1. Imaging Setup LOT possono essere assemblati in un compatto e leggero portatile campo dell&#39;architettura 8, e alternativamente come un microscopio optofluidic con la capacità di imaging in sezione. 9 In tale relazione, tuttavia, descriveremo la configurazione di base delle immagini per una panchina-top implementazione verso la tomografia di elettricità statica campioni. <ol><li> Modulo di illuminazione: In LOT, fonti di luce parzialmente coerente come emettitori di luce dei diodi (LED) può essere utilizzato. Per una maggiore flessibilità di sperimentazione, abbiamo utilizzato un monocromatore con una lampada allo xeno (Cornerstone T260, Newport Corp.). Il monocromatore è stata regolata per fornire un output con ~ larghezza spettrale 1-10 nm lunghezza d&#39;onda intorno a un centro di esempio 450-650 nm. Questa uscita parzialmente coerente viene poi accoppiato ad una fibra ottica multimodale (Thorlabs AFS105/125Y) per fornire luce parzialmente coerente al sistema. La fibra ottica è montato su uno stadio di rotazione motorizzata (ThorlabsPRM-1Z8 guidato da Thorlabs TDC001 controller) per cambiare l&#39;angolo di illuminazione. La fase motorizzato, con la sorgente luminosa collegata, è montato su una bidimensionale stadio lineare XY (Newport ILS50CC guidato da Newport MFA-controllori PPD), che è stato utilizzato per ottenere nel piano spostamenti della sorgente di luce ad un angolo determinato. </li><li> Detection: tomografia Lensfree ottica è una tecnologia scalabile tale che l&#39;array rivelatore può essere scelto secondo le esigenze delle applicazioni senza alterare significativamente la procedura di acquisizione di immagini o il trattamento dei dati. In questa relazione, abbiamo utilizzato un array di sensori CMOS con 5 megapixel, con una dimensione dei pixel di 2,2 micron (IDS Imaging, UI-1485LE-M). Il rilevatore viene utilizzato per registrare i ampio campo di vista (esempio 24 mm 2) immagini olografiche di campioni che sono stati posizionati direttamente sulla parte superiore della zona attiva del rivelatore. Se un doppio asse configurazione tomografia è attuato 7, il rivelatore uno dovrebbeLSO essere montato su una orizzontale (rispetto alla tabella ottico) stadio di rotazione (ad esempio Thorlabs RP-01) per ruotare il campione (insieme con il rivelatore) nel piano normale all&#39;asse ottico 7. </li></ol> 2. Preparazione del campione Mentre tomografia lensfree ottico può immagine una varietà di oggetti quali cellule e microrganismi, si illustreranno i principi fondamentali eseguendo tridimensionale microscopia di C. elegans campione. <ol><li> Usando un bisturi o una spatola, prendete un piccolo pezzo di agar dalla capsula di Petri contenente il C. elegans cultura. Un pezzo cubo di alcuni millimetri, lungo ogni dimensione conterrà centinaia di nematodi. </li><li> Posizionare il piccolo pezzo di agar in un flaconcino polipropilene contenente 1 ml deionizzata (DI). </li><li> Delicatamente su vortex per 0,5-1 min, e attendere per 10-15 minuti fino a quando i vermi strisciare fuori del pezzo agar in acqua deionizzata. </li><li> Allo scopo diimmobilizzare temporaneamente i vermi, aggiungere 1 ml di soluzione di 5-10 levamisole mM (Sigma-Aldrich) e attendere per 10 minuti. </li><li> Pipettare 5-10 microlitri di campione dal fondo della fiala, e sandwich tra due vetrini. In questo esempio, contenente un gran numero di vermi temporaneamente immobilizzati (ad esempio 50-100 vermi), possono essere immessi sul rivelatore per avviare acquisizione dati. </li></ol> 3. Data Acquisition Qui, riassumiamo le fasi di acquisizione delle immagini per un esperimento LOT tipico, che è stato automatizzato mediante un interfaccia personalizzata sviluppata LabView. <ol><li> Regolare l&#39;angolo iniziale della fase di rotazione a -50 °, dove 0 ° corrisponde alla posizione verticale della sorgente luminosa. </li><li> Regolare la posizione iniziale della fase XY a (0,0), che è la posizione iniziale. </li><li> Regolare il tempo di esposizione del rivelatore di utilizzare al meglio la gamma dinamica in modo tale che l&#39;immagine è luminosa come possibile senza saturated pixel. </li><li> Senza modificare l&#39;angolo di fase di rotazione, catturare immagini 9. Per ogni immagine, spostare lo stadio XY ad una nuova posizione in una griglia 3x3 quadrato tale che ciascuna immagine si sposta di circa un quarto di pixel rispetto al precedente. Acquisire più immagini ad ogni angolo, ad esempio in una griglia 6μ6, può migliorare la risoluzione a seconda del tipo di oggetto e di segnale-rumore (SNR) 10. </li><li> Regolare la posizione iniziale della fase XY a (0,0), che è la posizione iniziale. </li><li> Aumentare l&#39;angolo di fase di rotazione da incrementi di 2 °, fino a raggiungere 50 °. Dopo ogni incremento, cioè ad ogni