Vorremmo ringraziare Gaby Martins e gli organizzatori del workshop EMBO2010 3D Developmental Imaging dove questo lavoro è iniziato e tutti i collaboratori ai progetti Fiji e ImageJ.

1. 4D Time-lapse esperimenti di imaging Le impostazioni utilizzate per l&#39;acquisizione di immagini variano a seconda del materiale utilizzato. La capacità di microscopia confocale a sezione otticamente un campione dipende da una serie di fattori: la lunghezza d&#39;onda di eccitazione, dimensioni pinhole, apertura numerica dell&#39;obiettivo, l&#39;indice di rifrazione del campione e del mezzo in cui è incorporato il campione. La dimensione del foro stenopeico confocale selezionato determinerà lo spessore della sezione ottica raccolto. Un foro più piccolo produrrà una sezione ottica sottile aumentando la risoluzione asse z ma riducendo la quantità di luce catturata. Un foro più grande aumenta lo spessore della sezione ottica, riducendo la risoluzione asse z ma aumentando la quantità di luce catturata. Altri fattori da considerare durante la raccolta dei dati 4D prima correzione includono: <ol><li> Ottimizzare la velocità di scansione per rimuovere, o limite, deriva durante la cattura di un singolo punto temporale. Pertanto il tempo impiegato per la raccolta di immagini dovrebbe essere una piccola frazione dell&#39;intervallo tra punti temporali. </li><li> Strutture perfettamente ripetitive, o griglie, non sono adatti come strutture per la registrazione in quanto non è possibile determinare la correzione richiesta. Perline casualmente distribuiti o strutture irregolari permetterà la registrazione univoca. </li><li> Se si prevede di deriva, aumentare l&#39;area di scansione e limiti fuoco superiore e inferiore per assicurare l&#39;area di interesse rimane all&#39;interno della serie di immagini. </li><li> Oltre a garantire l&#39;opportuno risoluzione spaziale per risolvere le strutture di interesse, impostare la frequenza di campionamento per fornire la risoluzione temporale per gli eventi dinamici in fase di studio. Nota: Il tempo tra immagine punti temporali dovrebbe quindi essere almeno la metà del tempo-intervallo tra eventi che si ripetono regolarmente e diminuire per eventi irregolari. </li></ol> 2. Apertura del confocale Dataset Il pacchetto open source Fiji è una distribuzione del programma ImageJ, che contiene i plug-in pre-installati per eseguire numerosi processi sui dati raccolti dagli esperimenti di microscopia. Il software fornisce facile plug-in aggiornamento architettura e include una copia del corretto 3D deriva plug-in utilizzato per questo protocollo. Il software supporta l&#39;importazione di una vasta gamma di formati proprietari di immagine al microscopio attraverso l&#39;utilizzo di Bio-formati di importazione plug-in di Open Microscopia Ambiente. <ol><li> Installare il programma Fiji (http://fiji.sc/Downloads). </li><li> Caricare il set di dati acquisiti tramite il plugin Importatore LOCI Bio-formati. </li><li> Se il dataset è maggiore rispetto alla memoria assegnata al programma, selezionare l&#39;opzione &quot;Utilizza l&#39;opzione stack virtuale&quot; all&#39;interno della sezione gestione della memoria. </li></ol> 3. Correzione Drift di un oggetto 3D in Post Processing Nel corso di un prolungato tempo-lapseesperimento di un campione può muoversi anche quando incorporato. Per correggere qualsiasi movimento e consentire gli eventi di migrazione impressi da analizzare, post-elaborazione delle immagini può essere effettuata. Tutta l&#39;elaborazione dell&#39;immagine postale deve essere chiaramente descritta nella metodologia di qualsiasi analisi derivata da questo lavoro. <ol><li> Una volta che il dataset è stato caricato, eseguire il corretto 3D plug deriva. </li><li> Se ci sono canali di immagini multiple, selezionare il canale da utilizzare per registrare le immagini. Questo dovrebbe rappresentare idealmente una struttura statica all&#39;interno del campione piuttosto che elementi migratori o mobili. Tuttavia, se ciò non è possibile il canale di minor movimento dovrebbe essere scelto. </li><li> Se la RAM disponibile sul sistema utilizzato per questa analisi è inferiore a due volte la dimensione di dati originale, selezionare l&#39;opzione stack virtuale uso. Questo memorizzare il HyperStack registrato come una sequenza di immagini, piuttosto che salvare il file RAM. <ol><li> Selezionare una cartella per il plug-in per singola uscita corretto imafile Ges. Verrà creato un file di immagine separato per ogni canale in ciascuna posizione z. </li></ol></li><li> Il plugin sarà quindi condurre un&#39;analisi di correlazione di fase pair-wise tra ogni time-point per determinare la correzione richiesta che viene poi applicata al dataset. </li></ol>

