Current intraoperative imaging systems are typically not able to provide ‘sharp’ images over entire large areas or entire organs. Distinct structures such as tissue margins or groups of malignant cells are therefore often difficult to detect, especially under low signal-to-noise-ratio conditions. In this report, we introduce a noise suppressed multifocus image fusion algorithm, that provides detailed reconstructions even when images are acquired under sub-optimal conditions, such is the case for real time fluorescence intraoperative surgery. The algorithm makes use of the Anscombe transform combined with a multi-level stationary wavelet transform with individual threshold-based shrinkage. While the imaging system is integrated with a respiratory monitor triggering system, it can be easily adapted to any commercial imaging system. The developed algorithm is made available as a plugin for Osirix.
Intraoperative detection of small malignant fluorescent cells using the proposed noise suppressed multifocus image fusion system. Red/Yellow circles indicate small groups of malignant cells.
Intravital fluorescence microscopy, through extended penetration depth and imaging resolution, provides the ability to image at cellular and subcellular resolution in live animals, presenting an opportunity for new insights into in vivo biology. Unfortunately, physiological induced motion components due to respiration and cardiac activity are major sources of image artifacts and impose severe limitations on the effective imaging resolution that can be ultimately achieved in vivo. Here we present a novel imaging methodology capable of automatically removing motion artifacts during intravital microscopy imaging of organs and orthotopic tumors. The method is universally applicable to different laser scanning modalities including confocal and multiphoton microscopy, and offers artifact free reconstructions independent of the physiological motion source and imaged organ. The methodology, which is based on raw data acquisition followed by image processing, is here demonstrated for both cardiac and respiratory motion compensation in mice heart, kidney, liver, pancreas and dorsal window chamber.
During the inflammatory response that drives atherogenesis, macrophages accumulate progressively in the expanding arterial wall1,2. The observation that circulating monocytes give rise to lesional macrophages3–9 has reinforced the concept that monocyte infiltration dictates macrophage build-up. Recent work indicates, however, that macrophages do not depend on monocytes in some inflammatory contexts10. We therefore revisited the mechanism of macrophage accumulation in atherosclerosis. We show that murine atherosclerotic lesions experience a surprisingly rapid, 4-week, cell turnover. Replenishment of macrophages in these experimental atheromata depends predominantly on local macrophage proliferation rather than monocyte influx. The microenvironment orchestrates macrophage proliferation via the involvement of scavenger receptor (SR)-A. Our study reveals macrophage proliferation as a key event in atherosclerosis and identifies macrophage self-renewal as a therapeutic target for cardiovascular disease.
Intravital microscopy has emerged in the recent decade as an indispensible imaging modality for the study of the micro-dynamics of biological processes in live animals. Technical advancements in imaging techniques and hardware components, combined with the development of novel targeted probes and new mice models, have enabled us to address long-standing questions in several biology areas such as oncology, cell biology, immunology and neuroscience. As the instrument resolution has increased, physiological motion activities have become a major obstacle that prevents imaging live animals at resolutions analogue to the ones obtained in vitro. Motion compensation techniques aim at reducing this gap and can effectively increase the in vivo resolution. This paper provides a technical review of some of the latest developments in motion compensation methods, providing organ specific solutions.
Intravital microscopy; image stabilization; in vivo imaging; motion artifact and motion compensation
Macrophages frequently infiltrate tumors and can enhance cancer growth, yet the origins of the macrophage response are not well understood. Here we address molecular mechanisms of macrophage production in a conditional mouse model of lung adenocarcinoma. We report that over-production of the peptide hormone Angiotensin II (AngII) in tumor-bearing mice amplifies self-renewing hematopoietic stem cells (HSCs) and macrophage progenitors. The process occurred in the spleen but not the bone marrow, and was independent of hemodynamic changes. The effects of AngII required direct hormone ligation on HSCs, depended on S1P1 signaling, and allowed the extramedullary tissue to supply new tumor-associated macrophages throughout cancer progression. Conversely, blocking AngII production prevented cancer-induced HSC and macrophage progenitor amplification and thus restrained the macrophage response at its source. These findings indicate that AngII acts upstream of a potent macrophage amplification program and that tumors can remotely exploit the hormone’s pathway to stimulate cancer-promoting immunity.
