical properties, we analyzed the nature and transverse relaxation modes for MRSw detection. The study found that clustered MNPs are universally in a diffusion-limited fractal state (dimension of ~2.4). Importantly, a new model for transverse relaxation was constructed, that accurately recapitulates observed MRSw phenomena, and predicts the MRSw detection sensitivities and dynamic ranges.
Magnetic Relaxation Switching; Magnetic Nanoparticles; NMR; Biosensors
We used a molecular probe activated by protease cleavage to image expression of matrix metalloproteinases (MMPs) in the heart following myocardial infarction.
Methods and Results
We synthesized and characterized a near-infrared fluorescent (NIRF) probe that is activated by proteolytic cleavage by MMP2 and MMP9. The NIRF probe was injected into mice at various time-points up to 4 weeks after myocardial infarction induced by ligation of the left anterior descending artery. NIRF imaging of MMP activity increased in the infarct region with maximal expression at one to two weeks, persisting to four weeks. Zymography and real-time PCR analysis showed that MMP9 expression is increased at two to four days, while MMP2 expression is increased at one to two weeks. Dual label confocal microscopy showed colocalization of NIRF imaging with neutrophils on day 2, and flow cytometric analysis confirmed that NIRF signal is associated with leukocytes in the infarct zone.
This study demonstrates that the activity of MMPs in the myocardium may be imaged using specific activity-dependent molecular probes.
matrix metalloproteinases; Myocardial infarction; Imaging; Gelatinase; Remodelling
Magnetic stain. Bacteria are often classified into Gram-positive and Gram-negative strains by their visual staining properties using crystal violet (CV), a triarylmethane dye. Here we show, that bioorthogonal modification of crystal violet with transcyclooctene (TCO) can be used to render Gram-positive bacteria magnetic with magneto-nanoparticles-Tetrazine (MNP-Tz). This allows for class specific automated magnetic detection, magnetic separation or other magnetic manipulations.
Cycloaddition reaction; Bacteria; Nanoparticles; Dyes; Gram Stain
To date, while various diagnostic approaches for pathogen detection have been proposed, most are too expensive, lengthy or limited in specificity for clinical use. Nanoparticle systems with unique material properties, however, circumvent these problems and offer improved accuracy over current methods. Herein, we present novel magneto-DNA probes capable of rapid and specific profiling of pathogens directly in clinical samples. A nanoparticle hybridisation assay, involving ubiquitous and specific probes that target bacterial 16S rRNAs, was designed to detect amplified target DNAs using a miniaturised nuclear magnetic resonance device. Ultimately, the magneto-DNA platform allowed both universal and specific detection of various clinically relevant bacterial species, with sensitivity down to single bacteria. Furthermore, the assay was robust and rapid, simultaneously diagnosing a panel of 13 bacterial species in clinical specimens within 2 hours. The generic platform described could be used to rapidly identify and phenotype pathogens for a variety of applications.
bacteria; 16S rRNA; magnetic nanoparticles; μNMR
Small molecule imaging: Aurora kinase A (AKA) was imaged in live cells using an in cellulo bioorthogonal two-step reaction with a small molecule and a fluorescent reporter. The small molecule was localized to spindle poles and microtubules during metaphase, consistent with the localization of both endogenous and GFP-/RFP-tagged AKA. Using this approach, changes in AKA distribution during mitosis were also observed.
Cycloaddition reaction; cell cycle; live cell imaging; aurora kinase; cancer
Many cancers shed malignant cells into the circulation. Albeit their rare frequency, these cancer cells serve both diagnostic and therapeutic purposes. However, their purification, quantification and characterization remain challenging. Here, we present a low-cost, rapid microfluidic cell sorter (μFCS) device for the detection and molecular analysis of circulating tumor cells (CTCs). The μFCS employs a weir-shaped microfluidic structure to separate and capture CTCs from unprocessed whole blood cells based on their size difference. The system further allows on-chip culture and molecular profiling of captured cancer cells, and provides easy cell retrieval for subsequent proteomic and genetic analyses. Using a mouse model of cancer metastasis, we show that the μFCS can enrich CTCs from whole blood to unmask their cancer genetic signature. With its rapid processing speed and versatility for downstream analyses, this platform could have a wide range of potential applications in clinical cancer diagnosis.
biotechnology; cellular profiling; circulating tumor cells; genetic analysis; microfluidics
When resectable, invasive pancreatic ductal adenocarcinoma (PDAC) is most commonly treated with surgery and radiochemotherapy. Given the intricate local anatomy and locoregional mode of dissemination, achieving clean surgical margins can be a significant challenge. On the basis of observations that cathepsin E (CTSE) is overexpressed in PDAC and that an United States Food and Drug Administration (FDA)-approved protease inhibitor has high affinity for CTSE, we have developed a CTSE optical imaging agent [ritonavir tetramethyl-BODIPY (RIT-TMB)] for potential intraoperative use. We show nanomolar affinity [half maximal inhibitory concentration (IC50) of 39.9 ± 1.2 nM] against CTSE of the RIT-TMB in biochemical assays and intracellular accumulation and target-to-background ratios that allow specific delineation of individual cancer cells. This approach should be useful for more refined surgical staging, planning, and resection with curative intent.