<ol>
	<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Abramoff, M. D., Magalhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11 (7), 42-42 (2004).</li><li>Collins, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+for+Microscopy.">ImageJ for Microscopy.</a> BioTechniques. 43 (S1), 25-30 (2007).</li><li>Linkert, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metadata+matters:+access+to+image+data+in+the+real+world.">Metadata matters: access to image data in the real world.</a> The Journal of Cell Biology. 189 (5), 777-782 (2010).</li><li>Th&#233;venaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE transactions on image processing : a publication of the IEEE Signal Processing Society. 7 (1), 27-41 (1998).</li><li>Kuglin, C. D., Hines, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phase+correlation+image+alignment+method.">The phase correlation image alignment method.</a> Proceedings of the IEEE, International Conference on Cybernetics and Society. , 163-165 (1975).</li><li>Preibisch, S., Saalfeld, S., Tomancak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Globally+optimal+stitching+of+tiled+3D+microscopic+image+acquisitions.">Globally optimal stitching of tiled 3D microscopic image acquisitions.</a> Bioinformatics. 25 (11), 1463-1465 (2009).</li><li>Kaufmann, A., Mickoleit, M., Weber, M., Huisken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+mounting+enables+long-term+imaging+of+zebrafish+development+in+a+light+sheet+microscope.">Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.</a> Development. 139 (17), 3242-3247 (2012).</li><li>Andersen, E., Asuri, N., Clay, M., Halloran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+cell+motility+and+actin+cytoskeleton+of+individual+neurons+and+neural+crest+cells+in+zebrafish+embryos.">Live imaging of cell motility and actin cytoskeleton of individual neurons and neural crest cells in zebrafish embryos.</a> J VIs. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+Cell+Fate+Determination+within+the+Adaxial+Cell+Equivalence+Group+of+the+Zebrafish+Myotome.">Morphogenesis and Cell Fate Determination within the Adaxial Cell Equivalence Group of the Zebrafish Myotome.</a> PLoS Genetics. 8 (10), (2012).</li><li>Moore, C. A., Parkin, C. A., Bidet, Y., Ingham, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+the+Myoblast+city+homologues+Dock1+and+Dock5+and+the+adaptor+proteins+Crk+and+Crk-like+in+zebrafish+myoblast+fusion.">A role for the Myoblast city homologues Dock1 and Dock5 and the adaptor proteins Crk and Crk-like in zebrafish myoblast fusion.</a> Development. 134 (17), 3145-3153 (2007).</li><li>Higashijima, S., Okamoto, H., Ueno, N., Hotta, Y., Eguchi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+generation+of+transgenic+zebrafish+which+reliably+express+GFP+in+whole+muscles+or+the+whole+body+by+using+promoters+of+zebrafish+origin.">High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin.</a> Developmental Biology. 192 (2), 289-299 (1997).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

La nostra capacità di utilizzare software di post-elaborazione per correggere la deriva campione di set di dati derivati ​​da estese time-lapse esperimenti di microscopia è limitato da una serie di fattori. La capacità di discernere deriva contro movimento migratorio di un campione dipende marcatori cellulari utilizzati. Marcatori cellulari che sono sia ampiamente espresse all&#39;interno di un campione o non sono coinvolti in eventi migratori durante l&#39;acquisizione dell&#39;immagine garantiscono la migliore ...

Confocale è ampiamente usato in biologia cellulare e dello sviluppo di seguire i movimenti delle cellule e dei cambiamenti nella morfologia. Acquisizione di una serie di sezioni ottiche a diversi piani focali consente la generazione di un tridimensionale (3D) modello di un campione, che può essere esteso in quattro dimensioni (4D) creando una serie tempo-intervallo di set di dati 3D. La generazione di set di dati 4D permette la misurazione dettagliata dei movimenti delle cellule e dei comportamenti. In esperimenti di time-lapse a lungo termine è comune osservare il movimento del campione. Ciò può essere causato da lievi imprecisioni in fase di controllo hardware e le posizioni focali. Mentre in altri casi, la deriva è il risultato di movimenti indotti dalla crescita campione o flessibilità all&#39;interno del mezzo di montaggio del campione. I metodi esistono per compensare o limitare questi movimenti compresi i miglioramenti ai sistemi di messa a fuoco hardware e una maggiore rigidità del mezzo di montaggio. Tuttavia, questi approcci non possono essere applicati in molti casi a causa del l&#39;imaging set fino tenuta a fornire le condizioni adatte per il mantenimento e la crescita dei campioni. Esistono soluzioni software open source per la correzione del movimento in 2D nel corso del tempo, attraverso l&#39;utilizzo delle StackReg e TurboReg (http://bigwww.epfl.ch/thevenaz/stackreg/) 5 plugins in ImageJ o Fiji, ma questi non possono essere applicate ai dataset 4D. Per correggere la deriva del campione abbiamo sviluppato un plug-in (Correct deriva 3D) per utilizzare la piattaforma di imaging di elaborazione open-source, Fiji 1. Il nostro plug-in è in grado di eseguire fase di registrazione correlazione per correggere movimento che si verifica a causa di deriva campione tridimensionali esperimenti temporizzati. Correlazione di fase 6 è un metodo efficiente di calcolo per determinare traduzione tra immagini. Il plug-in qui descritto utilizza la libreria di fase-correlazione sviluppato da Preibisch et al. 7. In esperimenti multi-canale, il plug-in utilizza un canale di determinazionene la correzione richiesta. Questa correzione viene quindi applicata a tutti i canali supplementari conseguente registrazione del set di dati 4D. Nel sistema modello zebrafish è possibile effettuare time-lapse imaging in un periodo di molte ore o anche diversi giorni 8. Un metodo comune per il montaggio del zebrafish è di incorporare l&#39;embrione vivo anestetizzato in bassa agarosio punto di fusione (0,8-1,5%), limitando il suo movimento 9-11. Mentre il movimento è limitato crescita del campione si verifica ancora, con conseguente cellule all&#39;interno del campo visivo posizione spostamento. Al fine di seguire il movimento delle cellule all&#39;interno dell&#39;embrione è necessario correggere prima per il movimento dell&#39;intero campione. Questo protocollo è stato sviluppato con i campioni di zebrafish, ed è stato utilizzato per lo sviluppo di immagini somite 12, ma può essere applicato a qualsiasi confocale dataset 4D.

