Nous sommes reconnaissants au Professeur Richard Borgens, Centre de recherche paralysie, l&#39;Université de Purdue, de l&#39;aide dans le montage du système de sonde vibrante. Cette étude a été soutenue par IEN subventions du NIH pour 1R01EY019101 MZ et BR, et en partie par des subventions du California Institute of Regenerative Medicine RB1-01417, NSF MCB-0951199, et par une subvention sans restriction de la recherche pour prévenir la cécité, l&#39;UC Davis ophtalmologie.

<ol>
	<li>Reid, B., Nuccitelli, R., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+measurement+of+bioelectric+currents+with+a+vibrating+probe.">Non-invasive measurement of bioelectric currents with a vibrating probe.</a> Nat. Protoc. 2, 661-9282 (2007).</li><li>Reid, B., Song, B., McCaig, C. D., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+in+rat+cornea:+the+role+of+electric+currents.">Wound healing in rat cornea: the role of electric currents.</a> FASEB J. 19, 379-386 (2005).</li><li>Lois, N., Reid, B., Song, B., Zhao, M., Forrester, J. V., McCaig, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+currents+and+lens+regeneration+in+the+rat.">Electric currents and lens regeneration in the rat.</a> Exp. Eye Res. 90, 316-323 (2010).</li><li>Wang, E., Reid, B., Lois, N., Forrester, J. V., McCaig, C. D., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+inhibition+of+lens+epithelial+cell+proliferation:+an+additional+factor+in+secondary+cataract.">Electrical inhibition of lens epithelial cell proliferation: an additional factor in secondary cataract.</a> FASEB J. 19, 842-844 (2005).</li><li>Guo, A., Song, B., Reid, B., Gu, Y., Forrester, J. V., Jahoda, C., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+physiological+electric+fields+on+migration+of+human+dermal+fibroblasts.">Effects of physiological electric fields on migration of human dermal fibroblasts.</a> J. Invest. Derm. , (2010).</li><li>Reid, B., Song, B., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+currents+in+Xenopus+tadpole+tail+regeneration.">Electric currents in Xenopus tadpole tail regeneration.</a> Dev. Biol. 335, 198-207 (2009).</li><li>Zhao, M., Song, B., Pu, J., Wada, T., Reid, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+signals+control+wound+healing+through+phosphatidylinositol-3-OH+kinase-&#947;.">Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-&#947;.</a> 442, 457-460 (2006).</li><li>Reid, B., EO, G. r. a. u. e. -. H. e. r. n. a. n. d. e. z., Mannis, M. J., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+endogenous+electric+currents+in+human+corneal+wounds+-+a+novel+approach+of+bioelectric+stimulation+without+electrodes.">Modulating endogenous electric currents in human corneal wounds - a novel approach of bioelectric stimulation without electrodes.</a> Cornea. , (2010).</li><li>Nuccitelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ultrasensitive+vibrating+probe+for+measuring+steady+extracellular+currents.">An ultrasensitive vibrating probe for measuring steady extracellular currents.</a> J. Cell Biol. 63, 614-628 (1974).</li><li>Hotary, K. B., Nuccitelli, R., Robinson, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computerized+2-dimensional+vibrating+probe+for+mapping+extracellular+current+patterns.">A computerized 2-dimensional vibrating probe for mapping extracellular current patterns.</a> J. Neurosci. Meth. 43, 55-67 (1992).</li><li>Nuccitelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+ion+currents+and+DC+electric+fields+in+multicellular+animal+tissues.">Endogenous ion currents and DC electric fields in multicellular animal tissues.</a> Bioelectromagnetics Supplement. 1, 147-157 (1992).</li><li>Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioelectric+mechanisms+in+regeneration:+Unique+aspects+and+future+perspectives.">Bioelectric mechanisms in regeneration: Unique aspects and future perspectives.</a> Seminars in Cell Dev. Biol. 20, 543-556 (2009).</li><li>Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+fields+in+wound+healing+-+An+overriding+signal+that+directs+cell+migration.">Electric fields in wound healing - An overriding signal that directs cell migration.</a> Seminars in Cell Dev. Biol. 20, 674-682 (2009).</li></ol>

