RESUMEN
The spread of practical terahertz (THz) systems dedicated to the telecommunication, pharmacy, civil security, or medical markets requires the use of mainstream semiconductor technologies, such as complementary metal-oxide-semiconductor (CMOS) lines. In this paper, we discuss the operation of a CMOS-based free space all-electronic system operating near 250 GHz, exhibiting signal-to-noise ratio (SNR) with 62 dB in the direct detection regime for one Hz equivalent noise bandwidth. It combines the state-of-the-art detector based on CMOS field-effect-transistors (FET) and a harmonic voltage-controlled oscillator (VCO). Three generations of the oscillator circuit are presented, and the performance characterization techniques and their improvement are explained in detail. The manuscript presents different emitter-detector pair operation modalities, including spectroscopy and imaging.
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Semiconductores , Silicio , Electrónica , Óxidos , Relación Señal-RuidoRESUMEN
This work presents, to our knowledge, the first completely passive imaging with human-body-emitted radiation in the lower THz frequency range using a broadband uncooled detector. The sensor consists of a Si CMOS field-effect transistor with an integrated log-spiral THz antenna. This THz sensor was measured to exhibit a rather flat responsivity over the 0.1-1.5-THz frequency range, with values of the optical responsivity and noise-equivalent power of around 40 mA/W and 42 pW/ Hz , respectively. These values are in good agreement with simulations which suggest an even broader flat responsivity range exceeding 2.0 THz. The successful imaging demonstrates the impressive thermal sensitivity which can be achieved with such a sensor. Recording of a 2.3 × 7.5-cm 2 -sized image of the fingers of a hand with a pixel size of 1 mm 2 at a scanning speed of 1 mm/s leads to a signal-to-noise ratio of 2 and a noise-equivalent temperature difference of 4.4 K. This approach shows a new sensing approach with field-effect transistors as THz detectors which are usually used for active THz detection.
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Diagnóstico por Imagen , Cuerpo Humano , Radiación Terahertz , Diseño de Equipo , Humanos , Relación Señal-RuidoRESUMEN
Due to high associated costs and considerable time investments of cell-based screening, there is a strong demand for new technologies that enable preclinical development and tests of diverse biologicals in a cost-saving and time-efficient manner. For those reasons we developed the high-density cell array (HD-CA) platform, which miniaturizes cell-based screening in the form of preprinted and ready-to-run screening arrays. With the HD-CA technology, up to 24,576 samples can be tested in a single experiment, thereby saving costs and time for microscopy-based screening by 75%. Experiments on the scale of the entire human genome can be addressed in a real parallel manner, with screening campaigns becoming more comfortable and devoid of robotics infrastructure on the user side. The high degree of miniaturization enables working with expensive reagents and rare and difficult-to-obtain cell lines. We have also optimized an automated imaging procedure for HD-CA and demonstrate the applicability of HD-CA to CRISPR-Cas9- and RNAi-mediated phenotypic assessment of the gene function.
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Técnicas Citológicas/métodos , Genoma Humano , Sistemas CRISPR-Cas , Línea Celular , Endocitosis , Factor de Crecimiento Epidérmico/metabolismo , Humanos , Miniaturización , Fenotipo , Interferencia de ARN , RobóticaRESUMEN
α2ß1 integrin is one of the most important collagen-binding receptors, and it has been implicated in numerous thrombotic and immune diseases. α2ß1 integrin is a potent tumour suppressor, and its downregulation is associated with increased metastasis and poor prognosis in breast cancer. Currently, very little is known about the mechanism that regulates the cell-surface expression and trafficking of α2ß1 integrin. Here, using a quantitative fluorescence-microscopy-based RNAi assay, we investigated the impact of 386 cytoskeleton-associated or -regulatory genes on α2 integrin endocytosis and found that 122 of these affected the intracellular accumulation of α2 integrin. Of these, 83 were found to be putative regulators of α2 integrin trafficking and/or expression, with no observed effect on the internalization of epidermal growth factor (EGF) or transferrin. Further interrogation and validation of the siRNA screen revealed a role for KIF15, a microtubule-based molecular motor, as a significant inhibitor of the endocytic trafficking of α2 integrin. Our data suggest a novel role for KIF15 in mediating plasma membrane localization of the alternative clathrin adaptor Dab2, thus impinging on pathways that regulate α2 integrin internalization.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de la Mama/genética , Membrana Celular/metabolismo , Integrina alfa2beta1/metabolismo , Cinesinas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Reguladoras de la Apoptosis , Colágeno/metabolismo , Citoesqueleto/genética , Endocitosis/genética , Femenino , Pruebas Genéticas/métodos , Células HeLa , Humanos , Integrina alfa2beta1/genética , Cinesinas/genética , Microscopía Fluorescente , Metástasis de la Neoplasia , Unión Proteica/genética , Transporte de Proteínas/genética , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
High-throughput microscopy is an effective tool for rapidly collecting data on a large scale. However, high throughput comes at the cost of low spatial resolution. Here we introduce correlative light microscopy by combining fast automated widefield imaging, confocal microscopy and super-resolution microscopy. We demonstrate the potential of this approach for scalable experiments. The workflow consists of a robust approach for selecting cells of interest on a wide-field screening microscope at low resolution and subsequently re-localizing those cells with micrometer precision for confocal and super-resolution imaging. As a case study, we visualized and quantified cis- and trans-Golgi markers at increasing resolution.
