You do not have subscription access to articles in this section. Learn more about access.

  JoVE Biology

You do not have subscription access to articles in this section. Learn more about access.

  JoVE Neuroscience

You do not have subscription access to articles in this section. Learn more about access.

  JoVE Immunology and Infection

You do not have subscription access to articles in this section. Learn more about access.

  JoVE Clinical and Translational Medicine

You do not have subscription access to articles in this section. Learn more about access.

  JoVE Bioengineering

You do not have subscription access to articles in this section. Learn more about access.

  JoVE Applied Physics

You do not have subscription access to articles in this section. Learn more about access.

  JoVE Chemistry

You do not have subscription access to articles in this section. Learn more about access.

  JoVE Behavior

You do not have subscription access to articles in this section. Learn more about access.

  JoVE Environment


JoVE Science Education

General Laboratory Techniques

You do not have subscription access to videos in this collection. Learn more about access.

Basic Methods in Cellular and Molecular Biology

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms I

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms II

You have trial access to videos in this collection until May 31, 2014.

In JoVE (1)

Other Publications (13)

Articles by Heather A. Clark in JoVE

 JoVE Bioengineering

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

1Bioengineering Department, Northeastern University, 2Department of Pharmaceutical Sciences, Northeastern University

JoVE 2896

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

Other articles by Heather A. Clark on PubMed

Cholesterol-enriched Lipid Domains Can Be Visualized by Di-4-ANEPPDHQ with Linear and Nonlinear Optics

We present a membrane-staining dye, di-4-ANEPPDHQ, which differentiates liquid-ordered phases from liquid-disordered phases coexisting in model membranes under both linear and nonlinear microscopies. The dye's fluorescence emission spectrum is blue-shifted 60 nm in liquid-ordered phases compared with liquid-disordered phases, and shows strong second harmonic generation in the liquid-disordered phase compared with the liquid-ordered phase. The ease of staining and the ability of this single dye to detect both phases, should lead to broad applications in biophysical studies of lipid domains in model membranes and cells.

Species-specific Bacteria Identification Using Differential Mobility Spectrometry and Bioinformatics Pattern Recognition

As bacteria grow and proliferate, they release a variety of volatile compounds that can be profiled and used for speciation, providing an approach amenable to disease diagnosis through quick analysis of clinical cultures as well as patient breath analysis. As a practical alternative to mass spectrometry detection and whole cell pyrolysis approaches, we have developed methodology that involves detection via a sensitive, micromachined differential mobility spectrometer (microDMx), for sampling headspace gases produced by bacteria growing in liquid culture. We have applied pattern discovery/recognition algorithms (ProteomeQuest) to analyze headspace gas spectra generated by microDMx to reliably discern multiple species of bacteria in vitro: Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, and Mycobacterium smegmatis. The overall accuracy for identifying volatile profiles of a species within the 95% confidence interval for the two highest accuracy models evolved was between 70.4 and 89.3% based upon the coordinated expression of between 5 and 11 features. These encouraging in vitro results suggest that the microDMx technology, coupled with bioinformatics data analysis, has potential for diagnosis of bacterial infections.

Characterization and Application of a New Optical Probe for Membrane Lipid Domains

In this article, we characterize the fluorescence of an environmentally sensitive probe for lipid membranes, di-4-ANEPPDHQ. In large unilamellar lipid vesicles (LUVs), its emission spectrum shifts up to 30 nm to the blue with increasing cholesterol concentration. Independently, it displays a comparable blue shift in liquid-ordered relative to liquid-disordered phases. The cumulative effect is a 60-nm difference in emission spectra for cholesterol containing LUVs in the liquid-ordered state versus cholesterol-free LUVs in the liquid-disordered phase. Given these optical properties, we use di-4-ANEPPDHQ to image the phase separation in giant unilamellar vesicles with both linear and nonlinear optical microscopy. The dye shows green and red fluorescence in liquid-ordered and -disordered domains, respectively. We propose that this reflects the relative rigidity of the molecular packing around the dye molecules in the two phases. We also observe a sevenfold stronger second harmonic generation signal in the liquid-disordered domains, consistent with a higher concentration of the dye resulting from preferential partitioning into the disordered phase. The efficacy of the dye for reporting lipid domains in cell membranes is demonstrated in polarized migrating neutrophils.

