1. Imaging-Setup Vieles kann in einem kompakten und leichten tragbaren Feld-Architektur 8 zusammengebaut werden, und alternativ als optofluidischer Mikroskop mit Schnittbildverfahren Fähigkeit. 9 In diesem Bericht werden wir aber die Basic Imaging-Setup für eine Tisch-Implementierung in Richtung Tomographie von statischen beschreiben Proben. <ol><li> Beleuchtung Modul: LOT, teilweise kohärente Lichtquellen wie Licht emittierende Dioden (LEDs) verwendet werden. Für experimentelle Flexibilität, verwendeten wir einen Monochromator mit einer Xenon-Lampe (Cornerstone T260, Newport Corp.) Der Monochromator wurde eingestellt, um einen Ausgang mit ~ 1-10 nm spektrale Breite um eine Mittenwellenlänge von zB 450-650 nm bereitzustellen. Diese teilweise kohärente Ausgabe wird dann an einer Multimode-Lichtleitfaser (Thorlabs AFS105/125Y) teilweise kohärentes Licht auf das System liefern könnten. Die optische Faser wird auf einem Drehtisch montiert motorisierten (ThorlabsPRM-1Z8 durch Thorlabs TDC001 Regler angesteuert), um den Winkel der Beleuchtung ändern. Die motorisierten Tisch, wobei die Lichtquelle befestigt ist, auf einer zweidimensionalen linearen XY-Tisch (Newport ILS50CC von Newport MFA-Controllern gesteuert werden PPD), die verwendet werden, um Verschiebungen in der Ebene der Lichtquelle in einem bestimmten Winkel zu erreichen, wurde angebracht. </li><li> Detektion: Lensfree optischen Tomographie ist eine skalierbare Technologie, so dass die Detektoranordnung gemäß den Anforderungen der Anwendungen ohne wesentliche Änderung der Bildaufnahme, bzw. die Datenverarbeitungseinrichtung gewählt werden. In diesem Bericht beschäftigten wir einen CMOS-Sensor-Array mit 5 Megapixel, mit einer Pixelgröße von 2,2 um (IDS Imaging, UI-1485LE-M). Der Detektor wird verwendet, um die weiten Sichtfeld-Ansicht (zB 24 mm 2) holographische Bilder von Proben, die direkt auf der Oberseite des aktiven Bereichs des Detektors gebracht wurden aufgezeichnet. Wenn ein Zwei-Achsen-Tomographie-Setup durchzuführen ist 7, sollte der Detektor einlso auf einer horizontalen (mit Bezug auf die optische Tabelle) Rotationstisch (zB Thorlabs RP-01), um die Probe (zusammen mit dem Detektor) in der Ebene senkrecht zur optischen Achse drehbar gelagert werden. 7 </li></ol> 2. Probenvorbereitung Während lensfree optischen Tomographie abbilden kann eine Vielzahl von Objekten wie Zellen und Mikroorganismen, werden wir die grundlegenden Prinzipien, indem dreidimensionale Mikroskopie eines C veranschaulichen elegans Probe. <ol><li> Mit einem Skalpell oder einem Spatel, nehmen Sie ein kleines Stück Agar aus der Petrischale mit den C elegans Kultur. Eine kubische Stück von mehreren Millimetern entlang jeder Dimension enthält Hunderte von Nematoden. </li><li> Setzen Sie den kleinen Klumpen des Agar-Agar in einem Polypropylen-Fläschchen mit 1 ml deionisiertem Wasser (DI). </li><li> Vorsichtig vortexen für 0,5-1 min, und warten Sie 10-15 Minuten, bis die Würmer kriechen aus dem Agar-Stück in die DI-Wasser. </li><li> Umvorübergehend zu immobilisieren die Würmer, 1 ml von 5-10 mm Levamisol-Lösung (Sigma-Aldrich) und warten Sie 10 min. </li><li> Pipettieren 5-10 ul der Probe aus dem Boden des Fläschchens und Sandwich zwischen zwei Deckgläsern. Diese Probe, die eine große Anzahl von vorübergehend immobilisiert Schnecken (zB 50-100 Würmer), kann auf den Detektor mit der Datenerfassung beginnen abgegeben werden. </li></ol> 3. Data Acquisition Hier fassen wir die Bildaufnahme Schritte für eine typische LOT Experiment, das unter Verwendung einer benutzerdefinierten entwickelten LabView-Schnittstelle automatisiert wurde. <ol><li> Einstellen der ersten Drehwinkel Stufe bis -50 °, wobei 0 ° entspricht der vertikalen Position der Lichtquelle. </li><li> Stellen Sie die ursprüngliche Position der XY-Bühne auf (0,0), das ist der HOME-Position. </li><li> Stellen Sie die Belichtung des Detektors am besten nutzen seines Dynamikbereichs so daß das Bild so hell wie möglich ohne saturated Pixel. </li><li> Ohne Änderung des Winkels der Drehung Bühne, erobern 9 Bildern. Für jedes Bild, bewegen die XY-Stufe an eine neue Position in einem 3x3 quadratischen Raster so dass jeder Bildbereich um etwa ein Viertel eines Pixels verschiebt gegenüber dem vorherigen. Erfassen weitere Bilder in jedem Winkel, z. B. in einem 6μ6 Gitter, könnte Auflösung abhängig von der Art des Objekts und des Signal-Rausch-Verhältnis (SNR) zu verbessern. 