Repulsive guidance molecule (RGM) family members RGMa, RGMb/Dragon and RGMc/hemojuvelin were recently found to act as BMP co-receptors that enhance BMP signaling activity. While our previous studies have shown that hemojuvelin regulates hepcidin expression and iron metabolism through the BMP pathway, the role of the BMP signaling mediated by Dragon remains largely unknown. We have previously shown that Dragon is expressed in neural cells, germ cells and renal epithelial cells. Here, we demonstrate that Dragon is highly expressed in macrophages. Studies with RAW264.7 and J774 macrophage cell lines reveal that Dragon negatively regulates IL-6 expression in a BMP ligand dependent manner, via the p38 MAPK and Erk1/2 pathways but not the Smad1/5/8 pathway. We also generated Dragon knockout mice, and found that IL-6 is upregulated in macrophages and dendritic cells derived from whole lung tissue of these mice compared to respective cells derived from wild type littermates. These results indicate that Dragon is an important negative regulator of IL-6 expression in immune cells, and that Dragon-deficient mice may be a useful model for studying immune and inflammatory disorders.
To develop and validate a new fibrin targeted imaging agent that enables high-resolution near-infrared fluorescence (NIRF) imaging of deep venous thrombosis (DVT).
Near-infrared fluorescence (NIRF) imaging of fibrin could enable highly sensitive and noninvasive molecular imaging of thrombosis syndromes in vivo.
A fibrin-targeted peptide was conjugated to an NIR fluorophore Cy7, termed FTP11-Cy7. The NIRF peptide is based on a fibrin-specific imaging agent that has completed phase II clinical magnetic resonance imaging (MRI) trials. In vitro binding of FTP11-Cy7 to human plasma clots was assessed by fluorescence reflectance imaging (FRI). Next, FTP11-Cy7 was intravenously injected in mice with femoral DVT induced by topical 7.5% ferric chloride treatment. Intravital fluorescence microscopy (IVFM), and noninvasive fluorescence molecular tomography(FMT)-computed tomography (CT) were performed in mice (n = 32 total) with DVT, followed by histological analyses.
In vitro human clot-binding analyses showed a 6-fold higher NIRF clot target-to-background ratio (TBR) of FTP11-Cy7 than free Cy7 (6.3 ± 0.34 vs. 1.2 ± 0.03, p < 0.0001). The thrombus TBR of acute and sub-acute femoral DVT with FTP11-Cy7 obtained by IVFM was >400% higher than control free Cy7. Binding of FTP11-Cy7 to thrombi was blocked by a 100-fold excess of unlabeled competitor peptide both in vitro and in vivo (p < 0.001 for each). Histological analyses confirmed that FTP11-Cy7 specifically accumulated in thrombi. Noninvasive FMT-CT imaging of fibrin in jugular DVT via FMT-CT demonstrated strong NIRF signal in thrombi compared to sham-operated jugular veins (mean TBR = 3.5 ± 0.7 vs. 1.5 ± 0.3, p < 0.05).
The fibrin-targeted NIRF agent FTP11-Cy7 avidly and specifically binds human and murine thrombi, and enables sensitive, multimodal intravital and noninvasive NIRF molecular imaging detection of acute and sub-acute murine DVT in vivo.
fibrin; DVT; molecular imaging; fluorescence; optical imaging
Exaggerated and prolonged inflammation after myocardial infarction (MI) accelerates left ventricular remodeling. Inflammatory pathways may present a therapeutic target to prevent post-MI heart failure. However, the appropriate magnitude and timing of interventions are largely unknown, in part because noninvasive monitoring tools are lacking. We here employed nanoparticle-facilitated silencing of CCR2, the chemokine receptor that governs inflammatory Ly-6Chigh monocyte subset traffic, to reduce infarct inflammation in apoE−/− mice after MI. We used dual target PET/MRI of transglutaminase factor XIII (FXIII) and myeloperoxidase (MPO) activity to monitor how monocyte subset-targeted RNAi altered infarct inflammation and healing.