Transient cell therapy is an emerging drug class that requires new approaches for pharmacological monitoring during use. Human mesenchymal stem cells (MSCs) are a clinically-tested transient cell therapeutic that naturally secrete anti-inflammatory factors to attenuate immune-mediated diseases. MSCs were used as a proof-of-concept with the hypothesis that measuring the release of secreted factors after cell transplantation, rather than the biodistribution of the cells alone, would be an alternative monitoring tool to understand the exposure of a subject to MSCs. By comparing cellular engraftment and the associated serum concentration of secreted factors released from the graft, we observed clear differences between the pharmacokinetics of MSCs and their secreted factors. Exploration of the effects of natural or engineered secreted proteins, active cellular secretion pathways, and clearance mechanisms revealed novel aspects that affect the systemic exposure of the host to secreted factors from a cellular therapeutic. We assert that a combined consideration of cell delivery strategies and molecular pharmacokinetics can provide a more predictive model for outcomes of MSC transplantation and potentially other transient cell therapeutics.
nanoparticles; iron; oxidation; magnetism
We report a photoactivable nanoprobe for cell labeling and tracking. The nanoprobe enables all targeted cells to be imaged (at 680 nm) as well as specific cells to be photoactivated using 405 nm light. Photoactivated cells can then be tracked (at 525 nm) spatiotemporally in a separate channel over prolonged periods.
Photoactivation; nanoprobe; cell labeling; targeting; biomarkers
Longitudinal analyses of single cell lineages over prolonged periods have been challenging particularly in processes characterized by high cell turn-over such as inflammation, proliferation, or cancer. RGB marking has emerged as an elegant approach for enabling such investigations. However, methods for automated image analysis continue to be lacking. Here, to address this, we created a number of different multicolored poly- and monoclonal cancer cell lines for in vitro and in vivo use. To classify these cells in large scale data sets, we subsequently developed and tested an automated algorithm based on hue selection. Our results showed that this method allows accurate analyses at a fraction of the computational time required by more complex color classification methods. Moreover, the methodology should be broadly applicable to both in vitro and in vivo analyses.
LeGO cells; intravital imaging; automated cell analysis; RGB marking; tumor heterogeneity
Macrophages (MΦ) predominate among the inflammatory cells in rejecting allografts. These innate immune cells, in addition to allospecific T cells, can damage cardiomyocytes directly.
Methods and Results
We explored if sensitive PET/CT imaging of MΦ-avid nanoparticles detects rejection of heart allografts in mice. In addition, we employed the imaging method to follow the immunomodulatory impact of angiotensin converting enzyme inhibititor therapy (ACEi) on myeloid cells in allografts. Dextran nanoparticles were derivatized with the PET isotope copper-64 and imaged seven days after transplantation. C57/BL6 recipients of BALB/c allografts displayed robust PET signal (standard uptake value allograft, 2.8±0.3; isograft control, 1.7±0.2; p<0.05). Autoradiography and scintillation counting confirmed the in vivo findings. We then imaged the effects of ACEi (5mg/kg Enalapril). ACEi significantly decreased nanoparticle signal (p<0.05). Histology and flow cytometry showed a reduced number of myeloid cells in the graft, blood and lymph nodes, as well as diminished antigen presentation (p<0.05 versus untreated allografts). ACEi also significantly prolonged allograft survival (12 versus 7 days, p<0.0001).
Nanoparticle MΦ PET-CT detects heart transplant rejection and predicts organ survival by reporting on myeloid cells.
heart transplantation; macrophages; imaging; PET/CT
Reprogramming of cellular metabolism is a key event during tumorigenesis. Despite being known for decades (Warburg effect), the molecular mechanisms regulating this switch remained unexplored. Here, we identify SIRT6 as a novel tumor suppressor that regulates aerobic glycolysis in cancer cells. Importantly, loss of SIRT6 leads to tumor formation without activation of known oncogenes, while transformed SIRT6-deficient cells display increased glycolysis and tumor growth, suggesting that SIRT6 plays a role in both establishment and maintenance of cancer. Using a conditional SIRT6 allele, we show that SIRT6 deletion in vivo increases the number, size and aggressiveness of tumors. SIRT6 also functions as a novel regulator of ribosome metabolism by co-repressing MYC transcriptional activity. Lastly, SIRT6 is selectively downregulated in several human cancers, and expression levels of SIRT6 predict prognosis and tumor-free survival rates, highlighting SIRT6 as a critical modulator of cancer metabolism. Our studies reveal SIRT6 to be a potent tumor suppressor acting to suppress cancer metabolism.
