Microvesicles (MVs) are an increasingly important source for biomarker discovery and clinical diagnostics. The small size of MVs and their presence in complex biological environment, however, pose practical technical challenges, particularly when sample volumes are small. We herein present an acoustic nano-filter system that size-specifically separates MVs in a continuous and contact-free manner. The separation is based on ultrasound standing waves that exert differential acoustic force on MVs according to their size and density. By optimizing the design of the ultrasound transducers and underlying electronics, we were able to achieve a high separation yield and resolution. The “filter size-cutoff” can be controlled electronically in situ and enables versatile MV-size selection. We applied the acoustic nano-filter to isolate nanoscale (<200 nm) vesicles from cell culture media as well as MVs in stored red blood cell products. With the capacity for rapid and contact-free MV isolation, the developed system could become a versatile preparatory tool for MV analyses.
Nanotechnology; Microvesicles; Nanoparticle separation; Microfluidics; Acoustic wave
Accurate detection of recurrent same-site deep vein thrombosis (DVT) is a challenging clinical problem. As DVT formation and resolution are associated with a preponderance of inflammatory cells, we investigated whether noninvasive 18F-fluorodeoxyglucose (FDG)-PET imaging could identify inflamed, recently formed thrombi and thereby improve the diagnosis of recurrent DVT.
Methods and Results
We established a stasis-induced DVT model in murine jugular veins and also a novel model of recurrent stasis DVT in mice. C57BL/6 mice (n=35) underwent ligation of the jugular vein to induce stasis DVT. FDG-PET/CT was performed at DVT timepoints of day 2, 4, 7, 14, or 2+16 (same-site recurrent DVT at day 2 overlying a primary DVT at day 16). Antibody-based neutrophil depletion was performed in a subset of mice prior to DVT formation and FDG-PET/CT. In a clinical study, 38 patients with lower extremity DVT or controls undergoing FDG-PET were analyzed. Stasis DVT demonstrated that the highest FDG signal occurred at day 2, followed by a time-dependent decrease (p<0.05). Histological analyses demonstrated that thrombus neutrophils (p<0.01), but not macrophages, correlated with thrombus PET signal intensity. Neutrophil depletion decreased FDG signals in day 2 DVT compared to controls (p=0.03). Recurrent DVT demonstrated significantly higher FDG uptake than organized day 14 DVT (p=0.03). The FDG DVT signal in patients also exhibited a time-dependent decrease (p<0.01).
Noninvasive FDG-PET/CT identifies neutrophil-dependent thrombus inflammation in murine DVT, and demonstrates a time-dependent signal decrease in both murine and clinical DVT. FDG-PET/CT may offer a molecular imaging strategy to accurately diagnose recurrent DVT.
DVT; fluorodeoxyglucose; inflammation; recurrent DVT; PET; neutrophil
Supplemental Digital Content is available in the text.
Healing after myocardial infarction (MI) involves the biphasic accumulation of inflammatory Ly-6Chigh and reparative Ly-6Clow monocytes/macrophages. Excessive inflammation disrupts the balance between the 2 phases, impairs infarct healing, and contributes to left ventricle remodeling and heart failure. Lipoprotein-associated phospholipase A2 (Lp-PLA2), a member of the phospholipase A2 family of enzymes, produced predominantly by leukocytes, participates in host defenses and disease. Elevated Lp-PLA2 levels associate with increased risk of cardiovascular events across diverse patient populations, but the mechanisms by which the enzyme elicits its effects remain unclear. This study tested the role of Lp-PLA2 in healing after MI.
Methods and Results—
In response to MI, Lp-PLA2 levels markedly increased in the circulation. To test the functional importance of Lp-PLA2, we generated chimeric mice whose bone marrow–derived leukocytes were Lp-PLA2–deficient (bmLp-PLA2−/−). Compared with wild-type controls, bmLp-PLA2−/− mice subjected to MI had lower serum levels of inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1β, and IL-6, and decreased number of circulating inflammatory myeloid cells. Accordingly, bmLp-PLA2−/− mice developed smaller and less inflamed infarcts with reduced numbers of infiltrating neutrophils and inflammatory Ly-6Chigh monocytes. During the later, reparative phase, infarcts of bmLp-PLA2−/− mice contained Ly-6Clow macrophages with a skewed M2-prone gene expression signature, increased collagen deposition, fewer inflammatory cells, and improved indices of angiogenesis. Consequently, the hearts of bmLp-PLA2−/− mice healed more efficiently, as determined by improved left ventricle remodeling and ejection fraction.
