1. Configuration d&#39;imagerie LOT peut être assemblé dans un compact et léger sur le terrain portable architecture 8, et, alternativement, comme un microscope optofluidic avec la capacité d&#39;imagerie en coupe. 9 Dans ce rapport, cependant, nous allons décrire l&#39;installation d&#39;imagerie de base pour une mise en œuvre de paillasse vers la tomographie d&#39;électricité statique échantillons. <ol><li> Module d&#39;éclairage: dans le Lot, les sources de lumière partiellement cohérente comme émettrices de lumière des diodes électroluminescentes (DEL) peuvent être utilisés. Pour plus de flexibilité expérimentale, nous avons utilisé un monochromateur avec une lampe au xénon (Cornerstone T260, Newport Corp). Le monochromateur est ajusté pour fournir un signal de sortie avec ~ 1-10 nm largeur spectrale autour d&#39;un centre de longueur d&#39;onde par exemple 450-650 nm. Cette sortie partiellement cohérent est ensuite couplé à une fibre optique multimode (Thorlabs AFS105/125Y) pour délivrer la lumière partiellement cohérente au système. La fibre optique est monté sur une platine de rotation motorisée (ThorlabsPRM-1Z8 entraîné par Thorlabs TDC001 contrôleur) pour changer l&#39;angle d&#39;éclairage. L&#39;étape motorisé, avec la source de lumière fixée, est montée sur une scène bidimensionnelle linéaire XY (Newport ILS50CC entraîné par Newport MAE-PPD contrôleurs), qui a été utilisé pour réaliser des changements dans le plan de la source de lumière sous un angle donné. </li><li> Détection: Lensfree tomographie optique est une technologie s&#39;adaptent de telle sorte que le réseau de détecteurs peut être choisie en fonction des besoins des applications sans modifier sensiblement les étapes d&#39;acquisition d&#39;image ou le traitement des données. Dans ce rapport, nous avons utilisé un réseau de capteurs CMOS ayant 5 Mega Pixels, avec une taille de pixel de 2,2 microns (IDS Imaging, l&#39;assurance-chômage 1485LE-M). Le détecteur est utilisé pour enregistrer les larges champ de vue (par exemple 24 mm 2) des images holographiques d&#39;échantillons, qui ont été passées directement sur ​​le dessus de la zone active du détecteur. Si une installation de tomographie à double axe est à mettre en œuvre 7, le détecteur si unlso être monté sur une platine de rotation (par rapport à la table optique) horizontale (par exemple Thorlabs RP-01) pour faire tourner l&#39;échantillon (avec le détecteur) dans le plan normal à l&#39;axe optique. 7 </li></ol> 2. Préparation de l&#39;échantillon Alors que lensfree tomographie optique peut l&#39;image d&#39;une variété d&#39;objets tels que des cellules et des micro-organismes, nous allons illustrer les principes de base en effectuant trois dimensions microscopique d&#39;un C. elegans échantillon. <ol><li> L&#39;utilisation d&#39;un scalpel ou d&#39;une spatule, prendre un petit morceau d&#39;agar-agar de la boîte de Pétri contenant du C. elegans culture. Un morceau cube de quelques millimètres le long de chaque dimension contiendra des centaines de nématodes. </li><li> Placer le bloc d&#39;agar petite dans un flacon de polypropylène contenant 1 ml désionisée (DI) de l&#39;eau. </li><li> Doucement à vortex pour 0,51 mn, et attendre pendant 10-15 min jusqu&#39;à ce que les vers sortent de la pièce agar dans l&#39;eau DI. </li><li> Afin deimmobiliser temporairement les vers, ajouter 1 ml de solution mM lévamisole 5-10 (Sigma-Aldrich) et attendre pendant 10 min. </li><li> Pipeter pi 5-10 échantillon à partir du fond du flacon, et en sandwich entre deux lamelles. Cet échantillon, contenant un grand nombre de vers temporairement immobilisée (p.ex. 50-100 vers), peut être placé sur le détecteur à commencer l&#39;acquisition de données. </li></ol> 3. Acquisition de données Ici, nous résumons les étapes d&#39;acquisition d&#39;image pour une expérience typique de LOT, qui a été automatisée en utilisant une coutume développé LabView interface. <ol><li> Ajuster l&#39;angle initial de la phase de rotation à -50 °, où 0 ° correspondant à la position verticale de la source de lumière. </li><li> Ajustez la position initiale de la platine XY à (0,0), qui est la position HOME. </li><li> Réglez le temps d&#39;exposition du détecteur à utiliser au mieux sa gamme dynamique de telle sorte que l&#39;image est aussi brillant que possible sans sPixel aturated. </li><li> Sans changer l&#39;angle de la table de rotation, de capturer des images 9. Pour chaque image, déplacez le platine XY à une nouvelle position dans une grille de 3x3 carrés de sorte que chaque image se déplace d&#39;environ un quart de pixel par rapport à la précédente. Acquérir d&#39;autres images à chaque angle, par exemple dans une grille 6μ6, peut améliorer la résolution en fonction du type de l&#39;objet et le signal-sur-bruit (SNR). 