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Find video protocols related to scientific articles indexed in Pubmed.
Disposable platform provides visual and color-based point-of-care anemia self-testing.
J. Clin. Invest.
PUBLISHED: 08-26-2014
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Anemia, or low blood hemoglobin (Hgb) levels, afflicts 2 billion people worldwide. Currently, Hgb levels are typically measured from blood samples using hematology analyzers, which are housed in hospitals, clinics, or commercial laboratories and require skilled technicians to operate. A reliable, inexpensive point-of-care (POC) Hgb test would enable cost-effective anemia screening and chronically anemic patients to self-monitor their disease. We present a rapid, stand-alone, and disposable POC anemia test that, via a single drop of blood, outputs color-based visual results that correlate with Hgb levels.
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Modeling and validation of autoinducer-mediated bacterial gene expression in microfluidic environments.
Biomicrofluidics
PUBLISHED: 05-01-2014
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Biosensors exploiting communication within genetically engineered bacteria are becoming increasingly important for monitoring environmental changes. Currently, there are a variety of mathematical models for understanding and predicting how genetically engineered bacteria respond to molecular stimuli in these environments, but as sensors have miniaturized towards microfluidics and are subjected to complex time-varying inputs, the shortcomings of these models have become apparent. The effects of microfluidic environments such as low oxygen concentration, increased biofilm encapsulation, diffusion limited molecular distribution, and higher population densities strongly affect rate constants for gene expression not accounted for in previous models. We report a mathematical model that accurately predicts the biological response of the autoinducer N-acyl homoserine lactone-mediated green fluorescent protein expression in reporter bacteria in microfluidic environments by accommodating these rate constants. This generalized mass action model considers a chain of biomolecular events from input autoinducer chemical to fluorescent protein expression through a series of six chemical species. We have validated this model against experimental data from our own apparatus as well as prior published experimental results. Results indicate accurate prediction of dynamics (e.g., 14% peak time error from a pulse input) and with reduced mean-squared error with pulse or step inputs for a range of concentrations (10??M-30??M). This model can help advance the design of genetically engineered bacteria sensors and molecular communication devices.
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Noninvasive optical inhibition with a red-shifted microbial rhodopsin.
Nat. Neurosci.
PUBLISHED: 02-28-2014
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Optogenetic inhibition of the electrical activity of neurons enables the causal assessment of their contributions to brain functions. Red light penetrates deeper into tissue than other visible wavelengths. We present a red-shifted cruxhalorhodopsin, Jaws, derived from Haloarcula (Halobacterium) salinarum (strain Shark) and engineered to result in red light-induced photocurrents three times those of earlier silencers. Jaws exhibits robust inhibition of sensory-evoked neural activity in the cortex and results in strong light responses when used in retinas of retinitis pigmentosa model mice. We also demonstrate that Jaws can noninvasively mediate transcranial optical inhibition of neurons deep in the brains of awake mice. The noninvasive optogenetic inhibition opened up by Jaws enables a variety of important neuroscience experiments and offers a powerful general-use chloride pump for basic and applied neuroscience.
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Microfluidic thrombosis under multiple shear rates and antiplatelet therapy doses.
PLoS ONE
PUBLISHED: 01-01-2014
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The mainstay of treatment for thrombosis, the formation of occlusive platelet aggregates that often lead to heart attack and stroke, is antiplatelet therapy. Antiplatelet therapy dosing and resistance are poorly understood, leading to potential incorrect and ineffective dosing. Shear rate is also suspected to play a major role in thrombosis, but instrumentation to measure its influence has been limited by flow conditions, agonist use, and non-systematic and/or non-quantitative studies. In this work we measured occlusion times and thrombus detachment for a range of initial shear rates (500, 1500, 4000, and 10000 s(-1)) and therapy concentrations (0-2.4 µM for eptifibatide, 0-2 mM for acetyl-salicylic acid (ASA), 3.5-40 Units/L for heparin) using a microfluidic device. We also measured complete blood counts (CBC) and platelet activity using whole blood impedance aggregometry. Effects of shear rate and dose were analyzed using general linear models, logistic regressions, and Cox proportional hazards models. Shear rates have significant effects on thrombosis/dose-response curves for all tested therapies. ASA has little effect on high shear occlusion times, even at very high doses (up to 20 times the recommended dose). Under ASA therapy, thrombi formed at high shear rates were 4 times more prone to detachment compared to those formed under control conditions. Eptifibatide reduced occlusion when controlling for shear rate and its efficacy increased with dose concentration. In contrast, the hazard of occlusion from ASA was several orders of magnitude higher than that of eptifibatide. Our results show similar dose efficacy to our low shear measurements using whole blood aggregometry. This quantitative and statistically validated study of the effects of a wide range of shear rate and antiplatelet therapy doses on occlusive thrombosis contributes to more accurate understanding of thrombosis and to models for optimizing patient treatment.