Methods and Results
Flow cytometry, gene expression analysis and histology revealed reduced monocyte numbers and enhanced resolution of inflammation in infarcted hearts of apoE−/− mice that were treated with nanoparticle-encapsulated siRNA. To follow extracellular matrix crosslinking non-invasively, we developed a fluorine-18 labeled PET agent (18F-FXIII). Recruitment of MPO-rich inflammatory leukocytes was imaged using a molecular MRI sensor of MPO activity (MPO-Gd). PET/MRI detected anti-inflammatory effects of intravenous nanoparticle-facilitated siRNA therapy (75% decrease of MPO-Gd signal, p<0.05) while 18F-FXIII PET reflected unimpeded matrix crosslinking in the infarct. Silencing of CCR2 during the first week after MI improved ejection fraction on day 21 after MI from 29 to 35% (p<0.05).
CCR2 targeted RNAi reduced recruitment of Ly-6Chigh monocytes, attenuated infarct inflammation and curbed post-MI left ventricular remodeling.
myocardial infarction; remodeling; monocytes; RNAi; PET/MRI
Real-time imaging of moving organs and tissues at microscopic resolutions represents a major challenge in studying complex biology in live systems. Here, we present a new technique for imaging the beating murine heart at the single cell level, based on a novel stabilizer setup combined with a gating acquisition algorithm. The method allowed serial in vivo fluorescence imaging of the beating heart in live mice in both confocal and nonlinear modes for several hours. We demonstrate the utility of this technique for in vivo optical sectioning and dual-channel time-lapse fluorescence imaging of cardiac ischemia. The generic method could be adapted to other moving organs and thus broadly facilitate in vivo microscopic investigations.
Recent advances in the field of intravital imaging have for the first time allowed us to conduct pharmacokinetic and pharmacodynamic studies at the single cell level in live animal models. Due to these advances, there is now a critical need for automated analysis of pharmacokinetic data. To address this, we began by surveying common thresholding methods to determine which would be most appropriate for identifying fluorescently labeled drugs in intravital imaging. We then developed a segmentation algorithm that allows semi-automated analysis of pharmacokinetic data at the single cell level. Ultimately, we were able to show that drug concentrations can indeed be extracted from serial intravital imaging in an automated fashion. We believe that the application of this algorithm will be of value to the analysis of intravital microscopy imaging particularly when imaging drug action at the single cell level.
We have developed a multifaceted highly specific reporter for multimodal in vivo imaging and applied it for detection of brain tumors. A metabolically biotinylated, membrane-bound form of Gaussia luciferase was synthesized, termed mbGluc-biotin. We engineered glioma cells to express this reporter and showed that brain tumor formation can be temporally imaged by bioluminescence following systemic administration of coelenterazine. Brain tumors expressing this reporter had high sensitivity for detection by magnetic resonance and fluorescence tomographic imaging upon injection of streptavidin conjugated to magnetic nanoparticles or fluorophore, respectively. Moreover, single photon emission computed tomography showed enhanced imaging of these tumors upon injection with streptavidin complexed to 111In-DTPA-biotin. This work shows for the first time a single small reporter ( 40 kDa) which can be monitored with most available molecular imaging modalities and can be extended for single cell imaging using intravital microscopy, allowing real-time tracking of any cell expressing it in vivo.
The ability to detect rare cells (< 100 cells per ml of whole blood) and obtain quantitative measurements of specific biomarkers on single cells is increasingly important in basic biomedical research. Implementing such methodology for widespread use in the clinic, however, has been hampered by low cell density, small sample sizes, and requisite sample purification. To overcome these challenges, we have developed a microfluidic chip-based micro-Hall detector (μHD), which can directly measure single, immunomagnetically tagged cells in whole blood. The μHD can detect single cells even in the presence of vast numbers of blood cells and unbound reactants, and does not require any washing or purification steps. In addition, the high bandwidth and sensitivity of the semiconductor technology used in the μHD enables high-throughput screening (currently ~107 cells/min). The clinical utility of the μHD chip was demonstrated by detecting circulating tumor cells in whole blood of 20 ovarian cancer patients at higher sensitivity than currently possible with clinical standards. Furthermore, the use of a panel of magnetic nanoparticles, distinguished with unique magnetization properties and bio-orthogonal chemistry, allowed simultaneous detection of the biomarkers EpCAM, HER2/neu, and EGFR on individual cells. This cost-effective, single-cell analytical technique is well-suited to perform molecular and cellular diagnosis of rare cells in the clinic.