10 </li><li> Stellen Sie die ursprüngliche Position der XY-Bühne auf (0,0), das ist der HOME-Position. </li><li> Erhöhen Sie den Winkel der Drehung der Bühne in Schritten von 2 ° bis +50 ° erreicht. Nach jedem Inkrement bei jedem neuen Blickwinkel, wiederholen Sie die Schritte 3-5 dh. Die Winkelinkrementen können feinere oder gröbere abhängig von der Optimierung der Messzeit vs Bildauflösung. </li><li> (Optionaler Schritt, wenn ein Zwei-Achsen-Tomographie Schema genutzt werden soll) Drehen Sie den horizontalen Bühne, auf der der Detektor von 90 ° montiert ist. Dann,wiederholen Sie die Schritte 1-6. </li></ol> 4. Datenanalyse Nach den Schritten 1-6 in Abschnitt 3, ein Satz von 459 Bildern wird erworben, der 9 Sub-Pixel verschoben Bilder für jede der 51 verschiedenen Beleuchtungs enthält. Zunächst wird jeder Satz von 9 Bilder digital bearbeitet, mit Pixel-Super-Resolution-Algorithmen 10, zu einem hochauflösenden (HR) Projektion Hologramm pro Winkel zu erhalten. Es sei darauf hingewiesen, dass Pixel Super-Auflösungseffekt zur Überwindung der Begrenzung der Pixelgröße bezieht, als die Beugungsgrenze der räumlichen Auflösung der Bilder lensfree werden. Pixel super-aufgelösten Hologramme werden dann digital rekonstruiert 10.07 bis 51 Projektionsbilder erhalten. Dieser Satz von 51 Projektionsbilder dann rückprojiziert mit TomoJ, ein Plug-in für ImageJ (ein Open-Source Bildanalyse-Software). 11 Dieser Rückprojektion Betrieb gibt ein dreidimensionales Bild (Tomogramme) der Probe. Obwohl hier nicht implementiert,eine zweiachsige Tomographie Modell könnte auch verwendet werden, wie in Referenz beschrieben. 7. In diesem Schema wird ein zweiter Satz der Schichtbilder durch Drehen der Lichtquelle entlang einer orthogonalen Achse in Bezug auf die erste Drehachse, die mehrere räumliche Informationen über die Probe enthält, und verbessert die axiale Auflösung. 5. Repräsentative Ergebnisse Das große Feld-of-view (FOV) von lensfree optischen Tomographie ist in Abbildung 1 gezeigt. Wenn die Probe direkt auf der Oberseite des Detektor-Array platziert ist, können holographische Bilder der Objekte mit einem Sichtfeld von 24 mm 2, die weiter gesteigert werden kann anhand einer neuen Detektor-Arrays mit größeren aktiven Bereichen aufgezeichnet werden. Obwohl Pixelgröße des Detektors-Array begrenzt die Auflösung der aufgenommenen holographische Bilder, mildern Pixel Super-Resolution-Techniken dieses Problem. Abbildung 2 zeigt Pixel super-aufgelösten Hologramme entlangmit den rekonstruierten Bildern (also Bilder Projektion), die Sub-Mikrometer-räumliche Auflösung bieten, für drei verschiedene Winkel der Beleuchtung. Die Projektionsbilder kombiniert tomographische Bildrekonstruktionstechniken (zB gefilterten Rückprojektion) an Schichtaufnahmen (dh dreidimensionale Bilder) der Probe zu berechnen. 3 zeigt drei Schnittbilder in der xy-Ebene durch die vordere der Schnecke, wobei die Rachentubus ist nur in der Schicht durch z = 8 um, wie etwa ins zylindrische Struktur mit ~ 5 um Außendurchmesser erwartet sichtbar. Darüber hinaus ist die Querschnittsbild in der xz-Ebene (Einschub in Abbildung 3 oberes Feld) zeigt deutlich die Grenzen des Wurms und die Schlundsonde innen (spitz durch die Pfeile), was erfolgreiche 3D-Bildgebung des Rachens. <img src="/files/ftp_upload/4161/4161fig1.jpg" alt="1" /> <strong&gt; Abbildung 1 zeigt ein komplettes Feld-of-view (24 mm 2) holographische Bild aufgenommen mit dem lensfree optischen Tomographie (LOT) Setup für die vertikale Beleuchtung. Aufgrund der großen Abbildungsbereich LOT, können viele Würmer gleichzeitig mit einer einzigen Datenerfassung abgebildet würde. <img src="/files/ftp_upload/4161/4161fig2.jpg" alt="2" /> Abbildung 2 zeigt die Pixel super-aufgelösten Hologramme (linkes Bild) und die digital rekonstruierte Projektionsbilder (rechtes Bild) für eine C elegans-Wurm (abgeschnitten von einem großen Feld-of-view) an drei verschiedenen Beleuchtungswinkel. Jeder Vorsprung Bild enthält Informationen zu einem anderen Blickwinkel, die Berechnung der 3D-Struktur ermöglicht durch eine gefilterte Rückprojektion Betrieb. <img src="/files/ftp_upload/4161/4161fig3.jpg" alt="Abbildung 3" /> Abbildung 3 zeigt Computertomogramme für die C elegans Wurm oAbbildung 2 f. (Obere Reihe) zeigt ein Schnittbild des gesamten Wurm bei z = 3 um. Der Einschub zeigt eine Querschnittsansicht Bild von der vorderen der Schnecke. Maßstabsbalken des Einsatzes ist 50 um. (Untere Reihe) zeigt drei Schnittbilder durch die vordere des Wurms, was die optische Schnitte Fähigkeit lensfree optischen Tomographie. Pfeile in der Figur geben die gleichen Punkt auf dem Rachentubus der Schnecke. Ein Mikroskop-Bild (x40, 0,65-NA) wird auch für visuellen Vergleich zur Verfügung gestellt.

