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Find video protocols related to scientific articles indexed in Pubmed.
Removal of 8-oxo-GTP by MutT hydrolase is not a major contributor to transcriptional fidelity.
Nucleic Acids Res.
PUBLISHED: 10-07-2014
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Living in an oxygen-rich environment is dangerous for a cell. Reactive oxygen species can damage DNA, RNA, protein and lipids. The MutT protein in Escherichia coli removes 8-oxo-deoxyguanosine triphosphate (8-oxo-dGTP) and 8-oxo-guanosine triphosphate (8-oxo-GTP) from the nucleotide pools precluding incorporation into DNA and RNA. While 8-oxo-dGTP incorporation into DNA is mutagenic, it is not clear if 8-oxo-GTP incorporation into RNA can have phenotypic consequences for the cell. We use a bistable epigenetic switch sensitive to transcription errors in the Escherichia coli lacI transcript to monitor transient RNA errors. We do not observe any increase in epigenetic switching in mutT cells. We revisit the original observation of partial phenotypic suppression of a lacZamber allele in a mutT background that was attributed to RNA errors. We find that Lac(+) revertants can completely account for the increase in ?-galactosidase levels in mutT lacZamber cultures, without invoking participation of transient transcription errors. Moreover, we observe a fluctuation type of distribution of ?-galactosidase appearance in a growing culture, consistent with Lac(+) DNA revertant events. We conclude that the absence of MutT produces a DNA mutator but does not equally create an RNA mutator.
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A nutrient-tunable bistable switch controls motility in Salmonella enterica serovar Typhimurium.
MBio
PUBLISHED: 08-26-2014
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Many bacteria are motile only when nutrients are scarce. In contrast, Salmonella enterica serovar Typhimurium is motile only when nutrients are plentiful, suggesting that this bacterium uses motility for purposes other than foraging, most likely for host colonization. In this study, we investigated how nutrients affect motility in S. enterica and found that they tune the fraction of motile cells. In particular, we observed coexisting populations of motile and nonmotile cells, with the distribution being determined by the concentration of nutrients in the growth medium. Interestingly, S. enterica responds not to a single nutrient but apparently to a complex mixture of them. Using a combination of experimentation and mathematical modeling, we investigated the mechanism governing this behavior and found that it results from two antagonizing regulatory proteins, FliZ and YdiV. We also found that a positive feedback loop involving the alternate sigma factor FliA is required, although its role appears solely to amplify FliZ expression. We further demonstrate that the response is bistable: that is, genetically identical cells can exhibit different phenotypes under identical growth conditions. Together, these results uncover a new facet of the regulation of the flagellar genes in S. enterica and further demonstrate how bacteria employ phenotypic diversity as a general mechanism for adapting to change in their environment.
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Escherichia coli swimming is robust against variations in flagellar number.
Elife
PUBLISHED: 02-13-2014
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Bacterial chemotaxis is a paradigm for how environmental signals modulate cellular behavior. Although the network underlying this process has been studied extensively, we do not yet have an end-to-end understanding of chemotaxis. Specifically, how the rotational states of a cell's flagella cooperatively determine whether the cell 'runs' or 'tumbles' remains poorly characterized. Here, we measure the swimming behavior of individual E. coli cells while simultaneously detecting the rotational states of each flagellum. We find that a simple mathematical expression relates the cell's run/tumble bias to the number and average rotational state of its flagella. However, due to inter-flagellar correlations, an 'effective number' of flagella-smaller than the actual number-enters into this relation. Data from a chemotaxis mutant and stochastic modeling suggest that fluctuations of the regulator CheY-P are the source of flagellar correlations. A consequence of inter-flagellar correlations is that run/tumble behavior is only weakly dependent on number of flagella. DOI: http://dx.doi.org/10.7554/eLife.01916.001.
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Revisiting bistability in the lysis/lysogeny circuit of bacteriophage lambda.
PLoS ONE
PUBLISHED: 01-01-2014
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The lysis/lysogeny switch of bacteriophage lambda serves as a paradigm for binary cell fate decision, long-term maintenance of cellular state and stimulus-triggered switching between states. In the literature, the system is often referred to as "bistable." However, it remains unclear whether this term provides an accurate description or is instead a misnomer. Here we address this question directly. We first quantify transcriptional regulation governing lysogenic maintenance using a single-cell fluorescence reporter. We then use the single-cell data to derive a stochastic theoretical model for the underlying regulatory network. We use the model to predict the steady states of the system and then validate these predictions experimentally. Specifically, a regime of bistability, and the resulting hysteretic behavior, are observed. Beyond the steady states, the theoretical model successfully predicts the kinetics of switching from lysogeny to lysis. Our results show how the physics-inspired concept of bistability can be reliably used to describe cellular phenotype, and how an experimentally-calibrated theoretical model can have accurate predictive power for cell-state switching.