10 </li><li> Ajustez la position initiale de la platine XY à (0,0), qui est la position HOME. </li><li> Augmenter l&#39;angle de rotation de l&#39;étage par incréments de 2 °, jusqu&#39;à atteindre 50 °. Après chaque incrément, c&#39;est à dire à chaque nouvel angle, répétez les étapes 3-5. Les incréments angulaires peuvent être plus ou moins fin selon l&#39;optimisation du temps d&#39;acquisition par rapport à la résolution d&#39;imagerie. </li><li> (Étape optionnelle si un régime de tomographie par deux axes doit être utilisé) Tourner la scène horizontale sur laquelle le détecteur est monté à 90 °. Puis,répétez les étapes 1-6. </li></ol> 4. Analyse des données Après les étapes 1-6 dans la section 3, un ensemble de 459 images est acquise, qui contient 9 sous-pixels des images décalées pour chacune des 51 sous différents angles d&#39;illumination. Tout d&#39;abord, chaque série de 9 images est traitée numériquement, à l&#39;aide de pixels de super-résolution des algorithmes 10, pour obtenir un exemplaire haute résolution (HR) hologramme de projection par l&#39;angle. Il convient de noter que le pixel de super-résolution se réfère à surmonter la limitation de la taille du pixel, plutôt que la limite de diffraction, la résolution spatiale des images lensfree. Pixel hologrammes super-résolus sont ensuite numériquement reconstruite 7-10 pour obtenir 51 images de projection. Cet ensemble de 51 images de projection est alors rétro-projetée à l&#39;aide TomoJ, un plug-in pour ImageJ (un logiciel open source d&#39;analyse d&#39;images) 11. Cette opération de rétroprojection produit une image tridimensionnelle (tomographie) de l&#39;échantillon. Bien que n&#39;étant pas mis en œuvre ici,un système de tomographie à double axe pourrait également être utilisée comme décrit dans la réf. 7. Dans ce dispositif, un second ensemble de tomographies est obtenu en faisant tourner la source de lumière le long d&#39;un axe orthogonal par rapport à l&#39;premier axe de rotation, qui fournit des informations plus spatiale concernant l&#39;échantillon, et améliore la résolution axiale. 5. Les résultats représentatifs Le grand champ de vision (FOV) de lensfree tomographie optique est démontré dans la figure 1. Comme l&#39;échantillon est placé directement sur ​​la partie supérieure du détecteur-tableau, des images holographiques des objets peuvent être enregistrées sur une mm FOV de 24 2, qui peut encore être augmentée en utilisant des réseaux de détecteurs émergents avec de plus grandes zones actives. Bien que la taille des pixels du détecteur à réseau limite la résolution des images enregistrées holographiques, des pixels de super-résolution des techniques d&#39;atténuer ce problème. La figure 2 montre de pixels ultra-résolus hologrammes longavec les images reconstruites (images de projection par exemple) qui offrent des sub-micrométrique résolution spatiale, pour trois angles différents de l&#39;illumination. Les images de projection peuvent être combinés en utilisant des techniques de reconstruction tomographique d&#39;image (par exemple rétroprojection filtrée) pour calculer des tomographies (c.-à-images en trois dimensions) de l&#39;échantillon. La figure 3 montre trois images de coupe dans le plan xy par la partie antérieure du ver, où le Tube pharyngien n&#39;est visible que dans la tranche grâce à z = 8 um, comme prévu à partir de cette structure à peu près cylindrique dont le diamètre ~ 5 um extérieure. En outre, l&#39;image en coupe dans le plan xz (en médaillon dans la figure 3 panneau supérieur) montre clairement les limites de la vis sans fin et le tube pharyngé à l&#39;intérieur (souligné par les flèches), ce qui démontre avec succès l&#39;imagerie 3D du pharynx. <img src="/files/ftp_upload/4161/4161fig1.jpg" alt="Figure 1" /> <strong&gt; La figure 1 montre un plein champ de vue (24 mm 2) image holographique enregistré en utilisant la tomographie optique lensfree (LOT) de configuration pour l&#39;éclairage vertical. En raison de la zone d&#39;imagerie grand nombre de LOT, beaucoup de vers peuvent être simultanément imagé avec une étape d&#39;acquisition de données unique. <img src="/files/ftp_upload/4161/4161fig2.jpg" alt="Figure 2" /> La figure 2 montre le pixel super-résolues hologrammes (panneau de gauche) et les images de projection reconstruites numériquement (panneau de droite) pour un C. ver elegans (recadrée à partir d&#39;un grand champ de vue) à trois angles d&#39;éclairage différents. Chaque image de projection contient des informations sur un angle de vision différent, ce qui permet le calcul de la structure 3D à travers une rétroprojection filtrée opération. <img src="/files/ftp_upload/4161/4161fig3.jpg" alt="Figure 3" /> La figure 3 montre tomographies calculées pour la C. ver elegans oFigure f 2. (Rangée du haut) montre une image de tranche du ver ensemble, à z = 3 um. L&#39;encart montre une image transversale de la partie antérieure du ver. La barre d&#39;échelle de l&#39;encart est de 50 um. (Rangée du bas) montre trois images de coupe à travers la partie antérieure du ver, ce qui démontre la capacité optique de sectionnement de la tomographie optique lensfree. Flèches travers la figure indiquent le même point sur le tube pharyngé de la vis sans fin. Une image au microscope (x40, 0,65 NA) est également fourni pour une comparaison visuelle.