Lp-PLA2 augments the inflammatory response after MI and antagonizes healing by disrupting the balance between inflammation and repair, providing a rationale for focused study of ventricular function and heart failure after targeting this enzyme acutely in MI.
heart failure; inflammation; macrophages; monocytes; myocardial infarction
Sepsis is a frequently fatal condition characterized by an uncontrolled and harmful host reaction to microbial infection. Despite the prevalence and severity of sepsis, we lack a fundamental grasp of its pathophysiology. Here we report that the cytokine interleukin (IL)-3 potentiates inflammation in sepsis. Using a mouse model of abdominal sepsis, we show that innate response activator (IRA) B cells produce IL-3, which induces myelopoiesis of Ly-6Chigh monocytes and neutrophils, and fuels a cytokine storm. IL-3 deficiency protects mice against sepsis. In humans with sepsis, high plasma IL-3 levels associate with high mortality even after adjusting for prognostic indicators. Altogether, this study deepens our understanding of immune activation, identifies IL-3 as an orchestrator of emergency myelopoiesis, and reveals a new therapeutic target for treating sepsis.
The high incidence of acute and chronic kidney injury due to various environmental factors such as heavy metals or chemicals has been a major problem in developing countries. However, the diagnosis of kidney injury in these areas can be more challenging due to the lack of highly sensitive and specific techniques that can be applied in point-of-care settings. To address this, we have developed a technique called ‘micro-urine nanoparticle detection (μUNPD)’, that allows the detection of trace amounts of molecular markers in urine. Specifically, this technique utilizes an automated on-chip assay followed by detection with a hand-held device for the read-out. Using the μUNPD technology, the kidney injury markers KIM-1 and Cystatin C were detected down to concentrations of 0.1 ng/ml and 20 ng/ml respectively, which meets the cut-off range required to identify patients with acute or chronic kidney injury. Thus, we show that the μUNPD technology enables point of care and non-invasive detection of kidney injury, and has potential for applications in diagnosing kidney injury with high sensitivity in resource-limited settings.
We have developed a series of new ultrafluorogenic probes in the blue-green spectrum that display fluorescence enhancement exceeding 11,000-fold. These fluorogenic dyes integrate a coumarin fluorochrome with the bioorthogonal tetrazine-transcyclooctene (TCO) chemistry platform. By exploiting highly efficient through bond energy transfer (TBET), these probes exhibit the highest brightness enhancements reported thus far for any bioorthogonal fluorogenic dyes. No-wash, fluorogenic imaging of diverse targets including cell surface receptors in cancer cells, mitochondria, and the actin cytoskeleton is possible within seconds with minimal background signal and no appreciable non-specific binding, opening the possibility for in vivo sensing.
click chemistry; energy transfer; fluorescence probes; fluorogenic probes; tetrazines
Macrophages populate the steady-state myocardium. Previously, all macrophages were thought to arise from monocytes; however, it emerged that in several organs tissue-resident macrophages may self-maintain through local proliferation.
To study the contribution of monocytes to cardiac resident macrophages in steady-state, after macrophage depletion in CD11bDTR/+ mice and in myocardial infarction.
Methods and Results
Using in vivo fate mapping and flow cytometry, we estimated that during steady-state the heart macrophage population turns over in about one month. To explore the source of cardiac resident macrophages, we joined the circulation of mice using parabiosis. After 6 weeks, we observed blood monocyte chimerism of 35.3±3.4% while heart macrophages showed a much lower chimerism of 2.7±0.5% (p<0.01). Macrophages self renewed locally through proliferation: 2.1±0.3% incorporated BrdU 2 hours after a single injection and 13.7±1.4% heart macrophages stained positive for the cell cycle marker Ki67. The cells likely participate in defense against infection, as we found them to ingest fluorescently labeled bacteria. In ischemic myocardium, we observed that tissue resident macrophages died locally while some also migrated to hematopoietic organs. If the steady-state was perturbed by coronary ligation or diphtheria toxin-induced macrophage depletion in CD11bDTR/+ mice, blood monocytes replenished heart macrophages. However, in the chronic phase after myocardial infarction, macrophages residing in the infarct were again independent from the blood monocyte pool, returning to the steady-state situation.