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Sensitive, microliter PCR with consensus degenerate primers for Epstein Barr virus amplification.
Biomed Microdevices
PUBLISHED: 08-24-2013
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Sensitive identification of the etiology of viral diseases is key to implementing appropriate prevention and treatment. The gold standard for virus identification is the polymerase chain reaction (PCR), a technique that allows for highly specific and sensitive detection of pathogens by exponentially amplifying a specific region of DNA from as little as a single copy through thermocycling a biochemical cocktail. Today, molecular biology laboratories use commercial instruments that operate in 0.5-2 h/analysis using reaction volumes of 5-50 ?L contained within polymer tubes or chambers. Towards reducing this volume and maintaining performance, we present a semi-quantitative, systematic experimental study of how PCR yield is affected by tube/chamber substrate, surface-area-to-volume ratio (SA:V), and passivation methods. We perform PCR experiments using traditional PCR tubes as well as using disposable polymer microchips with 1 ?L reaction volumes thermocycled using water baths. We report the first oil encapsulation microfluidic PCR method without fluid flow and its application to the first microfluidic amplification of Epstein Barr virus using consensus degenerate primers, a powerful and broad PCR method to screen for both known and novel members of a viral family. The limit of detection is measured as 140 starting copies of DNA from a starting concentration of 3 × 10(5) copies/mL, regarded as an accepted sensitivity threshold for diagnostic purposes, and reaction specificity was improved as compared to conventional methods. Also notable, these experiments were conducted with conventional reagent concentrations, rather than commonly spiked enzyme and/or template mixtures. This experimental study of the effects of substrate, SA:V, and passivation, together with sensitive and specific microfluidic PCR with consensus degenerate primers represent advances towards lower cost and higher throughput pathogen screening.
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In vivo robotics: the automation of neuroscience and other intact-system biological fields.
Ann. N. Y. Acad. Sci.
PUBLISHED: 07-10-2013
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Robotic and automation technologies have played a huge role in in vitro biological science, having proved critical for scientific endeavors such as genome sequencing and high-throughput screening. Robotic and automation strategies are beginning to play a greater role in in vivo and in situ sciences, especially when it comes to the difficult in vivo experiments required for understanding the neural mechanisms of behavior and disease. In this perspective, we discuss the prospects for robotics and automation to influence neuroscientific and intact-system biology fields. We discuss how robotic innovations might be created to open up new frontiers in basic and applied neuroscience and present a concrete example with our recent automation of in vivo whole-cell patch clamp electrophysiology of neurons in the living mouse brain.
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Rapid, quantitative, reverse transcription PCR in a polymer microfluidic chip.
Biosens Bioelectron
PUBLISHED: 01-09-2013
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Quantitative PCR (qPCR) techniques have become invaluable, high-throughput tools to study gene expression. However, the need to measure gene expression patterns quickly and affordably, useful for applications such as stem cell biomanufacturing requiring real-time observation and control, has not been adequately met by rapid qPCR instrumentation to date. We report a reverse transcription, microfluidic qPCR system and its application to DNA and RNA amplification measurement. In the system, an environmental control fixture provides mechanical and thermal repeatability for an infrared laser to achieve both accurate and precise open-loop temperature control of 1 ?l reaction volumes in a low-cost polymer microfluidic chip with concurrent fluorescence imaging. We have used this system to amplify serial dilutions of ?-phage DNA (10(5)-10(7) starting copies) and RNA transcripts from the GAPDH housekeeping gene (5.45 ng total mouse embryonic stem cell RNA) and measured associated standard curves, efficiency (57%), repeatability (~1 cycle threshold), melting curves, and specificity. This microfluidic qRT-PCR system offers a practical approach to rapid analysis (~1 h), combining the cost benefits of small reagent volumes with the simplicity of disposable polymer microchips and easy setup.