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	<li>Schmitt, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+(OCT):+a+review,+J.+Sel.+Top.">Optical coherence tomography (OCT): a review, J. Sel. Top.</a> Quant. Elect. 5, 1205-1215 (1999).</li><li>Keller, P. J., Schmidt, A. D., Wittbrodt, J., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+zebrafish+early+embryonic+development+by+scanned+light+sheet+microscopy.">Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy.</a> Science. 322, 1065-1069 (2008).</li><li>Sharpe, J., Ahlgren, U., Perry, P., Hill, B., Ross, A., Hecksher-S&#248;rensen, J., Baldock, R., Davidson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Projection+Tomography+as+a+Tool+for+3D+Microscopy+and+Gene+Expression+Studies.">Optical Projection Tomography as a Tool for 3D Microscopy and Gene Expression Studies.</a> Science. 296, 541-545 (2002).</li><li>Sung, Y., Choi, W., Fang-Yen, C., Badizadegan, K., Dasari, R. R., Feld, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+diffraction+tomography+for+high+resolution+live+cell+imaging.">Optical diffraction tomography for high resolution live cell imaging.</a> Opt. Exp. 17, 266-277 (2009).</li><li>Debailleul, M., Simon, B., Georges, V., Haeberle, O., Lauer, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Holographic+microscopy+and+diffractive+microtomography+of+transparent+samples.">Holographic microscopy and diffractive microtomography of transparent samples.</a> Meas. Sci. Technol. 19, 074009 (2008).</li><li>Charri&#232;re, F., Pavillon, N., Colomb, T., Depeursinge, C., Heger, T. J., Mitchell, E. A. D., Marquet, P., Rappaz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+specimen+tomography+by+digital+holographic+microscopy:+Morphometry+of+testate+amoeba.">Living specimen tomography by digital holographic microscopy: Morphometry of testate amoeba.</a> Opt. Exp. 14, 7005-7013 (2006).</li><li>Isikman, S. O., Bishara, W., Mavandadi, S., Yu, S. W., Feng, S., Lau, R., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lens-free+optical+tomographic+microscope+with+a+large+imaging+volume+on+a+chip.">Lens-free optical tomographic microscope with a large imaging volume on a chip.</a> Proc. Nat. Acad. Sci. 108, 7296-7301 (2011).</li><li>Isikman, S. O., Bishara, W., Sikora, U., Yaglidere, O., Yeah, J., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field-portable+Lensfree+Tomographic+Microscope.">Field-portable Lensfree Tomographic Microscope.</a> Lab Chip. 11, 2222-2230 (2011).</li><li>Isikman, S. O., Bishara, W., Zhu, H., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optofluidic+tomography+on+a+chip.">Optofluidic tomography on a chip.</a> App. Phys. Lett. 98, 161109 (2011).</li><li>Bishara, W., Su, T. W., Coskun, A., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lensfree+on-chip+microscopy+over+a+wide+field-of-view+using+pixel+super-resolution.">Lensfree on-chip microscopy over a wide field-of-view using pixel super-resolution.</a> Opt. Exp. 18, 11181-11191 (2010).</li><li>Messaoudi, C., Boudier, T., Sorzano, C. O. S., Marco, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TomoJ:+tomography+software+for+three-dimensional+reconstruction+in+transmission+electron+microscopy.">TomoJ: tomography software for three-dimensional reconstruction in transmission electron microscopy.</a> BMC Bioinformatics. 8, 288 (2007).</li><li>Heng, X., Erickson, D., Baugh, L. R., Yaqoob, Z., Sternberg, P. W., Psaltis, D., Yang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optofluidic+Microscopy:+A+Method+for+Implementing+High+Resolution+Optical+Microscope+On+A+Chip.">Optofluidic Microscopy: A Method for Implementing High Resolution Optical Microscope On A Chip.</a> Lab on a Chip. 6, 1274-1276 (2006).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