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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>

Keine Interessenskonflikte erklärt.

Es ist wichtig zu betonen, dass die einzigartige Geometrie der lensfree On-Chip-holographische Mikroskopie die kritische Enabler zu Pixel Super-Resolution und tomographische Bildgebung zu erreichen ist. Da die aufgezeichneten Bilder werden nicht angenommen Projektionsbilder sein, wie in inkohärenten Kontaktabbildungsvorrichtung nähert 12, aber Vorsprung Hologramme, die Beugung des durchgelassenen Lichts, bis sie auf den Detektor auftrifft kann digital holografische Rekonstruktion korrigiert werden....

<table border="1"><tbody><tr><td> Name </td><td> Compay </td><td> Katalog # </td><td> Kommentar </td></tr><tr><td> Linear Kreuztische </td><td> Newport Corp </td><td> MFA-PP </td><td> Miniatur-Lineartisch </td></tr><tr><td> Motorisierte Drehung der Bühne </td><td> Thorlabs </td><td> PRM1Z8 </td><td> Motorisierte Präzisions-Berg </td></tr><tr><td> Multimode-Lichtleitfaser </td><td> Thorlabs </td><td> AFS105/125Y </td><td> Multimode-Glasfaser </td></tr><tr><td> Lichtquelle </td><td> Newport Corp </td><td> 6255 </td><td> Ozon-Xenon-Lampe </td></tr><tr><td> Monochromator </td><td> Newport Corp </td><td> 74100 </td><td> Cornerstone ™ 260 1/4 m Monochromator </td></tr><tr><td> CMOS-Sensor-Array </td><td> Aptina Inc. </td><td> MT9P031STC </td><td> 5 Megapixel CMOS Sensor </td></tr><tr><td> C. elegans Proben </td><td> Carolina Biosupply </td><td> 173500 </td><td> Wild-Typ C. elegans </td></tr><tr><td> Levamisol </td><td> Sigma Aldrich </td><td> L9756-5G </td><td> Tetramisole Hydrochlorid </td></tr></tbody></table>

