1. Configuração de imagens LOT pode ser montado em um compacto e leve arquitetura campo-portátil 8, e, alternativamente, como um microscópio optofluidic com a capacidade de imagem seccional. 9 Neste relatório, no entanto, vamos descrever a configuração de imagem de base para uma implementação de bancada para tomografia de estática As amostras. <ol><li> Módulo de iluminação: no Lote, fontes de luz parcialmente coerentes, tais como emissores de luz de diodos (LEDs) podem ser utilizados. Para uma flexibilidade experimental, foi utilizado um monocromador com uma lâmpada de Xenon (Cornerstone T260, Newport Corp.) O monocromador foi ajustado para fornecer uma saída com ~ largura 1-10 nm de comprimento de onda espectral em torno de um centro de, por exemplo 450-650 nm. Esta saída parcialmente coerente é, então, acoplado a uma fibra multimodo óptica (Thorlabs AFS105/125Y) para fornecer luz parcialmente coerente para o sistema. A fibra óptica é montada sobre uma fase de rotação motorizada (ThorlabsPRM-1Z8 impulsionado por Thorlabs TDC001 controlador) para alterar o ângulo de iluminação. A fase motorizada, com a fonte de luz ligado, é montado sobre um bidimensional fase XY linear (Newport ILS50CC impulsionado por Newport AMF-PPD controladores), que foi utilizado para alcançar no plano-turnos da fonte de luz num determinado ângulo. </li><li> Detecção: Lensfree tomografia óptica é uma tecnologia escalável de tal forma que a matriz do detector pode ser escolhido de acordo com os requisitos das aplicações sem alterar significativamente as etapas de aquisição de imagens ou o processamento de dados. Neste relatório, nós empregamos um sensor CMOS com 5 Mega Pixels, com um tamanho de pixel de 2,2 mM (IDS Imaging, UI-1485LE-M). O detector é utilizado para registar os amplo campo de visão (por exemplo 24 mm 2) imagens holográficas de amostras, que foram colocadas directamente no topo da área activa do detector. Se uma configuração tomografia de duplo eixo é para ser implementada 7, o detector deve umlso ser montado sobre uma fase de rotação horizontal (em relação à mesa óptica) (por exemplo Thorlabs RP-01) para rodar a amostra (em conjunto com o detector) no plano perpendicular ao eixo óptico. 7 </li></ol> 2. Preparação da Amostra Enquanto lensfree tomografia óptica pode imaginar uma variedade de objetos, tais como células e microorganismos, que irão ilustrar os princípios básicos através da realização de microscopia tridimensional de um C. elegans amostra. <ol><li> Usando um bisturi ou uma espátula, tomar um pequeno pedaço de agar a partir da placa de Petri contendo o C. elegans cultura. Um pedaço cúbico de vários milímetros ao longo de cada dimensão irá conter centenas de nemátodos. </li><li> Coloque o pequeno pedaço de agar em um frasco de polipropileno contendo 1 ml de água desionizada (DI). </li><li> Gentilmente vórtice para 0,5-1 min, e esperar por 10-15 minutos até que os vermes rastejar para fora da peça agar na água DI. </li><li> A fim deimobilizar temporariamente os vermes, adicionar 1 ml de solução de 5-10 mM levamisole (Sigma-Aldrich) e aguardar 10 min. </li><li> Pipetar 5-10 da amostra a partir do fundo do frasco, e sanduíche entre duas folhas de cobertura. Esta amostra, contendo um grande número de vermes temporariamente imobilizada (por exemplo, 50-100 vermes), pode ser colocado sobre o detector para iniciar a aquisição de dados. </li></ol> 3. Aquisição de Dados Aqui, vamos resumir as etapas de aquisição de imagens para um experimento típico LOT, que foi automatizado usando um costume desenvolvido LabView interface. <ol><li> Ajustar o ângulo inicial da fase de rotação a -50 °, em que 0 ° corresponde à posição vertical da fonte de luz. </li><li> Ajustar a posição inicial da fase de XY para (0,0), que é a posição inicial. </li><li> Ajustar o tempo de exposição do detector para utilizar melhor a sua gama dinâmica de tal modo que a imagem é tão brilhante quanto possível, sem qualquer saturated pixels. </li><li> Sem alterar o ângulo de fase de rotação, a captura de 9 imagens. Para cada imagem, mover a fase XY para uma nova posição em uma grade quadrada de 3x3 de tal modo que cada imagem desloca em cerca de um quarto de um pixel em relação à anterior. A aquisição de imagens em cada ângulo, por exemplo, em uma grade 6μ6, pode melhorar a resolução, dependendo do tipo de objecto e de sinal-para-ruído (SNR). 