nuovo angolo, ripetere i passaggi 3-5. Gli incrementi angolari possono essere più sottili o grossolane a seconda della ottimizzazione del tempo di acquisizione vs risoluzione delle immagini. </li><li> (Step opzionale se uno schema a doppio asse tomografia deve essere utilizzato) ruotando la fase orizzontale su cui è montato il rilevatore di 90 °. Quindi,ripetere i passaggi 1-6. </li></ol> 4. Analisi dei dati Seguendo i passaggi 1-6 nella sezione 3, una serie di immagini 459 è acquisita, che contiene 9 immagini trasferite sub-pixel per ciascuna delle 51 diversi angoli di illuminazione. In primo luogo, ogni serie di 9 immagini elaborate digitalmente, utilizzando pixel di risoluzione di algoritmi super-10, per ottenere uno ad alta risoluzione (HR) per l&#39;angolo di proiezione olografica. Va notato che pixel super-risoluzione si riferisce a superare la limitazione della dimensione dei pixel, piuttosto che il limite di diffrazione, la risoluzione spaziale di immagini lensfree. Pixel ologrammi super-risolti vengono poi ricostruiti digitalmente 7-10 di ottenere 51 immagini di proiezione. Questo insieme di 51 immagini di proiezione è quindi back-proiettato con TomoJ, un plug-in per ImageJ (open source software di analisi dell&#39;immagine) 11. Questo retroproiezione operazione genera un immagine tridimensionale (tomografie) del campione. Anche se non implementato qui,un doppio asse regime tomografia potrebbe essere utilizzato anche come descritto in rif. 7. In questo schema, un secondo insieme di tomogrammi è ottenuta ruotando la sorgente di luce lungo un asse ortogonale rispetto al primo asse di rotazione, che fornisce informazioni relative più spaziale del campione, e migliora la risoluzione assiale. 5. Risultati rappresentativi L&#39;ampio campo di vista (FOV) di tomografia lensfree ottico è mostrato nella Figura 1. Poiché il campione viene posto direttamente sulla sommità del rilevatore-matrice, immagini olografiche degli oggetti possono essere registrati su un FOV di 24 mm 2, che può essere ulteriormente aumentata mediante rilevatori a schiera emergenti con grandi aree attive. Sebbene dimensioni dei pixel del rivelatore-matrice limita la risoluzione delle immagini registrate olografiche, pixel super-risoluzione tecniche attenuare questo problema. Figura 2 mostra pixel super-risolti ologrammi lungocon le immagini ricostruite (cioè immagini di proiezione) che offrono sub-micrometrica risoluzione spaziale, per tre diversi angoli di illuminazione. Le immagini proiezione possono essere combinate con le tecniche di ricostruzione delle immagini tomografiche (per esempio filtrato retro-proiezione) per calcolare tomogrammi (ossia immagini tridimensionali) del campione. Figura 3 mostra tre immagini di fetta nel piano xy attraverso la parte anteriore della vite senza fine, in cui la tubo faringeo è visibile solo nella fetta attraverso z = 8 pm, come previsto da questa struttura approssimativamente cilindrica con diametro di 5 micron ~ esterno. Inoltre, la sezione trasversale immagine nel piano xz (inserto nella Figura 3 pannello superiore) mostra chiaramente i confini della vite e il tubo interno faringeo (indicato dalle frecce), dimostrando successo immagini 3D della faringe. <img src="/files/ftp_upload/4161/4161fig1.jpg" alt="Figura 1" /> <strong&gt; La figura 1 mostra un completo campo di vista (24 mm 2) un&#39;immagine olografica registrata usando la tomografia ottica lensfree (LOT) installazione per l&#39;illuminazione verticale. A causa della vasta area di esposizione della LOT, molti worm possono essere contemporaneamente ripreso con un singolo passo di acquisizione dati. <img src="/files/ftp_upload/4161/4161fig2.jpg" alt="Figura 2" /> La figura 2 mostra il super-pixel risolti ologrammi (pannello di sinistra) e le immagini di proiezione digitale ricostruito (pannello destro) per un C. elegans worm (ritagliata da un grande campo di vista) a tre diversi angoli di illuminazione. Ogni immagine di proiezione contiene informazioni riguardanti un diverso angolo visuale, che permette il calcolo della struttura 3D attraverso una retro-proiezione filtrata funzionamento. <img src="/files/ftp_upload/4161/4161fig3.jpg" alt="Figura 3" /> La Figura 3 mostra tomogrammi calcolati per la C. elegans verme of Figura 2. (Riga in alto) mostra una immagine fetta del worm intero z = 3 micron. L&#39;inserto mostra una sezione immagine dalla parte anteriore della vite senza fine. Barra di scala del riquadro è di 50 micron. (Riga in basso) mostra tre immagini di fetta attraverso la parte anteriore del verme, dimostrando la capacità di sezionamento ottico lensfree tomografia ottica. Frecce tutta la figura indicano lo stesso punto sul tubo faringeo del worm. Un&#39;immagine microscopio (x40, 0,65-NA) è anche fornita per confronto visivo.