Il est important de souligner que la géométrie unique de lensfree sur puce microscopie holographique est le catalyseur essentiel pour atteindre pixel de super-résolution et l&#39;imagerie tomographique. Étant donné que les images enregistrées ne sont pas supposées être des images de projection comme dans l&#39;imagerie de contact incohérente approche 12, mais des hologrammes de projection, la diffraction de la lumière transmise jusqu&#39;à ce qu&#39;il empiète sur le détecteur peut être...

<table border="1"><tbody><tr><td> Nom </td><td> Compay </td><td> Catalogue # </td><td> Commenter </td></tr><tr><td> Linéaires stades XY </td><td> Newport Corp </td><td> MFA-PP </td><td> Miniature scène linéaire </td></tr><tr><td> Platine de rotation motorisée </td><td> Thorlabs </td><td> PRM1Z8 </td><td> Motorisé de rotation de précision Mont </td></tr><tr><td> Fibre optique multimode </td><td> Thorlabs </td><td> AFS105/125Y </td><td> Fibre multimode </td></tr><tr><td> Source de lumière </td><td> Newport Corp </td><td> 6255 </td><td> La couche d&#39;ozone sans lampe au xénon </td></tr><tr><td> Monochromateur </td><td> Newport Corp </td><td> 74100 </td><td> Cornerstone 260 1/4 m Monochromateur </td></tr><tr><td> Réseau de capteurs CMOS </td><td> Aptina Inc </td><td> MT9P031STC </td><td> 5 mégapixels du capteur CMOS </td></tr><tr><td> C. elegans échantillon </td><td> Caroline Biosupply </td><td> 173500 </td><td> Wild-type C. elegans </td></tr><tr><td> Le lévamisole </td><td> Sigma-Aldrich </td><td> L9756-5G </td><td> Chlorhydrate tétramisole </td></tr></tbody></table>

lensfree on-chip tomographic microscopy employing multi-angle illumination and pixel super-resolution