In this study we show differential contribution of monocytes to heart macrophages during steady-state, after macrophage depletion or in the acute and chronic phase after myocardial infarction. We found that macrophages participate in the immunosurveillance of myocardial tissue. These data correspond with previous studies on tissue-resident macrophages and raise important questions on the fate and function of macrophages during the development of heart failure.
Macrophage; monocyte; heart; myocardial infarction; myocardial
A magnetic ligation method is utilized for the detection of microRNAs amongst a complex biological background without polymerase chain reaction or nucleotide modification. The sandwich probes assay can be adapted to analyze a panel of microRNAs associated with cardiovascular diseases in heart tissue samples.
microRNA; enzymatic ligation; magnetic labeling; diagnostics; magnetic resonance
Fluorine containing fluorochromes represent important validation agents for PET imaging agents as they can be easily rapidly validated in cells by fluorescence imaging. In particular, the 18F-labeled BODIPY-FL fluorophore has emerged as an important platform but little is known about alternative 18F-labeling strategies or labeling on red shifted fluorophores. Here we explore the acid-catalyzed 18F/19F exchange on a range of commercially available N-hydroxysuccinimidyl ester and maleimide BODIPY fluorophores. We show this method to be a simple and efficient 18F-labeling strategy for a diverse span of fluorescent compounds, including a BODIPY modified PARP-1 inhibitor, and amine- and thiol-reactive BODIPY fluorophores.
18F-Fluorine; Molecular Imaging; BODIPY; Fluorescence; Positron emission tomography (PET)
Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.
Accurate spatiotemporal assessment of extracellular vesicle (EV) delivery and cargo RNA translation requires specific and robust live-cell imaging technologies. Here we engineer optical reporters to label multiple EV populations for visualization and tracking of tumour EV release, uptake and exchange between cell populations both in culture and in vivo. Enhanced green fluorescence protein (EGFP) and tandem dimer Tomato (tdTomato) were fused at NH2-termini with a palmitoylation signal (PalmGFP, PalmtdTomato) for EV membrane labelling. To monitor EV-RNA cargo, transcripts encoding PalmtdTomato were tagged with MS2 RNA binding sequences and detected by co-expression of bacteriophage MS2 coat protein fused with EGFP. By multiplexing fluorescent and bioluminescent EV membrane reporters, we reveal the rapid dynamics of both EV uptake and translation of EV-delivered cargo mRNAs in cancer cells that occurred within 1-hour post-horizontal transfer between cells. These studies confirm that EV-mediated communication is dynamic and multidirectional between cells with delivery of functional mRNA.
Extracellular vesicles (EVs) act as a conduit for intercellular communication through the exchange of cellular materials without direct cell-to-cell contacts. Here the authors develop a multiplexed reporter system that allows monitoring of EV exchange, cargo delivery and protein translation between different cell populations.
Tumor-stromal interactions are a determining factor in cancer progression. In vivo, the interaction interface is associated with spatially-resolved distributions of cancer and stromal phenotypes. Here, we establish a micropatterned tumor-stromal assay (μTSA) with laser capture microdissection to control the location of co-cultured cells and analyze bulk and interfacial tumor-stromal signaling in driving cancer progression. μTSA reveals a spatial distribution of phenotypes in concordance with human estrogen receptor-positive (ER+) breast cancer samples, and heterogeneous drug activity relative to the tumor-stroma interface. Specifically, an unknown mechanism of reversine is shown in targeting tumor-stromal interfacial interactions using ER+ MCF-7 breast cancer and bone marrow-derived stromal cells. Reversine suppresses MCF-7 tumor growth and bone metastasis in vivo by reducing tumor stromalization including collagen deposition and recruitment of activated stromal cells. This study advocates μTSA as a platform for studying tumor microenvironmental interactions and cancer field effects with applications in drug discovery and development.
