To determine whether metabolic imaging with the use of fluorescence lifetime imaging microscopy (FLIM) identifies metabolic differences between normal oocytes and those with metabolic dysfunction.
Academic research laboratories.
Oocytes from mice with global knockout of Clpp (caseinolytic peptidase P; n = 52) were compared with wild-type (WT) oocytes (n = 55) as a model of severe oocyte dysfunction. Oocytes from old mice (1 year old; n = 29) were compared with oocytes from young mice (12 weeks old; n = 35) as a model of mild oocyte dysfunction.
MAIN OUTCOME MEASURE(S):
FLIM was used to measure the naturally occurring nicotinamide adenine dinucleotide dehydrogenase (NADH) and flavin adenine dinucleotide (FAD) autofluorescence in individual oocytes. Eight metabolic parameters were obtained from each measurement (4 per fluorophore): short (τ1) and long (τ2) fluorescence lifetime, fluorescence intensity (I), and fraction of the molecule engaged with enzyme (F). Reactive oxygen species (ROS) levels and blastocyst development rates were measured to assess illumination safety.
In Clpp-knockout oocytes compared with WT, FAD τ1 and τ2 were longer and I was higher, NADH τ2 was longer, and F was lower. In old oocytes compared with young ones, FAD τ1 was longer and I was lower, NADH τ1 and τ2 were shorter, and I and F were lower. FLIM did not affect ROS levels or blastocyst development rates.
FLIM parameters exhibit strong differentiation between Clpp-knockout versus WT, and old versus young oocytes, FLIM could potentially be used as a noninvasive tool to assess mitochondrial function in oocytes.
The observed MIC may depend on the number of bacteria initially inoculated into the assay. This phenomenon is termed the inoculum effect(IE) and is often most pronounced for β-lactams in strains expressing β-lactamase enzymes. The Clinical and Laboratory Standards Institute (CLSI)-recommended inoculum is 5 × 105 CFU ml-1 with an acceptable range of 2 × 105 to 8 × 105 CFU ml-1 IE testing is typically performed using an inoculum 100-fold greater than the CLSI-recommended inoculum. Therefore, it remains unknown whether the IE influences MICs during testing performed according to CLSI guidelines. Here, we utilized inkjet printing technology to test the IE on cefepime, meropenem, and ceftazidime-avibactam. First, we determined that the inkjet dispense volume correlated well with the number of bacteria delivered to microwells in 2-fold (R2 = 0.99) or 1.1-fold (R2 = 0.98) serial dilutions. We then quantified the IE by dispensing orthogonal titrations of bacterial cells and antibiotics. For cefepime-resistant and susceptible dose-dependent strains, a 2-fold increase in inoculum resulted in a 1.6 log2-fold increase in MIC. For carbapenemase-producing strains, each 2-fold reduction in inoculum resulted in a 1.26 log2-fold reduction in meropenem MIC. At the lower end of the CLSI-allowable inoculum range, minor error rates of 34.8% were observed for meropenem when testing a resistant-strain set. Ceftazidime-avibactam was not subject to an appreciable IE. Our results suggest that IE is sufficiently pronounced for meropenem and cefepime in multidrug-resistant Gram-negative pathogens to affect categorical interpretations during standard laboratory testing.
Electrical stimulation of the central and peripheral nervous systems - such as deep brain stimulation, spinal cord stimulation, and epidural cortical stimulation are common therapeutic options increasingly used to treat a large variety of neurological and psychiatric conditions. Despite their remarkable success, there are limitations which if overcome, could enhance outcomes and potentially reduce common side-effects. Micromagnetic stimulation (μMS) was introduced to address some of these limitations. One of the most remarkable properties is that μMS is theoretically capable of activating neurons with specific axonal orientations. Here, we used computational electromagnetic models of the μMS coils adjacent to neuronal tissue combined with axon cable models to investigate μMS orientation-specific properties. We found a 20-fold reduction in the stimulation threshold of the preferred axonal orientation compared to the orthogonal direction. We also studied the directional specificity of μMS coils by recording the responses evoked in the inferior colliculus of rodents when a pulsed magnetic stimulus was applied to the surface of the dorsal cochlear nucleus. The results confirmed that the neuronal responses were highly sensitive to changes in the μMS coil orientation. Accordingly, our results suggest that μMS has the potential of stimulating target nuclei in the brain without affecting the surrounding white matter tracts.