OBJECTIVE: The objective of this study is to assess lymphotropic nanoparticle-enhanced magnetic resonance imaging (LNMRI) in identifying malignant nodal involvement in patients with pancreatic ductal adenocarcinoma. METHODS: Magnetic resonance imaging was performed in 13 patients with known or high index of suspicion of pancreatic cancer and who were scheduled for surgical resection. Protocols included T2*-weighted imaging before and after administration of Ferumoxytol (Feraheme) for the evaluation of lymph node involvement. Eleven of the 13 patients underwent a Whipple procedure and lymph node dissection. Nodes that lacked contrast uptake were deemed malignant, and those that demonstrated homogeneous uptake were deemed benign. RESULTS: A total of 264 lymph nodes were resection, of which 17 were malignant. The sensitivity and specificity of LNMRI was 76.5% and 98.4% at a nodal level and 83.3% and 80% at a patient level. CONCLUSION: LNMRI demonstrated high sensitivity and specificity in patients with pancreatic ductal adenocarcinoma.
DNA barcoding is an attractive technology as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here, we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.
Until recently, the idea of observing life deep within the tissues of a living mouse, at a resolution sufficient to pick out cellular behaviors and molecular signals underlying them, remained a much-coveted dream. Now, a new era of intravital fluorescence microscopy has dawned. In this Primer, we review the technologies that made this revolution possible, and demonstrate how intravital imaging is beginning to provide quantitative and dynamic insights into cell biology, immunology, tumor biology and neurobiology.
The hepatocyte growth factor receptor (MET) is a receptor tyrosine kinase (RTK) that has emerged as an important cancer target. Consequently, a number of different inhibitors varying in specificity are currently in clinical development. However, to date, it has been difficult to visualize MET expression, intracellular drug distribution and small molecule MET inhibition. Using a bioorthogonal approach, we have developed two companion imaging drugs based on both mono- and polypharmacological MET inhibitors. We show exquisite drug and target co-localization that can be visualized at single-cell resolution. The developed agents may be useful chemical biology tools to investigate single-cell pharmacokinetics and pharmacodynamics of MET inhibitors.
To use molecular imaging targeting coagulation pathway and inflammation to 1) better understand the pathophysiology of silent brain ischemia (SBI) and 2) monitor the effects of factor XIIa inhibition.
Silent brain ischemia can be observed in patients who undergo invasive vascular procedures. Unlike acute stroke, the diffuse nature of SBI and its less tangible clinical symptoms make this disease difficult to diagnose and treat.
We induced SBI in mice by intra-arterial injection of fluorescently-labeled microbeads or fractionated clot into the carotid artery. After SBI induction, diffusion-weighted (DWI) magnetic resonance imaging (MRI) was performed to confirm the presence of microinfarcts in asymptomatic mice. Molecular imaging targeting the downstream factor XIII activity (SPECT-CT) at three hours and myeloperoxidase activity (MRI) on day three after SBI induction were performed, without and with the intravenous administration of a recombinant selective factor XIIa inhibitor derived from the hematophagous insect Triatoma infestans (rHA-Infestin-4). Statistical comparisons between two groups were evaluated by the Student’s t-test or the Mann-Whitney U test.
In SBI-induced mice, we found abnormal activation of the coagulation cascade (factor XIII activity) and increased inflammation (myeloperoxidase activity) close to where emboli lodge in the brain. rHA-Infestin-4 administration significantly reduced ischemic damage (53–85% reduction of infarct volume, p < 0.05) and pathological coagulation (35–39% reduction of factor XIII activity, p < 0.05) without increasing hemorrhagic frequency. Myeloperoxidase activity, when normalized to the infarct volume, did not significantly change with rHA-Infestin-4 treatment, suggesting that this treatment does not further decrease inflammation other than that resulted from the reduction in infarct volume.