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Genetic determinants and cellular constraints in noisy gene expression.
Science
PUBLISHED: 12-07-2013
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In individual cells, transcription is a random process obeying single-molecule kinetics. Often, it occurs in a bursty, intermittent manner. The frequency and size of these bursts affect the magnitude of temporal fluctuations in messenger RNA and protein content within a cell, creating variation or "noise" in gene expression. It is still unclear to what degree transcriptional kinetics are specific to each gene and determined by its promoter sequence. Alternative scenarios have been proposed, in which the kinetics of transcription are governed by cellular constraints and follow universal rules across the genome. Evidence from genome-wide noise studies and from systematic perturbations of promoter sequences suggest that both scenarios-namely gene-specific versus genome-wide regulation of transcription kinetics-may be present to different degrees in bacteria, yeast, and animal cells.
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The maternal-to-zygotic transition targets actin to promote robustness during morphogenesis.
PLoS Genet.
PUBLISHED: 11-01-2013
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Robustness is a property built into biological systems to ensure stereotypical outcomes despite fluctuating inputs from gene dosage, biochemical noise, and the environment. During development, robustness safeguards embryos against structural and functional defects. Yet, our understanding of how robustness is achieved in embryos is limited. While much attention has been paid to the role of gene and signaling networks in promoting robust cell fate determination, little has been done to rigorously assay how mechanical processes like morphogenesis are designed to buffer against variable conditions. Here we show that the cell shape changes that drive morphogenesis can be made robust by mechanisms targeting the actin cytoskeleton. We identified two novel members of the Vinculin/?-Catenin Superfamily that work together to promote robustness during Drosophila cellularization, the dramatic tissue-building event that generates the primary epithelium of the embryo. We find that zygotically-expressed Serendipity-? (Sry-?) and maternally-loaded Spitting Image (Spt) share a redundant, actin-regulating activity during cellularization. Spt alone is sufficient for cellularization at an optimal temperature, but both Spt plus Sry-? are required at high temperature and when actin assembly is compromised by genetic perturbation. Our results offer a clear example of how the maternal and zygotic genomes interact to promote the robustness of early developmental events. Specifically, the Spt and Sry-? collaboration is informative when it comes to genes that show both a maternal and zygotic requirement during a given morphogenetic process. For the cellularization of Drosophilids, Sry-? and its expression profile may represent a genetic adaptive trait with the sole purpose of making this extreme event more reliable. Since all morphogenesis depends on cytoskeletal remodeling, both in embryos and adults, we suggest that robustness-promoting mechanisms aimed at actin could be effective at all life stages.
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Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization.
Nat Protoc
PUBLISHED: 05-16-2013
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We present a protocol for measuring the absolute number of mRNA molecules from a gene of interest in individual, chemically fixed Escherichia coli cells. A set of fluorescently labeled oligonucleotide probes is hybridized to the target mRNA, such that each mRNA molecule is decorated by a known number of fluorescent dyes. Cells are then imaged using fluorescence microscopy. The copy number of the target mRNA is estimated from the total intensity of fluorescent foci in the cell, rather than from counting discrete spots as in other currently available protocols. Image analysis is performed using an automated algorithm. The measured mRNA copy number distribution obtained from many individual cells can be used to extract the parameters of stochastic gene activity, namely the frequency and size of transcription bursts from the gene of interest. The experimental procedure takes 2 d, with another 2-3 d typically required for image and data analysis.
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The Plasma Membrane Flattens Out to Fuel Cell-Surface Growth during Drosophila Cellularization.