Platinum(II) compounds, principally cisplatin and carboplatin, are commonly used front-line cancer therapeutics. Despite widespread use and the interest of developing new derivatives, including nanoformulations with improved properties, it has been difficult to visualize Pt compounds in live subjects, in real-time, and with subcellular resolution. Here we present four novel cisplatin- and carboplatin-derived fluorescent imaging compounds for quantitative intravital cancer imaging. We conjugate boron dipyromethene FL (BODIPY-FL) to PtII complexes for robust in vivo fluorescence, and show retained DNA-damaging and cytotoxic properties. We image pharmacokinetics and tumor uptake in a xenograft cancer mouse model. Finally, we present a genetic reporter of single-cell DNA damage for in vivo imaging, and simultaneously monitor Pt drug accumulation and resultant DNA damage in individual tumor cells, at subcellular resolution, and in real-time in a live animal model of cancer.
intravital imagingagents; pharmacology; fluorescent probes; DNA damage; pharmacokinetics
Targeting DNA repair pathways is a powerful strategy to treat cancers. To gauge efficacy in vivo, typical response markers include late stage effects such as tumor shrinkage, progression free survival, or invasive repeat biopsies. These approaches are often difficult to answer critical questions such as how a given drug affects single cell populations as a function of dose and time, distance from microvessels or how drug concentration (pharmacokinetics) correlates with DNA damage (pharmacodynamics). Here, we established a single-cell in vivo pharmacodynamic imaging read-out based on a truncated 53BP1 double-strand break reporter to determine whether or not poly(ADP-ribose) polymerase (PARP) inhibitor treatment leads to accumulation of DNA damage. Using this reporter, we show that not all PARP inhibitor treated tumors incur an increase in DNA damage. The method provides a framework for single cell analysis of cancer therapeutics in vivo.
Real-time monitoring of drug efficacy in glioblastoma multiforme (GBM) is a major clinical problem as serial re-biopsy of primary tumours is often not a clinical option. MGMT (O6-methylguanine DNA methyltransferase) and APNG (alkylpurine-DNA-N-glycosylase) are key enzymes capable of repairing temozolomide-induced DNA damages and their levels in tissue are inversely related to treatment efficacy. Yet, serial clinical analysis remains difficult, and, when done, primarily relies on promoter methylation studies of tumour biopsy material at the time of initial surgery. Here we present a microfluidic chip to analyse mRNA levels of MGMT and APNG in enriched tumour exosomes obtained from blood. We show that exosomal mRNA levels of these enzymes correlate well with levels found in parental cells and that levels change considerably during treatment of seven patients. We propose that if validated on a larger cohort of patients, the method may be used to predict drug response in GBM patients.
Predicting and monitoring chemotherapy response remains a challenge for glioma treatment. Here the authors show that a microfluidic device can isolate glioma-derived exosomes from patient blood and accurately determine the levels of mRNA of key enzymes important for chemoresponsiveness.
Intravital imaging microscopy (i.e., imaging in live animals at microscopic resolution) has become an indispensable tool for studying the cellular micro-dynamics in cancer, immunology and neurobiology. High spatial and temporal resolution, combined with large penetration depth and multi-reporter visualization capability make fluorescence intravital microscopy compelling for heart imaging. However, tissue motion caused by cardiac contraction and respiration critically limits its use. As a result, in vitro cell preparations or non-contracting explanted heart models are more commonly employed. Unfortunately, these approaches fall short of understanding the more complex host physiology that may be dynamic and occur over longer periods of time. In this review, we report on novel technologies, which have been recently developed by our group and others, aimed at overcoming motion-induced artifacts and capable of providing in vivo subcellular resolution imaging in the beating mouse heart. The methods are based on mechanical stabilization, image processing algorithms, gated/triggered acquisition schemes or a combination of both. We expect that in the immediate future all these methodologies will have considerable applications in expanding our understanding of the cardiac biology, elucidating cardiomyocyte function and interactions within the organism in vivo, and ultimately improving the treatment of cardiac diseases.
cardiac imaging; motion artifacts; laser scanning microscopy; motion compensation; image stabilization
Healing after myocardial infarction (MI) involves the biphasic accumulation of inflammatory Ly-6Chigh and reparative Ly-6Clow monocytes/macrophages (Mo/MΦ). According to one model, Mo/MΦ heterogeneity in the heart originates in the blood and involves the sequential recruitment of distinct monocyte subsets that differentiate to distinct macrophages. Alternatively, heterogeneity may arise in tissue from one circulating subset via local macrophage differentiation and polarization. The orphan nuclear hormone receptor, Nr4a1, is essential to Ly-6Clow monocyte production but dispensable to Ly-6Clow macrophage differentiation; dependence on Nr4a1 can thus discriminate between systemic and local origins of macrophage heterogeneity.