Imagerie tomographique a été un outil largement utilisé en médecine comme il peut fournir en trois dimensions (3D) des informations structurelles relatives des objets de tailles différentes échelles. Dans les échelles micrométriques et millimétriques, les modalités de microscopie optique trouver une utilisation croissante en raison de la nature non-ionisant de la lumière visible, et la disponibilité d&#39;un riche ensemble de sources d&#39;éclairage (telles que les lasers et électro-luminescentes des diodes) et des éléments de détection (tels que grand format CCD et CMOS détecteur-arrays). Parmi les optiques récemment développés modalités microscopie tomographique, on peut inclure la tomographie par cohérence optique, la tomographie par diffraction optique, la tomographie optique de projection et de la lumière-feuille de la microscopie. 1-6 Ces plates-formes fournissent des images en coupe de cellules, micro-organismes et les animaux modèles tels que C. elegans, le poisson-zèbre et d&#39;embryons de souris. Existants 3D imageurs optiques ont généralement des architectures relativement encombrants et complexes, ce qui limite ee la disponibilité de ces équipements à des laboratoires de pointe, et d&#39;empêcher leur intégration avec le laboratoire-sur-une-puce plates-formes et des puces microfluidiques. Pour fournir une solution de rechange microscope tomographique, nous avons récemment développé lensfree tomographie optique (LOT) comme une modalité tomographie optique à haut débit, compact et rentable. 7 rejets LOT l&#39;utilisation de lentilles et de volumineux composants optiques, et repose plutôt sur ​​multi-angle l&#39;éclairage et de calcul numérique pour obtenir la profondeur d&#39;imagerie à résolution de micro-objets sur une grande volume d&#39;imagerie. LOT peut spécimen biologique à l&#39;image d&#39;une résolution spatiale de moins de 1 mm x &lt;1 um x &lt;3 um dans le x, y et z dimensions, respectivement, au cours d&#39;une grande volume d&#39;imagerie de 15-100 mm 3, et peut être particulièrement utile pour lab-on-a-chip plates-formes.

Watch this Scientific Journal Video about Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution at JoVE.com

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Lensfree tomographie optique est une technique de microscopie en trois dimensions qui offre une résolution spatiale de moins de 1 um × &lt;1 um x &lt;3 microns en x, y et z dimensions, respectivement, au cours d&#39;une imagerie grand volume de 15-100 mm<sup&gt; 3</sup&gt;, Qui peut être particulièrement utile pour l&#39;intégration avec le laboratoire-sur-une-puce plates-formes.

Lensfree sur puce microscopie tomographique Employant multi-angle d&#39;éclairage et de Pixel Super-résolution

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

lensfree-on-chip-tomographic-microscopy-employing-multi-angle

Research

JoVE Journal

Bioengineering

11.4K vues.  University of California, Los Angeles. Lensfree tomographie optique est une technique de microscopie en trois dimensions qui offre une résolution spatiale de moins de 1 um × 

Vidéo: Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

Détection biomoléculaire employant le capteur interférométrique Réflectance Imaging (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Recherche

Bio-ingénierie

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration1,2 and efficient separation and extraction based on ionic mobility.3 For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation4-8 and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood.9-12
ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.13). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.
We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.

Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.

Sur la puce isotachophorèse pour la séparation des ions et purification des acides nucléiques

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

On-Chip phénotypage endothélium inflammatoire

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells.