Ochratoxin A: Its Cancer Risk and Potential for Exposure

Ochratoxin A (OA) is a naturally occurring mycotoxin known to contaminate a variety of foods and beverages. The cancer risk posed by OA was reviewed as relevant to human exposure, regulatory activities, and risk management efforts occurring worldwide, particularly in Europe. OA moves through the food chain and has been found in the tissues and organs of animals, including human blood and breast milk. Results from the National Toxicology Program's rodent bioassays show significantly increased incidence of mammary gland tumors in female rats and kidney tumors in male and female rats given OA orally. Liver tumors in female mice fed OA in the diet have also been observed. In humans, OA exposure has been most often associated with the kidney disease Balkan endemic nephropathy (BEN), symptoms of which include tumors of the kidney and urinary tract. No epidemiological studies have yet adequately evaluated the cancer risk of OA in human populations. Studies have shown OA to be genotoxic as well as immunotoxic, although its mode of action is not fully understood. Organizations and agencies in many countries are currently promulgating standards for OA in foods and beverages. Increased efforts in farm management and food safety are being made to mitigate the risks to public health posed by OA. The U.S. Food and Drug Administration (FDA) is currently evaluating data on OA levels in domestic and imported commodities but has not established official regulations or guidelines for OA in the U.S. food supply.

Fluorescent Ion-selective Nanosensors for Intracellular Analysis with Improved Lifetime and Size

We describe the synthesis and characterization of sodium-selective polymeric nanosensors that improves upon the lifetime and size of previous fiberless nanosensors. Sonication is used to form the polymer nanospheres that contain all the components needed for ion sensing. Even though the size is small (approximately 120 nm), the lifetime of these sensors in solution is on the order of a week. The surface coating has also been optimized for stability, biocompatibility, and ease of chemical modification.

Ion-selective Nano-optodes Incorporating Quantum Dots

Protection of Sensors for Biological Applications by Photoinitiated Chemical Vapor Deposition of Hydrogel Thin Films

We report photoinitiated chemical vapor deposition (piCVD), a gentle synthetic method for the preparation of ultrathin films (approximately 100 nm) of the hydrogel poly(hydroxyethyl methacrylate) (pHEMA). piCVD occurs near room temperature and requires only mild vacuum conditions. The deposited films swell rapidly and reversibly in buffer solution, and the swelling properties can be controlled via the deposition conditions. Analysis of the swelling data indicates that the mesh size of the hydrogel creates a selectively permeable coating. The mesh is large enough to allow small molecule analytes to permeate the film but small enough to prevent the transport of large biomolecules such as proteins. X-ray photoelectron spectroscopy (XPS) shows that the films decrease nonspecific adhesion of the protein albumin by nearly 8-fold over bare silicon. A dry process, piCVD is suitable for coating particles with diameters as small as 5 microm. The absence of solvents and plasmas in piCVD allows films to be directly synthesized on optode sensors without degradation of sensitivity or response time.

Visualizing Sodium Dynamics in Isolated Cardiomyocytes Using Fluorescent Nanosensors

Regulation of sodium flux across the cell membrane plays a vital role in the generation of action potentials and regulation of membrane excitability in cells such as cardiomyocytes and neurons. Alteration of sodium channel function has been implicated in diseases such as epilepsy, long QT syndrome, and heart failure. However, single cell imaging of sodium dynamics has been limited due to the narrow selection of fluorescent sodium indicators available to researchers. Here we report, the detection of spatially defined sodium activity during action potentials. Fluorescent nanosensors that measure sodium in real-time, are reversible and are completely selective over other cations such as potassium that were used to image sodium. The use of the nanosensors in vitro was validated by determining drug-induced activation in heterologous cells transfected with the voltage-gated sodium channel Na(V)1.7. Spatial information of sodium concentrations during action potentials will provide insight at the cellular level on the role of sodium and how slight changes in sodium channel function can affect the entirety of an action potential.