<ol>
	<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>

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È importante sottolineare che la geometria unica di lensfree on-chip microscopia olografico l&#39;attivatore è critica per ottenere pixel super-risoluzione e di imaging tomografico. Poiché le immagini registrate non vengono considerati come immagini di proiezione di immagini in contatto incoerente avvicina 12, ma ologrammi di proiezione, la diffrazione della luce trasmessa fino colpisca il rivelatore può essere corretto digitalmente da ricostruzione olografica. Pertanto, le variazioni nel campion...

<table border="1"><tbody><tr><td> Nome </td><td> Compay </td><td> Catalogo # </td><td> Commento </td></tr><tr><td> Lineari XY fasi </td><td> Newport Corp. </td><td> MAE-PP </td><td> Miniature Linear Stage </td></tr><tr><td> Rotazione stage motorizzato </td><td> Thorlabs </td><td> PRM1Z8 </td><td> Motorizzato precisione di rotazione Monte </td></tr><tr><td> Fibra ottica multimodale </td><td> Thorlabs </td><td> AFS105/125Y </td><td> Multimode Fiber </td></tr><tr><td> Sorgente luminosa </td><td> Newport Corp. </td><td> 6255 </td><td> Ozone-free lampada Xenon </td></tr><tr><td> Monocromatore </td><td> Newport Corp. </td><td> 74100 </td><td> Cornerstone 260 1/4 m Monochromator </td></tr><tr><td> Sensore CMOS serie </td><td> Aptina Inc. </td><td> MT9P031STC </td><td> 5 Megapixel del sensore CMOS </td></tr><tr><td> C. elegans campione </td><td> Carolina Biosupply </td><td> 173500 </td><td> Wild-tipo C. elegans </td></tr><tr><td> Levamisole </td><td> Sigma Aldrich </td><td> L9756-5G </td><td> Tetramisole cloridrato </td></tr></tbody></table>

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

Di imaging tomografico è stato uno strumento ampiamente utilizzato in medicina come può fornire tridimensionale (3D) informazioni strutturali per quanto riguarda oggetti di scale di misura diverse. In scale micrometri e millimetri, modalità microscopia ottica trovare uso sempre a causa della non ionizzanti natura della luce visibile, e la disponibilità di una vasta gamma di sorgenti di illuminazione (ad esempio laser e che emettono luce di diodi) e elementi di rilevazione, ad esempio grande formato CCD e CMOS-array detector). Tra i recentemente sviluppate modalità di microscopia ottica tomografiche, si può comprendere la tomografia a coerenza ottica, la tomografia a diffrazione ottica, tomografia a proiezione ottica e luce fogli microscopio. 1-6 Queste piattaforme forniscono immagini in sezione di cellule, microrganismi e animali modello come C. elegans, zebrafish ed embrioni di topo. Esistenti imager 3D ottici hanno generalmente architetture relativamente ingombranti e complessi, limitando °e la disponibilità di questi strumenti e laboratori avanzati, e impedendo la loro integrazione con lab-on-a-chip piattaforme e chip microfluidica. Per fornire un microscopio alternativa tomografica, abbiamo recentemente sviluppato lensfree tomografia ottica (LOT) come una modalità high-throughput, compatto e conveniente tomografia ottica. 7 LOT rigetti l&#39;uso di lenti e ingombranti componenti ottici, e si basa invece sul multi-angolo illuminazione e calcolo digitale per raggiungere profondità risolto imaging di micro-oggetti su un volume di immagini di grandi dimensioni. LOT può immagine campione biologico ad una risoluzione spaziale di &lt;1 um x &lt;1 um x &lt;3 um x, y e Z, rispettivamente, su un grande volume di imaging di 15-100 mm 3, e possono essere particolarmente utili per lab-on-a-chip piattaforme.

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 ottico tomografia è una tecnica tridimensionale microscopia che offre una risoluzione spaziale di &lt;1 um x &lt;1 um x &lt;3 pm di x, y e Z, rispettivamente, per un grande volume di imaging di 15-100 mm<sup&gt; 3</sup&gt;, Che può essere particolarmente utile per l&#39;integrazione con lab-on-a-chip piattaforme.

Lensfree On-chip Tomographic Microscopia Utilizzando Multi-angolo di illuminazione e Pixel Super-risoluzione

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 Views.  University of California, Los Angeles. Lensfree ottico tomografia è una tecnica tridimensionale microscopia che offre una risoluzione spaziale di 

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.

Biomolecolare Detection impiega il sensore di immagine interferometrica riflettanza (IRIS)

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

Ricerca

Bioingegneria

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 per separazione degli ioni e purificazione di acidi nucleici

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 Fenotipizzazione Endothelial infiammatoria

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 guida per Strutturato illuminazione TIRF Microscopia a alta velocità con colori multipli

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.

Film secco basati su Photoresist biosensore elettrochimico Microfluidic Platform: Fabbricazione di dispositivi, saggio su chip preparazione e funzionamento 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.

Fabbricazione e validazione di un sistema di organo-on-chip con elettrodi integrati direttamente quantificare transendoteliale resistenza elettrica

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.

Metanolo espressione indipendente di Pichia Pastoris che impiegano tecnologie di-repressione

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

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

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

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

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

CRISPR/Cas12a Multiplex Genome Editing di Saccharomyces cerevisiae e la creazione di Yeast Pixel Art

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;

Applicazioni per pannelli di illuminazione compatibili con microplacca open source

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

Building a Simple and Versatile Illumination System for Optogenetic Experiments

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

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

Costruire un sistema di illuminazione semplice e versatile per esperimenti optogenetici

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