10 </li><li> Ajustar a posição inicial da fase de XY para (0,0), que é a posição inicial. </li><li> Aumentar o ângulo de fase de rotação por incrementos de 2 °, até atingir 50 °. Após cada incremento, ou seja, em cada ângulo novo, repita os passos 3-5. Os incrementos angulares pode ser mais fina ou mais grossa, dependendo da otimização do tempo de aquisição versus resolução de imagem. </li><li> (Passo opcional se um esquema de tomografia de duplo eixo é para ser utilizado) Rodar a fase horizontal no qual o detector está montado de 90 °. Em seguida,repita os passos 1-6. </li></ol> 4. Análise de Dados Seguindo os passos 1-6 na Seção 3, um conjunto de 459 imagens é adquirido, que contém 9 sub-pixel imagens deslocadas para cada um dos 51 diferentes ângulos de iluminação. Primeiro, cada conjunto de 9 imagens é processados ​​digitalmente, utilizando de pixel super-resolução algoritmos 10, para se obter uma alta resolução (HR) holograma de projecção por ângulo. Deve notar-se que o pixel de super-resolução refere-se a ultrapassar a limitação do tamanho do pixel, em vez do que o limite de difracção, sobre a resolução espacial das imagens lensfree. Pixel hologramas super-resolvidas são, então, reconstruiu digitalmente 7-10 para obter 51 imagens de projeção. Este conjunto de 51 imagens de projeção é, então, volta-projetada usando TomoJ, um plug-in para ImageJ (um open source software de análise de imagem) 11. Esta operação volta projeção gera uma imagem tridimensional (tomografias) da amostra. Apesar de não ser implementado aqui,um esquema de tomografia de duplo eixo poderia também ser utilizada como descrito na ref. 7. Neste esquema, um segundo conjunto de tomogramas é obtido pela rotação da fonte de luz ao longo de um eixo ortogonal em relação ao eixo de rotação em primeiro lugar, que fornece informação mais espacial em relação à amostra, e melhora a resolução axial. 5. Os resultados representativos O grande campo de visão (FOV) de lensfree tomografia óptica é demonstrado na Figura 1. À medida que a amostra é colocada directamente sobre o topo do detector de matriz, as imagens holográficas de os objectos podem ser gravados ao longo de um milímetro FOV de 24 2, que pode ainda ser aumentada utilizando matrizes de detectores emergentes com maiores áreas activas. Embora o tamanho do pixel do detector de arranjo de-limita a resolução das imagens gravadas holográficos, de pixel de super-resolução técnicas mitigar este problema. A Figura 2 mostra de pixel super-resolvidos hologramas ao longocom as imagens reconstruídas (ou seja, projeção de imagens) que oferecem sub-micrômetro resolução espacial, por três diferentes ângulos de iluminação. As imagens de projecção podem ser combinados usando técnicas de reconstrução tomográficos de imagem (por exemplo, filtrada volta projecção-) para calcular tomogramas (isto é, três imagens tridimensionais) do espécime. Figura 3 mostra três imagens de corte no plano xy através da anterior do sem-fim, onde o tubo faríngeo é visível apenas na fatia através z = 8 mM, como esperado a partir desta estrutura cilíndrica com cerca de ~ 5 mM diâmetro exterior. Além disso, a imagem em corte transversal no plano xz (inserção na Figura 3 painel superior) mostra claramente as fronteiras do verme eo tubo interior da faringe (apontado pelas setas), demonstrando imagem 3D bem sucedida da faringe. <img src="/files/ftp_upload/4161/4161fig1.jpg" alt="A Figura 1" /> <strong&gt; Figura 1 mostra uma imagem do campo de visão (24 mm 2) completo holográfica gravados utilizando a tomografia óptica lensfree (LOT) para instalação de iluminação vertical. Devido à grande área de imagem de LOTE, vermes pode ser simultaneamente fotografada com uma única etapa de aquisição de dados. <img src="/files/ftp_upload/4161/4161fig2.jpg" alt="A Figura 2" /> A Figura 2 mostra o pixel super-resolvidos hologramas (painel esquerdo) e as imagens reconstruídas digitalmente projecção (painel da direita) para um C. elegans worm (cortada de um grande campo de visão) em três ângulos diferentes de iluminação. Cada imagem de projecção contém informação sobre um ângulo de visão diferente, que permite o cálculo da estrutura 3D através de uma operação de volta filtrada projecção. <img src="/files/ftp_upload/4161/4161fig3.jpg" alt="A Figura 3" /> A Figura 3 mostra tomogramas calculados para a C. elegans verme of Figura 2. (Linha superior) mostra uma imagem de corte do verme inteiro em z = 3 mm. A inserção mostra uma imagem em corte transversal a partir do anterior do sem-fim. Barra de escala da inserção é de 50 mM. (Linha inferior) mostra três imagens de corte através do anterior do verme, demonstrando a capacidade de seccionamento óptico da tomografia óptica lensfree. As setas ao longo da figura indicam o mesmo ponto no tubo da faringe do sem-fim. Uma imagem de microscópio (x40, 0,65-NA) é também proporcionada para comparação visual.

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É importante ressaltar que a geometria original de lensfree microscopia on-chip holográfico é o capacitador fundamental para alcançar pixels super-resolução e imagem tomográfica. Desde que as imagens gravadas não são consideradas como imagens de projeção de imagens em contato incoerente se aproxima de 12, mas hologramas de projeção, a difração da luz transmitida até que incide sobre o detector pode ser digitalmente corrigidas pela reconstrução holográfica. Portanto, as variações ...

<table border="1"><tbody><tr><td> Nome </td><td> Compay </td><td> Catalogo </td><td> Comentar </td></tr><tr><td> Lineares estágios XY </td><td> Newport Corp </td><td> MFA-PP </td><td> Stage Linear em miniatura </td></tr><tr><td> Fase de rotação motorizada </td><td> Thorlabs </td><td> PRM1Z8 </td><td> Precision Motorizado Rotação Monte </td></tr><tr><td> Fibra óptica multimodo </td><td> Thorlabs </td><td> AFS105/125Y </td><td> Fibra multimodo </td></tr><tr><td> Fonte de luz </td><td> Newport Corp </td><td> 6255 </td><td> Ozônio-livre da lâmpada Xenon </td></tr><tr><td> Monocromador </td><td> Newport Corp </td><td> 74100 </td><td> Cornerstone 260 1/4 m Monocromador </td></tr><tr><td> Matriz de sensor CMOS </td><td> Aptina Inc. </td><td> MT9P031STC </td><td> 5 Megapixels Sensor CMOS </td></tr><tr><td> C. elegans amostra </td><td> Carolina Biosupply </td><td> 173500 </td><td> De tipo selvagem C. elegans </td></tr><tr><td> Levamisole </td><td> Sigma Aldrich </td><td> L9756-5G </td><td> Cloridrato tetramisol </td></tr></tbody></table>