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Programming cells by multiplex genome engineering and accelerated evolution.
Nature
PUBLISHED: 03-06-2009
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The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales. Although in vitro and directed evolution methods have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-D-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.
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Automated whole-cell patch-clamp electrophysiology of neurons in vivo.
Nat. Methods
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Whole-cell patch-clamp electrophysiology of neurons is a gold-standard technique for high-fidelity analysis of the biophysical mechanisms of neural computation and pathology, but it requires great skill to perform. We have developed a robot that automatically performs patch clamping in vivo, algorithmically detecting cells by analyzing the temporal sequence of electrode impedance changes. We demonstrate good yield, throughput and quality of automated intracellular recording in mouse cortex and hippocampus.
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Microfluidic system for simultaneous optical measurement of platelet aggregation at multiple shear rates in whole blood.
Lab Chip
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Thrombosis is the pathological formation of platelet aggregates which occlude blood flow causing stroke and heart attack-the leading causes of death in developed nations. Instrumentation for diagnosing and exploring treatments for pathological platelet aggregation thus has the potential for major clinical impact. Most current thrombosis methods focus on single flow conditions, non-occlusive platelet adhesion, or low shear rates and so are limited in their application to comparative studies involving multiple, pathological test conditions (e.g., shear rate, stenotic geometries that mimic arteries, and rapid platelet accumulation to occlusion). The field could benefit from a low volume, high throughput, short analysis time, and low cost system while minimizing sample handling. We report on the design, fabrication, testing, and application of a microfluidic device and associated optical system for simultaneous measurement of platelet aggregation at multiple initial shear rates within four stenotic channels in label-free whole blood. Following computational design, requisite shear rates were achieved in the device by micro- surface milling a mold and subsequent PDMS casting. We applied the microfluidic system to measure platelet aggregation in whole porcine blood for shear rates spanning physiological to pathological flow conditions (500-13000 s(-1)). Real-time, non-contact, label-free, microscope-free measurements of platelet aggregation were acquired using an optical system comprising a 650 nm diode laser and a linear CCD. We observed fully occlusive platelet aggregation in less than 20 min above a threshold initial shear rate of 4000 s(-1), and no occlusive platelet aggregation below 1500 s(-1) (N = 86 trials). Accumulation of thrombus was consistent between laser intensity, light microscopy, histology, and mass flow rate measurements. The amount of blood volumes producing occlusion were dependent on shear rate. Times to occlusion were not found to be dependent on shear rate above the threshold level of 4000 s(-1). This microfluidic system enables measurement of the entire process of occlusive platelet thrombosis in whole, unlabeled blood, in vitro, at multiple shear rates. Such a system may be useful as a point-of-care diagnostic tool for studying anti-platelet therapies in individual blood samples from high-risk patients.
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Plug-and-play, infrared, laser-mediated PCR in a microfluidic chip.
Biomed Microdevices
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Microfluidic polymerase chain reaction (PCR) systems have set milestones for small volume (100 nL-5 ?L), amplification speed (100-400 s), and on-chip integration of upstream and downstream sample handling including purification and electrophoretic separation functionality. In practice, the microfluidic chips in these systems require either insertion of thermocouples or calibration prior to every amplification. These factors can offset the speed advantages of microfluidic PCR and have likely hindered commercialization. We present an infrared, laser-mediated, PCR system that features a single calibration, accurate and repeatable precision alignment, and systematic thermal modeling and management for reproducible, open-loop control of PCR in 1 ?L chambers of a polymer microfluidic chip. Total cycle time is less than 12 min: 1 min to fill and seal, 10 min to amplify, and 1 min to recover the sample. We describe the design, basis for its operation, and the precision engineering in the system and microfluidic chip. From a single calibration, we demonstrate PCR amplification of a 500 bp amplicon from ?-phage DNA in multiple consecutive trials on the same instrument as well as multiple identical instruments. This simple, relatively low-cost plug-and-play design is thus accessible to persons who may not be skilled in assembly and engineering.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.