Dev. Cell
PUBLISHED: 04-01-2013
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Cell-shape change demands cell-surface growth, but how growth is fueled and choreographed is still debated. Here we use cellularization, the first complete cytokinetic event in Drosophila embryos, to show that cleavage furrow ingression is kinetically coupled to the loss of surface microvilli. We modulate furrow kinetics with RNAi against the Rho1-GTPase regulator slam and show that furrow ingression controls the rate of microvillar depletion. Finally, we directly track the microvillar membrane and see it move along the cell surface and into ingressing furrows, independent of endocytosis. Together, our results demonstrate that the kinetics of the ingressing furrow regulate the utilization of a microvillar membrane reservoir. Because membranes of the furrow and microvilli are contiguous, we suggest that ingression drives unfolding of the microvilli and incorporation of microvillar membrane into the furrow. We conclude that plasma membrane folding/unfolding can contribute to the cell-shape changes that promote embryonic morphogenesis.
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A SUMOylation-dependent transcriptional subprogram is required for Myc-driven tumorigenesis.
Science
PUBLISHED: 12-08-2011
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Myc is an oncogenic transcription factor frequently dysregulated in human cancer. To identify pathways supporting the Myc oncogenic program, we used a genome-wide RNA interference screen to search for Myc-synthetic lethal genes and uncovered a role for the SUMO-activating enzyme (SAE1/2). Loss of SAE1/2 enzymatic activity drives synthetic lethality with Myc. Inactivation of SAE2 leads to mitotic catastrophe and cell death upon Myc hyperactivation. Mechanistically, SAE2 inhibition switches a transcriptional subprogram of Myc from activated to repressed. A subset of these SUMOylation-dependent Myc switchers (SMS genes) is required for mitotic spindle function and to support the Myc oncogenic program. SAE2 is required for growth of Myc-dependent tumors in mice, and gene expression analyses of Myc-high human breast cancers suggest that low SAE1 and SAE2 abundance in the tumors correlates with longer metastasis-free survival of the patients. Thus, inhibition of SUMOylation may merit investigation as a possible therapy for Myc-driven human cancers.
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Following cell-fate in E. coli after infection by phage lambda.
J Vis Exp
PUBLISHED: 10-26-2011
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The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant). We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells. Here, we describe the full procedure for performing the infection experiments described in our earlier work. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35°C to trigger viral DNA injection. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.
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Two-photon 3D FIONA of individual quantum dots in an aqueous environment.
Nano Lett.
PUBLISHED: 09-13-2011
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We report the first two-photon (2P) microscopy of individual quantum dots (QDs) in an aqueous environment with both widefield and point-scan excitations at nanometer accuracy. Thiol-containing reductants suppress QD blinking and enable measurement of the 36 nm step size of individual Myosin V motors in vitro. We localize QDs with an accuracy of 2-3 nm in all three dimensions by using a 9 × 9 matrix excitation hologram and an array detector, which also increases the 3D scan imaging rate by 80-fold. With this 3D microscopy we validate the LamB receptor distribution on E. coli and the endocytosis of EGF-receptors in breast cancer cells.
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Optical measurement of cycle-dependent cell growth.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 07-25-2011
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Determining the growth patterns of single cells offers answers to some of the most elusive questions in contemporary cell biology: how cell growth is regulated and how cell size distributions are maintained. For example, a linear growth in time implies that there is no regulation required to maintain homeostasis; an exponential pattern indicates the opposite. Recently, there has been great effort to measure single cells using microelectromechanical systems technology, and several important questions have been explored. However, a unified, easy-to-use methodology to measure the growth rate of individual adherent cells of various sizes has been lacking. Here we demonstrate that a newly developed optical interferometric technique, known as spatial light interference microscopy, can measure the cell dry mass of many individual adherent cells in various conditions, over spatial scales from micrometers to millimeters, temporal scales ranging from seconds to days, and cell types ranging from bacteria to mammalian cells. We found evidence of exponential growth in Escherichia coli, which agrees very well with other recent reports. Perhaps most importantly, combining spatial light interference microscopy with fluorescence imaging provides a unique method for studying cell cycle-dependent growth. Thus, by using a fluorescent reporter for the S phase, we measured single cell growth over each phase of the cell cycle in human osteosarcoma U2OS cells and found that the G2 phase exhibits the highest growth rate, which is mass-dependent and can be approximated by an exponential.
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Decision making in living cells: lessons from a simple system.
Annu Rev Biophys
PUBLISHED: 05-07-2011
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The life cycle of bacteriophage lambda serves as a simplified paradigm for cell-fate decisions. The ongoing quantitative, high-resolution experimental investigation of this life cycle has produced some important insights in recent years. These insights have to do with the way cells choose among alternative fates, how they maintain long-term memory of their gene-expression state, and how they switch from one stable state to another. The recent studies have highlighted the role of spatiotemporal effects in cellular processes and the importance of distinguishing chemical stochasticity from possible hidden variables in cellular decision making.