This study tested the role of Nr4a1 in MI in the context of the two Mo/MΦ accumulation scenarios.
Methods and Results
We show that Ly-6Chigh monocytes infiltrate the infarcted myocardium and, unlike Ly-6Clow monocytes, differentiate to cardiac macrophages. In the early, inflammatory phase of acute myocardial ischemic injury, Ly-6Chigh monocytes accrue in response to a brief Ccl2 burst. In the second, reparative phase, accumulated Ly-6Chigh monocytes give rise to reparative Ly-6Clow F4/80high macrophages that proliferate locally. In the absence of Nr4a1, Ly-6Chigh monocytes express heightened levels of Ccr2 on their surface, avidly infiltrate the myocardium, and differentiate to abnormally inflammatory macrophages, which results in defective healing and compromised heart function.
Ly-6Chigh monocytes orchestrate both inflammatory and reparative phases during MI and depend on Nr4a1 to limit their influx and inflammatory cytokine expression.
Monocyte; macrophage; myocardial infarction; nuclear hormone receptor; healing
Cellular up-regulation of multidrug
resistance protein 1 (MDR1)
is a common cause for resistance to chemotherapy; development of third
generation MDR1 inhibitors—several of which contain a common
underway. Efficacy of these agents has been difficult to ascertain,
partly due to a lack of pharmacokinetic reporters for quantifying
inhibitor localization and transport dynamics. Some of the recent
third generation inhibitors have a pendant heterocycle, for example,
a chromone moiety, which we hypothesized could be converted to a fluorophore.
Following synthesis and teasing of a small set of analogues, we identified
one lead compound that can be used as a cellular imaging agent that
exhibits structural similarity and behavior akin to the latest generation
of MDR1 inhibitors.
Here we evaluated a series of Si-derivatized rhodamine (SiR) dyes for their ability to visualize a model drug in live cells. We show that a charge neutral SiR derivative (but not others) can indeed be used to follow the intracellular location of the model therapeutic drug in GFP cells
The aim of the study was to test wether silencing of the transcription factor Interferon Regulatory Factor 5 (IRF5) in cardiac macrophages improves infarct healing and attenuates post-MI remodeling.
In healing wounds, M1➛M2 macrophage phenotype transition supports resolution of inflammation and tissue repair. Persistence of inflammatory M1 macrophages may derail healing and compromise organ functions. The transcription factor IRF5 promotes genes associated with M1 macrophages.
Here we used nanoparticle-delivered siRNA to silence the transcription factor IRF5 in macrophages residing in myocardial infarcts (MI) and in surgically induced skin wounds in mice.
Infarct macrophages expressed high levels of IRF5 during the early inflammatory wound healing stages (day 4 after coronary ligation) whereas expression of the transcription factor decreased during the resolution of inflammation (day 8). Following in vitro screening, we identified an siRNA sequence that, when delivered by nanoparticles to wound macrophages, efficiently suppressed expression of IRF5 in vivo. Reduction of IRF5 expression, a factor that regulates macrophage polarization, reduced inflammatory M1 macrophage markers, supported resolution of inflammation, accelerated cutaneous and infarct healing and attenuated development of post-MI heart failure after coronary ligation as measured by protease targeted FMT-CT imaging and cardiac MRI (p<0.05 respectively).
This work identifies a new therapeutic avenue to augment resolution of inflammation in healing infarcts by macrophage phenotype manipulation. This therapeutic concept may be used to attenuate post-MI remodeling and heart failure.
Atherosclerotic lesions grow via the accumulation of leukocytes and oxidized lipoproteins in the vessel wall. Leukocytes can attenuate or augment atherosclerosis through the release of cytokines, chemokines, and other mediators. Deciphering how leukocytes develop, oppose and complement each other’s function, and shape the course of disease, can illuminate understanding of atherosclerosis. Innate response activator (IRA) B cells are a recently described population of GM-CSF-secreting cells of hitherto unknown function in atherosclerosis.