Nous décrivons un faible coût, de base, mais très sensible du système sonde vibrante pour mesurer de façon non invasive de courant électrique dans une variété de systèmes biologiques. Modifications possibles <ol><li> Si platine / iridium électrodes (instruments de précision du monde; # cat PTM23B20) sont utilisés au lieu de l&#39;acier inoxydable, puis l&#39;étape de placage à l&#39;or peut être éliminé. </li></ol> Applications <p class="jove_conten...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Eligoy-Stainless Electrode</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>SSM33A70</td>
<td>76 mm, 7 M&Omega;, 1-2&mu;m tip</td>
</tr>
<tr>
<td>Gold R30 connector</td>
<td>&nbsp;</td>
<td>www.vectorelect.com</td>
<td>R30</td>
<td>Re-usable</td>
</tr>
<tr>
<td>Silver-loaded epoxy</td>
<td>&nbsp;</td>
<td>3M</td>
<td>SL65</td>
<td>Mix 1-part Resin with 1-part Hardener</td>
</tr>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>SZ40</td>
<td>Magnification x6 to x40</td>
</tr>
<tr>
<td>Potassium dicyanoaurate (KAu(CN)2)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>379867</td>
<td>CAUTION: Toxic</td>
</tr>
<tr>
<td>Chloroplatinic acid hydrate (H2PtCl6 x 6H2O)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>520896</td>
<td>CAUTION: Toxic</td>
</tr>
<tr>
<td>Lead(II) acetate trihydrate (Pb(CH3CO2)2 x 3H2O)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>185191</td>
<td>CAUTION: Toxic</td>
</tr>
<tr>
<td>Nano-Amp power source</td>
<td>&nbsp;</td>
<td>Made in-house</td>
<td>-</td>
<td>Powered by six 1.5 V (AAA) batteries</td>
</tr>
<tr>
<td>3-dimensional micro-positioner</td>
<td>&nbsp;</td>
<td>Line Tool Co.</td>
<td>Model H</td>
<td>&nbsp;</td>
<tr>
<td>Lock-in amplifier</td>
<td>&nbsp;</td>
<td>Stanford Research Systems</td>
<td>SR530</td>
<td>&nbsp;</td>
<tr>
<td>Digital I/O interface</td>
<td>&nbsp;</td>
<td>National instruments</td>
<td>PCI-6220</td>
<td>&nbsp;</td>
<tr>
<td>Shielded Connector Block with BNC connections</td>
<td>&nbsp;</td>
<td>National instruments</td>
<td>BNC-2110</td>
<td>&nbsp;</td>
<tr>
<td>Strathclyde Electrophysiology Software</td>
<td>&nbsp;</td>
<td>University of Strathclyde Institute of Pharmacy and Biomedical Sciences, UK</td>
<td>WinWCP V4.1.5</td>
<td>Free download from: <a href="http://spider.science.strath.ac.uk/sipbs/software_ses.htm">http://spider.science.strath.ac.uk/sipbs/software_ses.htm</a></td>
</tr>
<tr>
<td>Calibration Chamber</td>
<td>&nbsp;</td>
<td>Made in-house</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Constant Current Calibrator</td>
<td>&nbsp;</td>
<td>Vibrating Probe Company</td>
<td>&nbsp;</td>
<td>Powered by one 9 V (PP3) battery</td>
</tr>		</tbody>
		</table>
		</div>