This article provides an in depth guide for the assembly and operation of a structured illumination microscope operating with total internal reflection fluorescence illumination (TIRF-SIM) to image dynamic biological processes with optical super-resolution in multiple colors.

Un guide pour structuré Illumination FRBR Microscopie à haute vitesse avec plusieurs couleurs

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

Feuil sec axée sur la résine photosensible de biocapteurs microfluidiques électrochimiques plate-forme : Fabrication de dispositifs, préparation de dosage sur puce et fonctionnement du système

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Fabrication et Validation d’un système d’orgue-on-chip avec électrodes intégrées à quantifier directement transendothéliale résistance électrique

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab and industrial scale. However, due to its toxicity and flammability, there is a desire to avoid methanol while maintaining the high productivity of P. pastoris. Small scale bioreactor cultivations (0.5-5 L working volume) are commonly used to evaluate a strain and its protein production characteristics since microscale cultivation in deep well plates can be hardly controlled or relies on expensive equipment. Furthermore, traditional protocols for the cultivation and induction of P. pastoris were established for constitutive expression or methanol induction and so far, no reliable protocols were described to screen P. pastoris expression strains with derepressible promoters in (controlled and monitored) parallel cultivations. To simplify such initial cultivations to characterize and compare new protein production strains, we established a simple shake flask cultivation system for methanol free expression that simulates bioreactor conditions including a constant slow glycerol feed and online monitoring, thereby coming closer to the real conditions in bioreactors compared to mostly applied small scale batch cultivations. To drive recombinant protein expression in P. pastoris, the carbon source repressed promoters PDC and PDF were applied. Polymer discs with embedded carbon source, releasing a constant amount of glycerol, assured a feed rate delivering the necessary energy for maintaining the promoters active while keeping the biomass generation low.

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

This method for the first time describes reliable experimental procedures for efficient inducible methanol-free recombinant protein production in Pichia pastoris (Komagataella phaffii), employing the carbon source regulated promoters PDCand PDF in small scale experiments while cultivation parameters can be monitored online, and a constant glycerol feed is applied.

Méthanol Expression indépendante de Pichia Pastoris employant des Technologies de la répression

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab ...

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single CRISPR RNA (crRNA) array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S.&#160;cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently colored carotenoid producing yeast strains that were constructed.

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

The CRISPR/Cas12a system in combination with a single crRNA array enables efficient multiplex editing of the S.&#160;cerevisiae genome at multiple loci simultaneously. This is demonstrated by constructing carotenoid producing yeast strains which are subsequently used to create yeast pixel art.

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art CRISPR

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

Applications pour les panneaux d'éclairage Microplate-Compatible open Source

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...

Building a Simple and Versatile Illumination System for Optogenetic Experiments

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical control allows for an unprecedented ability to dissect function and holds the potential for enabling novel genetic therapies. However, optogenetic experiments require adequate light sources with spatial, temporal, or intensity control, often a bottleneck for researchers. Here we detail how to build a low-cost and versatile LED illumination system that is easily customizable for different available optogenetic tools. This system is configurable for manual or computer control with adjustable LED intensity. We provide an illustrated step-by-step guide for building the circuit, making it computer-controlled, and constructing the LEDs. To facilitate the assembly of this device, we also discuss some basic soldering techniques and explain the circuitry used to control the LEDs. Using our open-source user interface, users can automate precise timing and pulsing of light on a personal computer (PC) or an inexpensive tablet. This automation makes the system useful for experiments that use LEDs to control genes, signaling pathways, and other cellular activities that span large time scales. For this protocol, no prior expertise in electronics is required to build all the parts needed or to use the illumination system to perform optogenetic experiments.

This protocol describes how to perform optogenetic experiments for controlling gene expression with red and far-red light using PhyB and PIF3. Included are step-by-step instructions for building a simple and flexible illumination system, which enables the control of gene expression or other optogenetics with a computer.

Construire un système d’éclairage simple et polyvalent pour les expériences optogénétiques

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical ...