1. Imágenes de instalación Muchas cosas pueden ser montados en un compacto y ligero portátil de la arquitectura de campo 8, y, alternativamente, como un microscopio con la capacidad de formación de imágenes optofluídico sección 9. En este informe, sin embargo, vamos a describir la configuración de imagen básica para una aplicación de sobremesa hacia la tomografía de la electricidad estática muestras. <ol><li> Módulo de iluminación: en Lot, fuentes de luz parcialmente coherente, como emisores de luz de diodos (LED) se puede utilizar. Para una mayor flexibilidad experimental, se utilizó un monocromador con una lámpara de xenón (Cornerstone T260, Newport Corp.). El monocromador se ajustó para proporcionar una salida con ancho ~ 1-10 nm espectral alrededor de una longitud de onda central de, por ejemplo 450-650 nm. Esta salida parcialmente coherente se acopla entonces a una fibra óptica multimodo (Thorlabs AFS105/125Y) para entregar luz parcialmente coherente con el sistema. La fibra óptica está montada en una etapa de rotación motorizada (ThorlabsPRM-1Z8 impulsado por Thorlabs TDC001 controlador) para cambiar el ángulo de iluminación. La etapa motorizada, con la fuente de luz adjunta, está montado en una etapa bidimensional lineal XY (Newport ILS50CC impulsado por Newport PPD AMF-controladores), que fue utilizado para alcanzar en el plano, los desplazamientos de la fuente de luz en un ángulo dado. </li><li> Detección: tomografía Lensfree óptica es una tecnología escalable de tal manera que el conjunto de detectores pueden ser elegidos de acuerdo con los requisitos de las aplicaciones sin cambiar significativamente los pasos de adquisición de la imagen o el procesamiento de datos. En este informe, se empleó un conjunto de sensores CMOS que tiene 5 megapíxeles, con un tamaño de píxel de 2,2 micras (IDS Imaging, IU-1485LE-M). El detector se utiliza para registrar los anchos de campo de visión (por ejemplo 24 mm 2) imágenes holográficas de muestras, que se colocan directamente sobre la parte superior de la zona activa del detector. Si una configuración tomografía de doble eje que se aplicará 7, el detector debe unaLSO ser montado en una etapa de rotación horizontal (con respecto a la mesa óptica) (por ejemplo Thorlabs RP-01) para girar la muestra (junto con el detector) en el plano normal al eje óptico. 7 </li></ol> 2. Preparación de la muestra Si bien la tomografía óptica lensfree pueden tomar imágenes de una variedad de objetos, tales como células y microorganismos, que se ilustran los principios básicos mediante la realización de la microscopía en tres dimensiones de un C. elegans muestra. <ol><li> Con un bisturí o una espátula, tomar un pequeño trozo de agar de la placa de Petri que contiene el C. elegans cultura. Un trozo cúbico de varios milímetros a lo largo de cada dimensión contendrá cientos de nematodos. </li><li> Coloque el pequeño trozo de agar en un vial de polipropileno que contiene 1 ml desionizada (DI). </li><li> Suavemente vórtice durante 0,5-1 minutos, y esperar durante 10-15 minutos hasta que los gusanos se arrastran fuera de la pieza de agar en el agua desionizada. </li><li> Parainmovilizar temporalmente los gusanos, añadir 1 ml de solución 5.10 mM levamisol (Sigma-Aldrich) y esperar durante 10 minutos. </li><li> Pipetear l 5-10 de la muestra desde el fondo del vial, y emparedado entre dos cubreobjetos. Esta muestra, que contiene un gran número de gusanos temporalmente inmovilizada (por ejemplo, 50-100 gusanos), se puede colocar en el detector para iniciar la adquisición de datos. </li></ol> 3. Adquisición de Datos Aquí, un resumen de los pasos de adquisición de imágenes para un experimento de lote típico, que fue automatizado mediante el uso de una costumbre desarrollada LabView interfaz. <ol><li> Ajustar el ángulo inicial de la fase de rotación a -50 °, donde 0 ° corresponde a la posición vertical de la fuente de luz. </li><li> Ajuste la posición inicial de la fase XY para (0,0), que es la posición inicial. </li><li> Ajustar el tiempo de exposición del detector de utilizar mejor su rango dinámico de tal manera que la imagen es tan brillante como sea posible sin ningún tipo de saturated píxeles. </li><li> Sin cambiar el ángulo de la fase de rotación, la captura de imágenes 9. Para cada imagen, mover la etapa XY para una nueva posición en una cuadrícula de 3x3 cuadrado de tal manera que cada imagen se desplaza por aproximadamente una cuarta parte de un pixel en comparación con la anterior. La adquisición más imágenes en cada ángulo, por ejemplo, en una rejilla 6μ6, podría mejorar la resolución en función del tipo del objeto y de señal a ruido (SNR). 10 </li><li> Ajuste la posición inicial de la fase XY para (0,0), que es la posición inicial. </li><li> Aumentar el ángulo de la fase de rotación en incrementos de 2 °, hasta llegar a 50 °. Después de cada incremento, es decir, en cada nuevo ángulo, repita los pasos 3-5. Los incrementos angulares pueden ser más fino o más gruesa en función de la optimización de tiempo de adquisición de imágenes de resolución vs. </li><li> (Paso opcional si un régimen de tomografía de doble eje se va a utilizar) Girar la etapa horizontal sobre el que está montado el detector en 90 °. Entonces,repetir los pasos 1-6. </li></ol> 4. Análisis de Datos Siguiendo los pasos 1-6 en la sección 3, un conjunto de 459 imágenes que se adquiere, que consta de 9 sub-pixel para imágenes desplazadas cada una de las 51 diferentes ángulos de iluminación. En primer lugar, cada grupo de 9 imágenes se procesan digitalmente, utilizando píxeles algoritmos de super resolución 10, para obtener una alta resolución (HR) holograma de proyección por ángulo. Cabe señalar que píxel de super-resolución se refiere a la superación de la limitación del tamaño de pixel, en lugar del límite de difracción, en la resolución espacial de imágenes lensfree. Pixel hologramas super resueltos luego se reconstruyó digitalmente 7-10 para obtener 51 imágenes de la proyección. Este conjunto de 51 imágenes de proyección se encuentra de nuevo-proyectaron a partir de TomoJ, un plug-in para ImageJ (una imagen de código abierto de software de análisis). 11 Esta operación de retroproyección genera una imagen en tres dimensiones (tomografías) de la muestra. Aunque no se han aplicado aquí,un régimen de tomografía de doble eje también podría ser utilizado como se describe en la referencia. 7. En este esquema, un segundo conjunto de tomogramas se obtiene haciendo girar la fuente de luz a lo largo de un eje ortogonal con respecto al eje de rotación primero, que proporciona información más espacial con respecto a la muestra, y mejora la resolución axial. 5. Los resultados representativos El gran campo de visión (FOV) de la tomografía lensfree óptico se demuestra en la Figura 1. A medida que la muestra se coloca directamente sobre la parte superior del detector de matriz, las imágenes holográficas de los objetos se pueden grabar sobre una mm FOV de 24 2, que puede incrementarse aún más utilizando emergentes conjuntos de detectores con grandes áreas activas. Aunque el tamaño de pixel del detector de matriz limita la resolución de las imágenes holográficas grabados, píxel de super-resolución técnicas mitigar este problema. Figura 2 muestra super-pixel-resueltos a lo largo de hologramascon las imágenes reconstruidas (es decir, imágenes de proyección) que ofrecen sub-micrómetro resolución espacial, para tres diferentes ángulos de iluminación. Las imágenes de la proyección se puede combinar con técnicas de reconstrucción tomográfica de imágenes (por ejemplo, de retroproyección filtrada) para calcular las tomografías (es decir, imágenes en tres dimensiones) de la muestra. La Figura 3 muestra tres imágenes de división en el plano xy a través de la parte anterior del gusano, donde el tubo faríngeo sólo es visible en el corte a través de z = 8 m, como se esperaba a partir de esta estructura más o menos cilíndrica con un diámetro de 5 micras ~ exterior. Además, la imagen de la sección transversal en el plano xz (inserto en la Figura 3 panel superior) muestra claramente los límites del gusano y el tubo interior faríngea (señalado por las flechas), demostrando exitosa de imágenes 3D de la faringe. <img src="/files/ftp_upload/4161/4161fig1.jpg" alt="Figura 1" /> <strong&gt; La figura 1 muestra un completo campo de visión (24 mm 2) imagen holográfica grabado con la tomografía óptica lensfree (LOT) de instalación para iluminación vertical. Debido a la gran área de imagen de lote, muchos gusanos pueden ser simultáneamente imágenes con un paso de adquisición de datos clave. <img src="/files/ftp_upload/4161/4161fig2.jpg" alt="Figura 2" /> La Figura 2 muestra el pixel super-resueltos hologramas (panel izquierdo) y las imágenes de proyección reconstruidas digitalmente (panel derecho) para un C. gusano elegans (recortada de un gran campo de visión) a los tres ángulos de iluminación diferentes. Cada imagen de proyección contiene información relativa a un ángulo de visión diferente, que permite el cálculo de la estructura en 3D a través de una de retroproyección filtrada operación. <img src="/files/ftp_upload/4161/4161fig3.jpg" alt="Figura 3" /> La Figura 3 muestra tomogramas calculadas para la C. elegans gusano of Figura 2. (Fila superior) muestra una imagen de corte del gusano entero en z = 3 m. El recuadro muestra una imagen de corte transversal de la parte anterior del gusano. Barra de escala de la inserción es de 50 micras. (Fila inferior) muestra tres imágenes de cortar a través de la parte anterior del gusano, lo que demuestra la capacidad óptica de seccionamiento de la tomografía óptica lensfree. Las flechas en toda la figura indican el mismo punto en el tubo faríngeo del gusano. Una imagen de microscopio (x40, 0.65-NA) también se proporciona para la comparación visual.

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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>

No hay conflictos de interés declarado.

Es importante destacar que la geometría única de lensfree en el chip microscopio holográfico es el elemento crítico para lograr super-pixel de resolución y las imágenes tomográficas. Puesto que las imágenes grabadas no se supone que son imágenes de proyección de imagen como en contacto se aproxima a 12 incoherente, pero hologramas de proyección, la difracción de la luz transmitida hasta que choca con el detector puede ser corregida digitalmente por reconstrucción holográfica. Por lo tan...

<table border="1"><tbody><tr><td> Nombre </td><td> Compay </td><td> Catálogo # </td><td> Comentario </td></tr><tr><td> Lineales etapas XY </td><td> Newport Corp. </td><td> AMF-PP </td><td> Etapa lineal miniatura </td></tr><tr><td> La rotación motorizada fase </td><td> Thorlabs </td><td> PRM1Z8 </td><td> Precisión de rotación motorizada de montaje </td></tr><tr><td> Fibra óptica multimodo </td><td> Thorlabs </td><td> AFS105/125Y </td><td> La fibra multimodo </td></tr><tr><td> Fuente de luz </td><td> Newport Corp. </td><td> 6255 </td><td> Libre de ozono de la lámpara de xenón </td></tr><tr><td> Monocromador </td><td> Newport Corp. </td><td> 74100 </td><td> Cornerstone 260 4.1 m Monocromador </td></tr><tr><td> Conjunto de sensores CMOS </td><td> Aptina Inc. </td><td> MT9P031STC </td><td> 5 megapíxeles del sensor CMOS </td></tr><tr><td> C. elegans muestra </td><td> Carolina Biosupply </td><td> 173500 </td><td> Wild-tipo C. elegans </td></tr><tr><td> El levamisol </td><td> Sigma Aldrich </td><td> L9756-5G </td><td> Tetramisol clorhidrato de </td></tr></tbody></table>