In calcific aortic valve disease (CAVD), microcalcifications originating from nanoscale calcifying vesicles disrupt the aortic valve (AV) leaflets, which consist of three (biomechanically) distinct layers: the fibrosa, spongiosa, and ventricularis. CAVD has no pharmacotherapy and lacks in vitro models as a result of complex valvular biomechanical features surrounding resident mechanosensitive valvular interstitial cells (VICs). We measured layer-specific mechanical properties of the human AV and engineered a three-dimensional (3D)-bioprinted CAVD model that recapitulates leaflet layer biomechanics for the first time. Human AV leaflet layers were separated by microdissection, and nanoindentation determined layer-specific Young’s moduli. Methacrylated gelatin (GelMA)/methacrylated hyaluronic acid (HAMA) hydrogels were tuned to duplicate layer-specific mechanical characteristics, followed by 3D-printing with encapsulated human VICs. Hydrogels were exposed to osteogenic media (OM) to induce microcalcification, and VIC pathogenesis was assessed by near infrared or immunofluorescence microscopy. Median Young’s moduli of the AV layers were 37.1, 15.4, and 26.9 kPa (fibrosa/spongiosa/ventricularis, respectively). The fibrosa and spongiosa Young’s moduli matched the 3D 5% GelMa/1% HAMA UV-crosslinked hydrogels. OM stimulation of VIC-laden bioprinted hydrogels induced microcalcification without apoptosis. We report the first layer-specific measurements of human AV moduli and a novel 3D-bioprinted CAVD model that potentiates microcalcification by mimicking the native AV mechanical environment. This work sheds light on valvular mechanobiology and could facilitate high-throughput drug-screening in CAVD.
Antibiotic resistance is compromising our ability to treat bacterial infections. Clinical microbiology laboratories guide appropriate treatment through antimicrobial susceptibility testing (AST) of patient bacterial isolates. However, increasingly, pathogens are developing resistance to a broad range of antimicrobials, requiring AST of alternative agents for which no commercially available testing methods are available. Therefore, there exists a significant AST testing gap in which current methodologies cannot adequately address the need for rapid results in the face of unpredictable susceptibility profiles. To address this gap, we developed a multicomponent, microscopy-based AST (MAST) platform capable of AST determinations after only a 2 h incubation. MAST consists of a solid-phase microwell growth surface in a 384-well plate format, inkjet printing-based application of both antimicrobials and bacteria at any desired concentrations, automated microscopic imaging of bacterial replication, and a deep learning approach for automated image classification and determination of antimicrobial minimal inhibitory concentrations (MICs). In evaluating a susceptible strain set, 95.8% were within ±1 and 99.4% were within ±2, twofold dilutions, respectively, of reference broth microdilution MIC values. Most (98.3%) of the results were in categorical agreement. We conclude that MAST offers promise for rapid, accurate, and flexible AST to help address the antimicrobial testing gap.
Chemical clocks usually achieve well-defined temporal delays through concentration thresholding coupled to the production, degradation, activation, or inhibition of downstream effectors. In this way, the stochastic dynamics of many individual molecules yield essentially deterministic bulk behavior through ensemble averaging. As a result, their temporal evolution is governed by ensemble dynamics rather than by the behavior of an individual molecule or complex. Here, we present a general approach for the design of single-molecule clocks that permits quasi-deterministic control over the lifetime of single molecular interactions without any external synchronization. By coupling the dissociation of a bimolecular complex to a series of irreversible chemical steps, we interpose a well-defined time delay between binding and dissociation. The number and speed of irreversible steps can be varied to systematically tune both the lifetimes of complexes and the precision of the time delay, raising the prospect of localized timekeeping in nanoscale systems and devices.
Microscopic interpretation of stained smears is one of the most operator-dependent and time intensive activities in the clinical microbiology laboratory. Here, we investigated application of an automated image acquisition and convolutional neural network (CNN)-based approach for automated Gram stain classification. Using an automated microscopy platform, uncoverslipped slides were scanned with a 40x dry objective, generating images of sufficient resolution for interpretation. We collected 25,488 images from positive blood culture Gram stains prepared during routine clinical workup. These images were used to generate 100,213 crops containing Gram-positive cocci in clusters, Gram-positive cocci in chains/pairs, Gram-negative rods, or background (no cells). These categories were targeted for proof-of-concept development as they are associated with the majority of bloodstream infections. Our CNN model achieved classification accuracy of 94.9% on a test set of image crops. Receiver operating characteristic curve (ROC) analysis indicated a robust ability to differentiate between categories with area under the curve >0.98 for each. After training and validation, we applied the classification algorithm to new images collected from 189 whole slides without human intervention. Sensitivity/specificity was 98.4/75.0% for Gram-positive cocci in chains/pairs; 93.2/97.2% for Gram-positive cocci in clusters; and 96.3/98.1% for Gram-negative rods. Taken together, our data support proof-of-concept for a fully automated classification methodology for blood-culture Gram-stains. Importantly, the algorithm was highly adept at identifying image crops with organisms and could be used to present prescreened, classified crops to technologists to accelerate smear review. This concept could potentially be extended to all Gram stain interpretive activities in the clinical laboratory.
The need to identify the most viable embryo following in vitro fertilization (IVF) was established early in the history of human IVF. The stalwart of identifying the best embryos has been morphology. Other techniques have however seen wide acceptance, including the use of preimplantation genetic screening, even though concerns exist over the invasive nature of the technique. Alternatively, noninvasive assessment technologies have tried to determine an embryo's viability through measurements of factors in the media or by imaging of the embryo. We present data showing that the metabolic blueprint of an embryo is linked to viability, and argue that analysis of metabolic function, using either spent medium or by novel microscopies, could provide the basis for selecting the embryo with the highest viability. This review therefore asks, "Will noninvasive methods surpass invasive for assessing gametes and embryos?" We examine the current state of research on noninvasive technologies, including novel optical methods, and conclude noninvasive embryo viability assessment will assist in embryo selection for transfer.