Focal intracerebral clotting and inflammatory activity are part of the pathophysiology underlying SBI. Inhibiting factor XIIa with rHA-Infestin-4 may present a safe and effective treatment to decrease the morbidity from SBI.
Silent brain ischemia; coagulation; inflammation; imaging; infestin
Assessment of thrombus inflammation in vivo could provide new insights into deep vein thrombosis (DVT) resolution. Here we develop and evaluate two integrated fluorescence molecular-structural imaging strategies to quantify DVT-related inflammation and architecture, and to assess the effect of thrombus inflammation on subsequent DVT resolution in vivo.
Methods and Results
Murine DVT were created with topical 5% FeCl3 application to thigh or jugular veins (n=35). On day 3, mice received macrophage and matrix metalloproteinase (MMP) activity fluorescence imaging agents. On day 4, integrated assessment of DVT inflammation and architecture was performed using confocal fluorescence intravital microscopy (IVM). Day 4 analyses showed robust relationships among in vivo thrombus macrophages, MMP activity, and FITC-dextran deposition (r>0.70, p<0.01). In a serial two-timepoint study, mice with DVT underwent IVM at day 4 and at day 6. Analyses revealed that the intensity of thrombus inflammation at day 4 predicted the magnitude of DVT resolution at day 6 (p<0.05). In a second approach, noninvasive fluorescence molecular tomography-computed tomography (FMT-CT) was employed, and detected macrophages within jugular DVT (p<0.05 vs. sham-controls).
Integrated fluorescence molecular-structural imaging demonstrates that the DVT-induced inflammatory response can be readily assessed in vivo, and can inform the magnitude of thrombus resolution.
deep vein thrombosis; inflammation; molecular imaging; intravital microscopy; post-thrombotic syndrome
The sonic hedgehog (Shh) pathway has an established role in pancreatic cancer (pancreatic adenocarcinoma [PDAC]). We tested whether magnetic resonance imaging measures of vascular volume fraction (VVF) using magnetic iron oxide nanoparticles are sensitive to the antiangiogenic effect of targeted Shh therapies in a PDAC xenograft model.
Pancreatic adenocarcinoma xenograft lines were subcutaneously implanted into nude mice (n = 19 samples within 4 groups). Therapies were targeted to 3 loci of the Shh signaling pathway (anti-Shh antibody, cyclopamine, or forskolin). Magnetic resonance imaging (4.7-T Bruker Pharmascan) was performed (after 1 week of intraperitoneal therapy) before and after intravenous injection of MION-47. Vascular volume fraction was quantified as ΔR2 (from multicontrast T2 sequences) and normalized to an assumed VVF in muscle of 3%. Linear regression compared VVF to histological indices including microvessel density (MVD), viable gland density (VGD), and proliferative index (PI).
In response to anti-Hh treatment, tumors showed a decrease in VGD, PI, MVD, and VVF compared with controls (P < 0.001). Vascular volume fraction was compared with histological indicators of response: PI (R2 = 0.88; P < 0.05), VGD (R2 = 0.87; P< 0.05), and MVD (R2 = 0.85; P < 0.05).
Magnetic resonance imaging VVF using magnetic iron oxide nanoparticles may serve as a noninvasive measure of biological response to Shh PDAC therapy with easy translation to the clinic.
angiogenesis; magnetic resonance imaging; nanoparticle; pancreatic cancer; sonic hedgehog
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.
In vivo imaging is often severely compromised by cardiovascular and respiratory motion. Highly successful motion compensation techniques have been developed for clinical imaging (e.g. magnetic resonance imaging) but the use of more advanced techniques for intravital microscopy is largely unexplored. Here, we implement a sequential cardiorespiratory gating scheme (SCG) for averaged microscopy. We show that SCG is very efficient in eliminating motion artifacts, is highly practical, enables high signal-to-noise ratio (SNR) in vivo imaging, and yields large field of views. The technique is particularly useful for high-speed data acquisition or for imaging scenarios where the fluorescence signal is not significantly above noise or background levels.
(180.0180) Microscopy; (170.2520) Fluorescence microscopy; (170.5810) Scanning microscopy; (170.3880) Medical and biological imaging
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