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

Imágenes tomográficas ha sido una herramienta ampliamente utilizada en la medicina, ya que puede dar en tres dimensiones (3D) la información estructural de las escalas de los objetos de diferentes tamaños. En las escalas micrométricas y milimétricas, las modalidades de microscopía óptica encontrar un uso cada vez mayor debido a la naturaleza no ionizante de la luz visible, y la disponibilidad de un amplio conjunto de fuentes de iluminación (por ejemplo, los láseres y diodos emisores de luz) y los elementos de detección (por ejemplo, de gran formato CCD y CMOS de las matrices de detectores). Entre los recientemente desarrollados modalidades de microscopía óptica tomográficos, se puede incluir tomografía de coherencia óptica, la tomografía por difracción óptica, la tomografía por proyección óptica y microscopía de luz hojas. 1-6 Estas plataformas proporcionan imágenes de la sección de células, microorganismos y animales modelo como C. elegans, el pez cebra y embriones de ratón. Existentes imágenes ópticas 3D en general, tienen arquitecturas relativamente voluminosos y complejos, lo que limita ªe la disponibilidad de estos equipos a los laboratorios avanzados, e impidiendo su integración con lab-on-a-chip plataformas y los chips de microfluidos. Para ofrecer una alternativa microscopio de tomografía, hemos desarrollado recientemente lensfree tomografía óptica (LOT) como una modalidad de la tomografía de alto rendimiento, compacto y rentable óptica. 7 descartes LOT el uso de lentes y componentes ópticos voluminosos, y en su lugar se basa en multi-ángulo la iluminación y la computación digital para alcanzar la profundidad de imagen con resolución de micro-objetos de más de un volumen de imágenes de gran tamaño. Mucho puede la muestra biológica de la imagen con una resolución espacial de &lt;1 m X &lt;1 m x menos de 3 m en la x, y y z dimensiones, respectivamente, sobre un volumen de imágenes de gran cantidad de 15-100 mm 3, y puede ser particularmente útil para lab-on-a-chip plataformas.

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 tomografía óptica es una técnica de microscopía tridimensional que ofrece una resolución espacial de &lt;1 m × m &lt;1 × menos de 3 micras x, y y z dimensiones, respectivamente, más una gran imagen-volumen de 15-100 mm<sup&gt; 3</sup&gt;, Que puede ser particularmente útil para la integración con laboratorio en un chip plataformas.

Lensfree en el chip tomográfica Microscopía empleo de multi-ángulo de iluminación y píxeles de super-resolución

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 vistas.  University of California, Los Angeles. Lensfree tomografía óptica es una técnica de microscopía tridimensional que ofrece una resolución espacial de 

Video: 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.

Biomoleculares de detección utilizando el sensor de reflectancia de imágenes interferométricas (IRIS)

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

Investigación

Bioingeniería

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.

El chip Isotachophoresis para separación de iones y purificación de ácidos nucleicos

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 fenotipificación inflamatoria endotelial

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.

Una guía para la iluminación estructurada de microscopía TIRF a alta velocidad con colores múltiples

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.

Seca de la película basada en la fotoresistencia el Biosensor electroquímico de microfluidos plataforma: Fabricación de dispositivo, preparación del análisis de la en-viruta y el funcionamiento del sistema

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.

Fabricación y validación de un sistema de órganos-en-viruta con electrodos integrados directamente cuantificar Transendothelial resistencia eléctrica

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.

Expresión independiente de metanol por Pichia Pastoris empleando tecnologías de la represión

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 Genoma Edición de Saccharomyces cerevisiae y la creación de Arte de píxeles de levadura

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;

Aplicaciones para paneles de iluminación compatibles con microplacas de código abierto

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.

Construyendo un sistema de iluminación simple y versátil para experimentos optogenéticos

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