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

Tomographischen Bildgebung wurde ein weit verbreitetes Instrument in der Medizin, wie sie dreidimensionale (3D) Struktur-Informationen über Objekte unterschiedlicher Größe skaliert bieten kann. In Mikrometer und Millimeterskalen finden optische Mikroskopie Modalitäten zunehmend aufgrund der nicht-ionisierender Natur des sichtbaren Lichts, und die Verfügbarkeit eines umfassenden Satzes von Lichtquellen (wie Laser und Licht emittierende Dioden-) und Detektorelemente (z. B. großformatigen CCD-und CMOS-Detektor-Arrays). Unter den neu entwickelten optischen Mikroskopie tomographischen Modalitäten, kann man auch die optische Kohärenztomographie, beugungsoptisch Tomographie, optische Projektion und Licht-Tomographie-Blatt-Mikroskopie. 6.1 Diese Plattformen bieten Schnittbildverfahren von Zellen, Mikroorganismen und Tiere wie Modell C elegans, Zebrafisch und Maus-Embryonen. Vorhandene optische 3D-Imager haben in der Regel relativ sperrige und komplexe Architekturen, die Begrenzung the Verfügbarkeit dieser Anlagen zu modernsten Labore, und behindert ihre Integration in Lab-on-a-Chip-Plattformen und Mikrofluidik-Chips. Um eine Alternative tomographischen Mikroskop zu schaffen, haben wir kürzlich entwickelt lensfree optischen Tomographie (LOT) als High-Throughput-, kompakte und kostengünstige optische Tomographie Modalität. 7 LOT verwirft den Einsatz von Linsen und optischen Komponenten sperrig, und statt dessen setzt auf Multi-Angle- Beleuchtung und digitale Berechnung zu tiefenaufgelöste Abbildung von Mikro-Objekte über ein großes Volumen Bildgebung zu erreichen. LOT abbilden kann biologischen Probe mit einer räumlichen Auflösung von &lt;1 um x &lt;1 um x &lt;3 um in der x, y und z jeweils über einen großen Bildgebungsvolumen von 15-100 mm 3, und ist besonders nützlich Lab-on-a-Chip-Plattformen.

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 optischen Tomographie ist eine dreidimensionale Mikroskopie-Technik, die eine räumliche Auflösung von &lt;1 um bietet × &lt;1 um x &lt;3 &amp;mgr; m in x-, y-und z-Dimensionen jeweils über einen großen Imaging-Volumen von 15-100 mm<sup&gt; 3</sup&gt; Kann das besonders nützlich sein für die Integration in Lab-on-a-Chip-Plattformen.

Lensfree On-Chip-tomographischen Mikroskopie beschäftigt Multi-Angle-Beleuchtung und Super-Pixel-Auflösung

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 Aufrufe.  University of California, Los Angeles. Lensfree optischen Tomographie ist eine dreidimensionale Mikroskopie-Technik, die eine räumliche Auflösung von 

Serie: 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.

Biomolekulare Erkennung Einsatz der Interferometrische Reflectance Imaging Sensor (IRIS)

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

Forschung

Biotechnik

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.

On-chip Isotachophorese für Separierung von Ionen und Aufreinigung von Nukleinsäuren

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-Endothelial Entzündliche Phänotypisierung

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.

Ein Leitfaden für Strukturierte Beleuchtung TIRF Mikroskopie bei hoher Geschwindigkeit mit mehreren Farben

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.

Trockenen Film Photoresist-basierte elektrochemische mikrofluidischen Biosensor Plattform: Gerät Fertigung, On-Chip-Test Vorbereitung und Systembetrieb

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.

Herstellung und Validierung von einer Orgel-on-Chip-System mit integrierten Elektroden, direkt Transendothelial elektrischen Widerstand zu quantifizieren

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.

Methanol unabhängigen Ausdruck von Pichia Pastoris de-Unterdrückung Technologien

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 Genom Bearbeitung von Saccharomyces cerevisiae und die Schaffung von Hefe Pixel Kunst

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;

Anwendungen für 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 ...

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.

Aufbau eines einfachen und vielseitigen Beleuchtungssystems für optogenetische Experimente

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