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General properties of transcriptional time series in Escherichia coli.
Nat. Genet.
PUBLISHED: 04-05-2011
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Gene activity is described by the time series of discrete, stochastic mRNA production events. This transcriptional time series shows intermittent, bursty behavior. One consequence of this temporal intricacy is that gene expression can be tuned by varying different features of the time series. Here we quantify copy-number statistics of mRNA from 20 Escherichia coli promoters using single-molecule fluorescence in situ hybridization in order to characterize the general properties of these transcriptional time series. We find that the degree of burstiness is correlated with gene expression level but is largely independent of other parameters of gene regulation. The observed behavior can be explained by the underlying variation in the duration of bursting events. Using Shannons mutual information function, we estimate the mutual information transmitted between an outside stimulus, such as the extracellular concentration of inducer molecules, and intracellular levels of mRNA. This suggests that the outside stimulus transmits information reflected in the properties of transcriptional time series.
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Single-virus tracking reveals a spatial receptor-dependent search mechanism.
Biophys. J.
PUBLISHED: 02-07-2011
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Viral infection begins with the binding of a virus to a specific target on the surface of the host cell, followed by viral genome delivery into the host and a continuation of the infection process. Before binding occurs, the virus must first find its receptor by a process whose details are largely unknown. We applied high-resolution fluorescence microscopy and single-particle tracking to elucidate the target-finding process in bacteriophage ? as it infects an Escherichia coli cell. By monitoring the motion of individual viruses through the early stages of infection, we identified a unique spatial focusing process that allows a virus to arrive from its initial random landing site to its destination at the cell pole. The search process is governed by the interaction between the virus and the LamB receptors, and by the spatial organization of the receptor network on the cell surface. Our findings allowed us to develop a theoretical model for the target-finding process that reproduces the key features observed in experiment. We discuss the possible implications of our findings for the process of viral receptor-finding in higher systems.
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FliZ induces a kinetic switch in flagellar gene expression.
J. Bacteriol.
PUBLISHED: 10-08-2010
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FliZ is an activator of class 2 flagellar gene expression in Salmonella enterica. To understand its role in flagellar assembly, we investigated how FliZ affects gene expression dynamics. We demonstrate that FliZ participates in a positive-feedback loop that induces a kinetic switch in class 2 gene expression.
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Lysogen stability is determined by the frequency of activity bursts from the fate-determining gene.
Mol. Syst. Biol.
PUBLISHED: 07-06-2010
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The ability of living cells to maintain an inheritable memory of their gene-expression state is key to cellular differentiation. Bacterial lysogeny serves as a simple paradigm for long-term cellular memory. In this study, we address the following question: in the absence of external perturbation, how long will a cell stay in the lysogenic state before spontaneously switching away from that state? We show by direct measurement that lysogen stability exhibits a simple exponential dependence on the frequency of activity bursts from the fate-determining gene, cI. We quantify these gene-activity bursts using single-molecule-resolution mRNA measurements in individual cells, analyzed using a stochastic mathematical model of the gene-network kinetics. The quantitative relation between stability and gene activity is independent of the fine details of gene regulation, suggesting that a quantitative prediction of cell-state stability may also be possible in more complex systems.
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Decision making at a subcellular level determines the outcome of bacteriophage infection.
Cell
PUBLISHED: 01-25-2010
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When the process of cell-fate determination is examined at single-cell resolution, it is often observed that individual cells undergo different fates even when subject to identical conditions. This "noisy" phenotype is usually attributed to the inherent stochasticity of chemical reactions in the cell. Here we demonstrate how the observed single-cell heterogeneity can be explained by a cascade of decisions occurring at the subcellular level. We follow the postinfection decision in bacteriophage lambda at single-virus resolution, and show that a choice between lysis and lysogeny is first made at the level of the individual virus. The decisions by all viruses infecting a single cell are then integrated in a precise (noise-free) way, such that only a unanimous vote by all viruses leads to the establishment of lysogeny. By detecting and integrating over the subcellular "hidden variables," we are able to predict the level of noise measured at the single-cell level.
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Lambda-prophage induction modeled as a cooperative failure mode of lytic repression.