Fluorescent Nano-optodes for Glucose Detection

We have designed fluorescent nanosensors based on ion-selective optodes capable of detecting small molecules. By localizing the sensor components in a hydrophobic core, these nanosensors are able to monitor dynamic changes in concentration of the model analyte, glucose. The nanosensors demonstrated this response in vitro and also when injected subcutaneously into mice. The response of the nanosensors tracked changes in blood glucose levels in vivo that were comparable to measurements taken using a glucometer. The development of these nanosensors offers an alternative, minimally invasive tool for monitoring glucose levels in such fields as diabetes research. Furthermore, the extension of the ion-selective optode sensor platform to small molecule detection will allow for enhanced monitoring of physiological processes.

Nanosensors and Nanomaterials for Monitoring Glucose in Diabetes

Worldwide, diabetes is a rapidly growing problem that is managed at the individual level by monitoring and controlling blood glucose levels to minimize the negative effects of the disease. Because of limitations in diagnostic methods, significant research efforts are focused on developing improved methods to measure glucose. Nanotechnology has impacted these efforts by increasing the surface area of sensors, improving the catalytic properties of electrodes and providing nanoscale sensors. Here, we discuss developments in the past several years on both nanosensors that directly measure glucose and nanomaterials that improve glucose sensor function. Finally, we discuss challenges that must be overcome to apply these developments in the clinic.

Ion-selective Optodes Measure Extracellular Potassium Flux in Excitable Cells

Optodes have been used for detection of ionic concentrations and fluxes for several years. However, their uses in biomedical applications have not yet been fully explored. This study investigates optodes as a potential sensor platform for monitoring cellular ion flux with attendant implications in the field of drug screening and toxicology. A prototype system was developed to quantitatively measure extracellular potassium flux from a monolayer of cardiomyocytes. Optodes were created and immobilized on a glass coverslip for fluorescent imaging. The system detected potassium (K(+) ) ion flux during the repolarization phase of the cardiac action potential and further detected a decrease in the magnitude of the flux in the presence of a known K(+) channel inhibitor by optically monitoring local K(+) ion concentrations during field stimulation of the cardiomyocyte monolayer.

Microworm Optode Sensors Limit Particle Diffusion to Enable in Vivo Measurements

There have been a variety of nanoparticles created for in vivo uses ranging from gene and drug delivery to tumor imaging and physiological monitoring. The use of nanoparticles to measure physiological conditions while being fluorescently addressed through the skin provides an ideal method toward minimally invasive health monitoring. Here we create unique particles that have all the necessary physical characteristics to serve as in vivo reporters, but with minimized diffusion from the point of injection. These particles, called microworms, have a cylindrical shape coated with a biocompatible porous membrane that possesses a large surface-area-to-volume ratio while maintaining a large hydrodynamic radius. We use these microworms to create fluorescent sodium sensors for use as in vivo sodium concentration detectors after subcutaneous injection. However, the microworm concept has the potential to extend to the immobilization of other types of polymers for continuous physiological detection or delivery of molecules.

The Design and Development of Fluorescent Nano-optodes for in Vivo Glucose Monitoring

The advent of fluorescent nanosensors has enabled intracellular monitoring of several physiological analytes, which was previously not possible with molecular dyes or other invasive techniques. We have extended the capability of these sensors to include the detection of small molecules with the development of glucose-sensitive nano-optodes. Herein, we discuss the design and development of glucose-sensitive nano-optodes, which have been proven functional both in vitro and in vivo.

simple hit counter