Laser-exposed plasmonic substrates permeabilize the plasma membrane of cells when in close contact to deliver cell-impermeable cargo. While studies have determined the cargo delivery efficiency and viability of laser-exposed plasmonic substrates, morphological changes in a cell have not been quantified. We porated myoblast C2C12 cells on a plasmonic pyramid array using a 532-nm laser with 850-ps pulse length and time-lapse fluorescence imaging to quantify cellular changes. We obtain a poration efficiency of 80%, viability of 90%, and a pore radius of 20 nm. We quantified area changes in the plasma membrane attached to the substrate (10% decrease), nucleus (5 - 10% decrease), and cytoplasm (5 - 10% decrease) over 1 h after laser treatment. Cytoskeleton fibers show a change of 50% in the alignment, or coherency, of fibers, which stabilizes after 10 mins. We investigate structural and morphological changes due to the poration process to enable the safe development of this technique for therapeutic applications.
Micro-magnetic stimulation (μMS) is an emerging neurostimulation technology that promises to revolutionize the therapeutic stimulation of the human nervous system. μMS uses sub-millimeter sized coils that can be implemented in the central nervous system to elicit neuronal activation using magnetically induced electric currents. By their microscopic size, μMS coils can be acutely implanted in deep brain structures to deliver therapeutic stimulation with effects analogous to those achieved by state-of-the-art deep brain stimulation (DBS). However, μMS technology has inherent advantages that make it particularly appealing for clinical applications. Specifically, μMS induces a focal electric current in the tissue, limiting the extent of activation to a few hundred microns. We recently demonstrated the feasibility of using μMS to elicit neuronal activation in vitro , as well as the possibility of activating neuronal circuitry on the system level in rodents . As μMS is a novel technology, its mechanism(s) of nerve activation, induced field characteristics, and optimum topological features are yet to be explored. In this regard, numerical simulations play a crucially important role, because they provide an insight into spatial distribution of induced electric fields, which in turn, dictate the dynamics of nerve stimulation. Here we report results of numerical simulations to predict the nerve-stimulation performance of different μMS geometries.
Efficiently delivering functional cargo to millions of cells on the time scale of minutes will revolutionize gene therapy, drug discovery, and high-throughput screening. Recent studies of intracellular delivery with thermoplasmonic structured surfaces show promising results but in most cases require time- or cost-intensive fabrication or lead to unreproducible surfaces. We designed and fabricated large-area (14 × 14 mm), photolithography-based, template-stripped plasmonic substrates that are nanosecond laser-activated to form transient pores in cells for cargo entry. We optimized fabrication to produce plasmonic structures that are ultrasmooth and precisely patterned over large areas. We used flow cytometry to characterize the delivery efficiency of cargos ranging in size from 0.6 to 2000 kDa to cells (up to 95% for the smallest molecule) and viability of cells (up to 98%). This technique offers a throughput of 50000 cells/min, which can be scaled up as necessary. This technique is also cost-effective as each large-area photolithography substrate can be used to deliver cargo to millions of cells, and switching to a nanosecond laser makes the setup cheaper and easier to use. The approach we present offers additional desirable features: spatial selectivity, reproducibility, minimal residual fragments, and cost-effective fabrication. This research supports the development of safer genetic and viral disease therapies as well as research tools for fundamental biological research that rely on effectively delivering molecules to millions of living cells.
Calcific aortic valve disease (CAVD) is the most prevalent valvular heart disease in the Western population, claiming 17000 deaths per year in the United States and affecting 25% of people older than 65 years of age. Contrary to traditional belief, CAVD is not a passive, degenerative disease but rather a dynamic disease, where initial cellular changes in the valve leaflets progress into fibrotic lesions that induce valve thickening and calcification. Advanced thickening and calcification impair valve function and lead to aortic stenosis (AS). Without intervention, progressive ventricular hypertrophy ensues, which ultimately results in heart failure and death. Currently, aortic valve replacement (AVR), surgical or transcatheter, is the only effective therapy to treat CAVD. However, these costly interventions are often delayed until the late stages of the disease. Nonetheless, 275000 are performed per year worldwide, and this is expected to triple by 2050. Given the current landscape, next-generation therapies for CAVD are needed to improve patient outcome and quality of life. Here, we first provide a background on the aortic valve (AV) and the pathobiology of CAVD as well as highlight current directions and future outlook on the development of functional 3D models of CAVD in vitro We then consider an often-overlooked aspect contributing to CAVD: miRNA (mis)regulation. Therapeutics could potentially normalize miRNA levels in the early stages of the disease and may slow its progression or even reverse calcification. We close with a discussion of strategies that would enable the use of miRNA as a therapeutic for CAVD. This focuses on an overview of controlled delivery technologies for nucleic acid therapeutics to the valve or other target tissues.