measurement of bioelectric current with a vibrating probe

Les champs électriques, générés par le transport actif des ions, sont présents dans de nombreux systèmes biologiques et servent souvent d&#39;importantes fonctions dans les tissus et organes. Par exemple, ils jouent un rôle important dans la direction de la migration des cellules lors de la cicatrisation. Nous décrivons ici la fabrication et l&#39;utilisation de sondes ultra vibrant pour mesurer les courants électriques extracellulaire. La sonde est un isolant, fil de métal aiguisé avec une petite pointe de noir de platine (30-35 um), qui peut détecter des courants ioniques dans le pA / cm 2 dans du sérum physiologique large. La sonde est mise en vibration à environ 200 Hz par un piézoélectriques Bender. En présence d&#39;un courant ionique, la sonde détecte une différence de tension entre les extrêmes de son mouvement. Un lock-dans des filtres amplificateurs à bruit parasite en verrouillant à la fréquence de la sonde de vibration. Les données sont enregistrées sur ordinateur. La sonde est étalonnée en début et en fin d&#39;expériences dans une solution saline appropriée, en utilisant une chambre qui applique un courant de 1,5 pA exactement / cm 2. Nous décrivons comment faire des sondes, configurer le système et les calibrer. Nous démontrons également la technique de mesure la cornée, et de montrer quelques résultats représentatifs à partir de spécimens différents (cornée, peau, du cerveau).

Watch this Scientific Journal Video about Measurement of Bioelectric Current with a Vibrating Probe at JoVE.com

Measurement of Bioelectric Current with a Vibrating Probe

La fabrication, l&#39;étalonnage et l&#39;utilisation de la non-invasive des sondes vibrant pour mesurer le courant bioélectrique dans divers systèmes biologiques est décrite.

Mesure du courant bioélectrique avec une sonde vibrante

Electric fields, generated by active transport of ions, are present in many biological systems and often serve important functions in tissues and organs. ...

measurement-of-bioelectric-current-with-a-vibrating-probe

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14.1K vues.  University of California, Davis. La fabrication, l'étalonnage et l'utilisation de la non-invasive des sondes vibrant pour mesurer le courant bioélectrique dans divers systèmes biologiques est décrite.

Vidéo: Measurement of Bioelectric Current with a Vibrating Probe

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

Mesure de la cohésion globale par tensiométrie surface du tissu

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

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Bio-ingénierie

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

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.

Nanoparticules fluorescentes pour la mesure de la concentration d&#39;ions dans les systèmes biologiques

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Enquêter sur les systèmes récepteur-ligand de la cellulosome avec base-AFM unique molécule Spectroscopie de Force

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

Fabrication microfluidique de fibres polymères et biohybrides avec une taille et une forme redessinées

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC)&#160;to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using &#8220;off-the-shelf&#8221; microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) &#956;m in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

Microscopie optique quantitative: mesure de biophysique cellulaire Caractéristiques avec un microscope optique standard

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

Une technique microfluidique à la sonde cellulaire Déformabilité

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.

Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces.

Surface de mesure potentielle de bactéries utilisant Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. ...

Fabrication of a Dipole-assisted Solid Phase Extraction Microchip for Trace Metal Analysis in Water Samples

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water samples. A brief overview of the evolution of chip-based SPE techniques is provided. This is followed by an introduction to specific polymeric materials and their role in SPE. To develop an innovative dipole-assisted SPE technique, a chlorine (Cl)-containing SPE functionality was implanted into a poly(methyl methacrylate) (PMMA) microchip. Herein, diverse analytical techniques including contact angle analysis, Raman spectroscopic analysis, and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) analysis were employed to validate the utility of the implantation protocol of the C-Cl moieties on the PMMA. The analytical results of the X-ray absorption near-edge structure (XANES) analysis also demonstrated the feasibility of the Cl-containing PMMA used as an extraction medium by virtue of the dipole-ion interactions between the highly electronegative C-Cl moieties and the positively charged metal ions.

The fabrication protocol of a dipole-assisted solid phase extraction microchip for the trace metal analysis is presented.

Fabrication d&#39;une extraction en phase solide Microchip Dipole-assistée pour analyse de traces métalliques dans des échantillons d&#39;eau

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

Modélisation soustractive rapide des couches de cellules vivantes avec une sonde microfluidique

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

Installation optimisée et protocole d’imagerie de domaines magnétiques sur mesure In Situ de hystérésis

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...