Phys Rev E Stat Nonlin Soft Matter Phys
PUBLISHED: 09-01-2009
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We analyze a system-level model for lytic repression of lambda phage in E. coli using reliability theory, showing that the repressor circuit comprises four redundant components whose failure mode is prophage induction. Our model reflects the specific biochemical mechanisms involved in regulation, including long-range cooperative binding, and its detailed predictions for prophage induction in E. coli under ultraviolet radiation are in good agreement with experimental data.
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High-resolution, long-term characterization of bacterial motility using optical tweezers.
Nat. Methods
PUBLISHED: 05-15-2009
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We present a single-cell motility assay, which allows the quantification of bacterial swimming in a well-controlled environment, for durations of up to an hour and with a temporal resolution greater than the flagellar rotation rates of approximately 100 Hz. The assay is based on an instrument combining optical tweezers, light and fluorescence microscopy, and a microfluidic chamber. Using this device we characterized the long-term statistics of the run-tumble time series in individual Escherichia coli cells. We also quantified higher-order features of bacterial swimming, such as changes in velocity and reversals of swimming direction.
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Quantitative transcription factor binding kinetics at the single-molecule level.
Biophys. J.
PUBLISHED: 01-27-2009
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We investigated the binding interaction between the bacteriophage lambda-repressor CI and its target DNA using total internal reflection fluorescence microscopy. Large stepwise changes in the intensity of the red fluorescent protein fused to CI were observed as it associated with and dissociated from individually labeled single-molecule DNA targets. The stochastic association and dissociation were characterized by Poisson statistics. Dark and bright intervals were measured for thousands of individual events. The exponential distribution of the intervals allowed direct determination of the association and dissociation rate constants (k(a) and k(d), respectively). We resolved in detail how k(a) and k(d) varied as a function of three control parameters: the DNA length L, the CI dimer concentration, and the binding affinity. Our results show that although interactions with nonoperator DNA sequences are observable, CI binding to the operator site is not dependent on the length of flanking nonoperator DNA.
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Visualizing Escherichia coli sub-cellular structure using sparse deconvolution Spatial Light Interference Tomography.
PLoS ONE
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Studying the 3D sub-cellular structure of living cells is essential to our understanding of biological function. However, tomographic imaging of live cells is challenging mainly because they are transparent, i.e., weakly scattering structures. Therefore, this type of imaging has been implemented largely using fluorescence techniques. While confocal fluorescence imaging is a common approach to achieve sectioning, it requires fluorescence probes that are often harmful to the living specimen. On the other hand, by using the intrinsic contrast of the structures it is possible to study living cells in a non-invasive manner. One method that provides high-resolution quantitative information about nanoscale structures is a broadband interferometric technique known as Spatial Light Interference Microscopy (SLIM). In addition to rendering quantitative phase information, when combined with a high numerical aperture objective, SLIM also provides excellent depth sectioning capabilities. However, like in all linear optical systems, SLIMs resolution is limited by diffraction. Here we present a novel 3D field deconvolution algorithm that exploits the sparsity of phase images and renders images with resolution beyond the diffraction limit. We employ this label-free method, called deconvolution Spatial Light Interference Tomography (dSLIT), to visualize coiled sub-cellular structures in E. coli cells which are most likely the cytoskeletal MreB protein and the division site regulating MinCDE proteins. Previously these structures have only been observed using specialized strains and plasmids and fluorescence techniques. Our results indicate that dSLIT can be employed to study such structures in a practical and non-invasive manner.
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Chemotactic adaptation kinetics of individual Escherichia coli cells.
Proc. Natl. Acad. Sci. U.S.A.
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Escherichia coli chemotaxis serves as a paradigm for the way living cells respond and adapt to changes in their environment. The chemotactic response has been characterized at the level of individual flagellar motors and in populations of swimming cells. However, it has not been previously possible to quantify accurately the adaptive response of a single, multiflagellated cell. Here, we use our recently developed optical trapping technique to characterize the swimming behavior of individual bacteria as they respond to sudden changes in the chemical environment. We follow the adaptation kinetics of E. coli to varying magnitudes of step-up and step-down changes in concentration of chemoattractant. We quantify two features of adaptation and how they vary with stimulus strength: abruptness (the degree to which return to prestimulus behavior occurs within a small number of run/tumble events) and overshoot (the degree of excessive response before the return to prestimulus behavior). We also characterize the asymmetry between step-up and step-down responses, observed at the single-cell level. Our findings provide clues to an improved understanding of chemotactic adaptation.
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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.

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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.