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

Tomográfico de imagem tem sido uma ferramenta amplamente utilizado em medicina como ele pode fornecer informação tridimensional (3D) estrutural sobre objectos de escalas de tamanho diferentes. Em escalas micrómetro e milímetros, as modalidades de microscopia óptica encontrar utilização crescente, devido à natureza não-ionizante de luz visível, e da disponibilidade de um amplo conjunto de fontes de iluminação (tais como lasers e emissores de luz-diodos) e elementos de detecção (tais como grande formato CCD e CMOS detector de matrizes). Entre os recentemente desenvolvidos modalidades de microscopia óptica tomográficos, pode-se incluir tomografia de coerência óptica, a tomografia de difração óptica, tomografia projeção óptica e luz folhas de microscopia. 1-6 Estas plataformas fornecer imagens seccional de células, microorganismos e animais modelo, como C. elegans zebrafish, e embriões de camundongos. 3D existente imagers ópticos geralmente têm arquiteturas relativamente volumosos e complexos, limitando ªe disponibilidade destes equipamentos para laboratórios avançados, e impedindo a sua integração com lab-on-a-chip plataformas e chips microfluídicos. Para fornecer um microscópio tomográfica alternativa, que recentemente desenvolveu a tomografia óptica lensfree (LOT) como uma modalidade de tomografia de alto rendimento, compactos e de baixo custo óptica. 7 LOT descarta o uso de lentes e componentes ópticos volumosos e, em vez se baseia em multi-ângulo iluminação e computação digital para alcançar profundidade resolvido imagens de micro-objetos sobre um volume grande de imagens. Muita coisa pode espécime biológico imagem com uma resolução espacial de 1 m &lt;x &lt;1 m x &lt;3 mm no X, Y e Z, respectivamente dimensões, mais de um volume grande de imagens de 15-100 mm 3, e pode 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 óptico tomografia é uma técnica de microscopia tridimensional que oferece uma resolução espacial de menos de 1 uM × menos de 1 uM × menos de 3 M em X, Y e dimensões Z, respectivamente, ao longo de um grande volume de imagiologia de 15-100 milímetros<sup&gt; 3</sup&gt;, Que pode ser particularmente útil para a integração com lab-on-a-chip plataformas.

Lensfree On-chip microscopia tomográfica Empregando Multi-ângulo de iluminação e Pixel Super-resolução

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 visualizações.  University of California, Los Angeles. Lensfree óptico tomografia é uma técnica de microscopia tridimensional que oferece uma resolução espacial de menos de 1 uM × menos de 1 uM × menos de 3 M em X, Y e dimensões Z, respectivamente, ao longo de um grande volume de imagiologia de 15-100 milímetros 3, Que pode ser particularmente útil para a integração com lab-on-a-chip plataformas.

Vídeo: 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.

Detecção de Biomolecular empregando o sensor de imagem Interferometric Reflectância (IRIS)

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

Bioengenharia

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 Isotachophoresis para separação dos íons e purificação de ácidos nucléicos

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 fenotipagem endotelial Inflamatória

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.

Um Guia para Estruturado iluminação TIRF microscopia de alta velocidade com várias cores

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.

Película seca baseada em fotorresiste eletroquímica Microfluidic Biosensor plataforma: Fabricação de dispositivo, ensaio em-microplaqueta preparação e operação de 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.

Fabricação e validação de um sistema de órgãos-em-microplaqueta com eléctrodos integrados para quantificar diretamente a resistência elétrica de Transendothelial

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.

Expressão independente de metanol por Pichia Pastoris , empregando tecnologias de repressão

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.

Edição do genoma de CRISPR/Cas12a multiplex de Saccharomyces cerevisiae e a criação da arte do pixel do fermento

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;

Aplicações para painéis de iluminação compatíveis com microplate de código aberto

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.

Construindo um sistema de iluminação simples e versátil para experimentos optogenéticos

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