Methods and Results
Here we show that IRA B cells arise during atherosclerosis in mice and humans. In response to high cholesterol diet, IRA B cell numbers increase preferentially in secondary lymphoid organs via Myd88-dependent signaling. Mixed chimeric mice lacking B cell-derived GM-CSF develop smaller lesions with fewer macrophages and effector T cells. Mechanistically, IRA B cells promote the expansion of classical dendritic cells, which then generate IFNγ-producing TH1 cells. This IRA B cell-dependent TH1 skewing manifests in an IgG1 to IgG2c isotype switch in the immunoglobulin response against oxidized lipoproteins.
GM-CSF-producing IRA B cells alter adaptive immune processes and shift the leukocyte response toward a TH1-associated mileu that aggravates atherosclerosis.
atherosclerosis; immunology; B cells; Dendritic cells; T cells; Granulocyte macrophage colony-stimulating factor
Advances in nanotechnology and microfluidics are enabling the analysis of small amounts of human cells. We tested whether recently developed micro-nuclear magnetic resonance (μNMR) technology could be leveraged for diagnosing pulmonary malignancy using fine needle aspirate (FNA) of primary lesions and/or peripheral blood samples. We enrolled a cohort of 35 patients referred for CT biopsy of primary pulmonary nodules, liver or adrenal masses and concurrently obtained FNA and peripheral blood samples. FNA sampling yielded sufficient material for μNMR analysis in 91% of cases and had a sensitivity and specificity of 91.6 and 100% respectively. Interestingly, among blood samples with positive circulating tumor cells (CTC), μNMR analysis of each patient's peripheral blood led to similar diagnosis (malignant vs benign) and differential diagnosis (lung malignancy subtype) in 100% and 90% (18/20) of samples, respectively. μNMR appears to be valuable, non-invasive adjunct in the diagnosis of lung cancer.
Bioorthogonal click chemistry; Circulating Tumor Cell; μNMR; Iron-oxide nanoparticles; Point of care diagnosis
Exosomes show potential for cancer diagnostics because they transport molecular contents of the cells from which they originate. Detection and molecular profiling of exosomes is technically challenging and often requires extensive sample purification and labeling. Here we describe a label-free, high-throughput approach for quantitative analyses of exosomes. Our nano-plasmonic exosome (nPLEX) assay is based on transmission surface plasmon resonance through periodic nanohole arrays. Each array is functionalized with antibodies to enable profiling of exosome surface proteins and proteins present in exosome lysates. We show that this approach offers improved sensitivity over previous methods, enables portable operation when integrated with miniaturized optics and allows retrieval of exosomes for further study. Using nPLEX to analyze ascites samples from ovarian cancer patients, we find that exosomes derived from ovarian cancer cells can be identified by their expression of CD24 and EpCAM, suggesting the potential of exosomes for diagnostics.
exosomes; surface plasmon resonance; periodic nanohole arrays; cancer; label-free sensing
The use of spectrally distinguishable fluorescent dyes enables imaging of multiple targets. However, in two-photon microscopy the number of fluorescent labels with distinct emission spectra that can be effectively excited and resolved is constrained by the confined tuning range of the excitation laser and the broad and overlapping nature of fluorophore two-photon absorption spectra. This limitation effectively reduces the number of available imaging channels. Here, we demonstrate that two-photon steady state anisotropy imaging (2PSSA) offers the capability to resolve otherwise unresolvable fluorescent tracers both in live cells and in mouse tumor models. This approach expands the number of biological targets that can be imaged simultaneously, increasing the total amount of information that can be obtained through imaging.
Sensitive, rapid and phenotype-specific enumeration of pathogens is essential for the diagnosis of infectious disease, monitoring of food chains, and for defense against bioterrorism. Microbiological culture and genotyping, techniques that sensitively and selectively detect bacteria in laboratory settings, have limited application in clinical environments due to high cost, slow response times, and the need for specially trained staff and laboratory infrastructure. To address these challenges, we developed a microfluidic chip-based micro-Hall (μHall) platform capable of measuring single, magnetically tagged bacteria directly in clinical specimens with minimal sample processing. We demonstrated the clinical utility of the μHall chip by enumerating Gram-positive bacteria using a two-step bioorthogonal labeling procedure. The overall detection limit of the system was similar to that of culture tests (~10 bacteria), but the assay time was 50-times faster. This low-cost, single-cell analytical technique is especially well-suited to diagnose infectious diseases in resource-limited clinical settings.
μHall; bacterial detection; magnetic nanoparticles