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Aparato de Golgi/ultraestructura , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Animales , Línea Celular , Procesamiento de Imagen Asistido por Computador/métodos , Riñón/citología , RatasRESUMEN
miRNA cluster miR-17-92 is known as oncomir-1 due to its potent oncogenic function. miR-17-92 is a polycistronic cluster that encodes 6 miRNAs, and can both facilitate and inhibit cell proliferation. Known targets of miRNAs encoded by this cluster are largely regulators of cell cycle progression and apoptosis. Here, we show that miRNAs encoded by this cluster and sharing the seed sequence of miR-17 exert their influence on one of the most essential cellular processes - endocytic trafficking. By mRNA expression analysis we identified that regulation of endocytic trafficking by miR-17 can potentially be achieved by targeting of a number of trafficking regulators. We have thoroughly validated TBC1D2/Armus, a GAP of Rab7 GTPase, as a novel target of miR-17. Our study reveals regulation of endocytic trafficking as a novel function of miR-17, which might act cooperatively with other functions of miR-17 and related miRNAs in health and disease.
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Endocitosis/genética , Proteínas Activadoras de GTPasa/metabolismo , MicroARNs/metabolismo , Secuencia de Bases , Proliferación Celular , Regulación hacia Abajo/genética , Factor de Crecimiento Epidérmico/metabolismo , Proteínas Activadoras de GTPasa/química , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , MicroARNs/genética , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Transporte de Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de LDL/metabolismo , Reproducibilidad de los ResultadosRESUMEN
We applied fluorescence microscopy-based quantitative assays to living cells to identify regulators of endoplasmic reticulum (ER)-to-Golgi trafficking and/or Golgi complex maintenance. We first validated an automated procedure to identify factors which influence Golgi-to-ER relocalization of GalT-CFP (ß1,4-galactosyltransferase I-cyan fluorescent protein) after brefeldin A (BFA) addition and/or wash-out. We then tested 14 proteins that localize to the ER and/or Golgi complex when overexpressed for a role in ER-to-Golgi trafficking. Nine of them interfered with the rate of BFA-induced redistribution of GalT-CFP from the Golgi complex to the ER, six of them interfered with GalT-CFP redistribution from the ER to a juxtanuclear region (i.e. the Golgi complex) after BFA wash-out and six of them were positive effectors in both assays. Notably, our live-cell approach captures regulator function in ER-to-Golgi trafficking, which was missed in previous fixed cell assays, as well as assigns putative roles for other less characterized proteins. Moreover, we show that our assays can be extended to RNAi and chemical screens.
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Bioensayo/métodos , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Animales , Células Cultivadas , Riñón/citología , Microscopía Fluorescente , Transporte de Proteínas , RatasRESUMEN
The Golgi complex is the central organelle in the secretory membrane trafficking and its organization strongly depends upon the flow of coming and leaving material. The principles of cargo transfer to, through, and away from the Golgi complex were investigated in numerous studies. However, the knowledge of how the Golgi complex responses to changes in diverse trafficking events (e.g., ER exit block) on a molecular level is far from being complete. In order to identify regulatory molecules playing a role in the dynamic organization of the Golgi complex, we established a fluorescent microscopy-based quantitative assay to measure rates of the Golgi redistribution and assembly after addition and washout of BFA, respectively. At first, we tested our system under the condition of over-expression of GFP-tagged proteins. We measured their influence upon BFA-induced effects in a format, suitable for large-scale studies in living cell, namely cell arrays. The approach can be applied for large-scale RNA interference studies as well as for chemical screening.