Observing drug responses in the tumor microenvironment in vivo can be technically challenging. As a result, cellular responses to molecularly targeted cancer drugs are often studied in cell culture, which does not accurately represent the behavior of cancer cells growing in vivo. Using high resolution microscopy and fluorescently labeled genetic reporters for apoptosis, we developed an approach to visualize drug-induced cell death at single cell resolution in vivo. Stable expression of the mitochondrial intermembrane protein IMS-RP was established in human breast and pancreatic cancer cells. Automated image analysis was then used to quantify release of IMS-RP into the cytoplasm upon apoptosis and irreversible mitochondrial permeabilization. Both breast and pancreatic cancer cells showed higher basal apoptotic rates in vivo than in culture. To study drug-induced apoptosis, we exposed tumor cells to navitoclax (ABT-263), an inhibitor of Bcl-2, Bcl-xL, and Bcl-w, both in vitro and in vivo. Although the tumors responded to Bcl-2 inhibition in vivo, inducing apoptosis in around 20% of cancer cells, the observed response was much higher in cell culture. Together, our findings demonstrate an imaging technique that can be used to directly visualize cell death within the tumor microenvironment in response to drug treatment.
apoptosis; chemotherapy; imaging; breast cancer; pancreatic cancer
Myeloid cell content in atherosclerotic plaques associates with rupture and thrombosis. Thus, imaging of lesional monocyte and macrophages (Mo/Mϕ) could serve as a biomarker of disease progression and therapeutic intervention.
To noninvasively assess plaque inflammation with dextran nanoparticle-facilitated hybrid PET/MR imaging.
Methods and Results
Using clinically approved building blocks, we systematically developed 13nm polymeric nanoparticles consisting of crosslinked short chain dextrans which were modified with desferoxamine for zirconium-89 radiolabeling (89Zr-DNP) and a near infrared fluorochrome (VT680) for microscopic and cellular validation. Flow cytometry of cells isolated from excised aortas showed DNP uptake predominantly in Mo/Mϕ (76.7%) and lower signal originating from other leukocytes such as neutrophils and lymphocytes (11.8% and 0.7%, p<0.05 versus Mo/Mϕ). DNP colocalized with the myeloid cell marker CD11b on immunohistochemistry. PET/MRI revealed high uptake of 89Zr-DNP in the aortic root of ApoE−/− mice (standard uptake value, ApoE−/− mice versus wild type controls, 1.9±0.28 versus 1.3±0.03, p<0.05), corroborated by ex vivo scintillation counting and autoradiography. Therapeutic silencing of the monocyte-recruiting receptor CCR2 with siRNA decreased 89Zr-DNP plaque signal (p<0.05) and inflammatory gene expression (p<0.05).
Hybrid PET/MR imaging with a 13nm DNP enables noninvasive assessment of inflammation in experimental atherosclerotic plaques and reports on therapeutic efficacy of anti-inflammatory therapy.
PET/MRI; inflammation; atherosclerosis; molecular imaging; nanoparticles
Oligonucleotide hybridization was used as a cell-labeling method to significantly amplify the loading of magnetic probes onto target cells. The method utilized short oligonucleotides as the binding agents between antibodies and superparamagnetic iron oxide. This method not only enabled multiplexed analysis, but also allowed detection of multiple markers on a single sample containing only scant cell numbers.
magnetic labeling; oligonucleotide hybridization; magnetic resonance; multiplexed analysis; signal amplification
Pharmacokinetic analysis at the organ level provides insight into how drugs distribute throughout the body but cannot explain how drugs work at the cellular level. Here we demonstrate in vivo single cell pharmacokinetic imaging of PARP-1 inhibitors (PARPi) and model drug behavior under varying conditions. We visualize intracellular kinetics of PARPi distribution in real time, showing that PARPi reaches its cellular target compartment, the nucleus, within minutes in vivo both in cancer and normal cells in various cancer models. We also use these data to validate predictive finite element modeling. Our theoretical and experimental data indicate that tumor cells are exposed to sufficiently high PARPi concentrations in vivo and suggest that drug inefficiency is likely related to proteomic heterogeneity or insensitivity of cancer cells to DNA repair inhibition. This suggests that single cell pharmacokinetic imaging and derived modeling improves our understanding of drug action at single cell resolution in vivo.
Mutually orthogonal tetrazine–transcyclooctene and azide–cyclooctyne cycloaddition reactions were used simultaneously for the bioorthogonal labeling of two different live cell populations in the same culture. These small-molecule probes show good chemical reactivity and can be readily incorporated into biological systems.
azides; bioorthogonal reactions; cycloaddition; imaging; tetrazines