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	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A5040</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Low gelling temperature agarose</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9414-25G</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Dumont #4 Forceps</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">0208-4-PO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable 3mL graduated</td>
			<td rowspan="2" style="width:71px;">Samco</td>
			<td rowspan="2" style="width:119px;">212</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Polyethylene transfer pipette</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">9cm bacterial grade Petri dishes</td>
			<td style="width:71px;">Greiner Bio One</td>
			<td style="width:119px;">632180</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fluorinated ethylene propylene (FEP) tubing</td>
			<td style="width:71px;">Bola</td>
			<td style="width:119px;">S1815-04</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Zeiss LSM-710 Confocal microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">W Plan-Apochromat 20x/1.0 DIC Objective</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">421452-9600-000</td>
		</tr>
	</tbody>
</table>

sample drift correction following 4d confocal time-lapse imaging

La generazione di quattro dimensioni (4D) dataset confocale; costituito da sequenze di immagini 3D nel tempo; fornisce un&#39;eccellente metodologia per catturare comportamenti cellulari coinvolti nei processi di sviluppo. La possibilità di monitorare e seguire i movimenti delle cellule è limitata dai movimenti di esempio che si verificano a causa di deriva del campione o, in alcuni casi, la crescita durante l&#39;acquisizione dell&#39;immagine. Inseguimento cellule in dataset colpite da drift e / o la crescita sarà integrare questi movimenti in una qualsiasi analisi della posizione della cella. Ciò può provocare il movimento apparente strutture statiche all&#39;interno del campione. Quindi prima di tracciamento delle cellule, la sua deriva campione deve essere corretto. Uso open source Fiji distribuzione 1 di ImageJ 2,3 e gli strumenti LOCI incorporati 4, abbiamo sviluppato il corretto 3D deriva plug-in per rimuovere movimento campione erronea in dataset confocale. Questo protocollo compensa in modo efficace per la traduzione campione o alterazioni po focalezione utilizzando fase correlazione registrare ogni time-point di una quadridimensionali dataset confocale, pur mantenendo la capacità di visualizzare e misurare i movimenti delle cellule nel corso prolungati esperimenti di time-lapse.

In zebrafish di sviluppo, le cellule muscolari veloci fondono in fibre multinucleate da 19 ore dopo la fecondazione (20 - fase somite) 13. Per visualizzare il movimento di nuclei e fusione di cellule muscolari noi svolte 4D confocale time-lapse utilizzando un ceppo transgenico che esprime la proteina fluorescente verde (GFP) sotto il controllo del promotore scheletrico α-actina di etichettare tutti del muscolo celle 14 e iniettato RNA codificante la proteina fluorescente rossa mCherry etichettato ...

Watch this Scientific Journal Video about Sample Drift Correction Following 4D Confocal Time-lapse Imaging at JoVE.com

Sample Drift Correction Following 4D Confocal Time-lapse Imaging

Microscopia time-lapse consente la visualizzazione dei processi di sviluppo. Crescita o deriva dei campioni durante l&#39;acquisizione dell&#39;immagine riduce la capacità di seguire con precisione e misurare i movimenti delle cellule durante lo sviluppo. Descriviamo l&#39;uso di software di elaborazione di immagini open source per correggere tridimensionale deriva campione nel tempo.

Correzione Drift seguente esempio 4D confocale Time-lapse imaging

The generation of four-dimensional (4D) confocal datasets; consisting of 3D image sequences over time; provides an excellent methodology to capture ...

sample-drift-correction-following-4d-confocal-time-lapse-imaging

Research

JoVE Journal

Bioengineering

16.3K Views.  Monash University.  Microscopia time-lapse consente la visualizzazione dei processi di sviluppo. Crescita o deriva dei campioni durante l'acquisizione dell'immagine riduce la capacità di seguire con precisione e misurare i movimenti delle cellule durante lo sviluppo. Descriviamo l'uso di software di elaborazione di immagini open source per correggere tridimensionale deriva campione nel tempo. 

Video: Sample Drift Correction Following 4D Confocal Time-lapse Imaging

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Imaging bioluminescenza per la valutazione delle risposte immunitarie A seguito di impianto di tessuto cardiaco Composti (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

Ricerca

Bioingegneria

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

Video-tasso di Microscopia confocale e scansione Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

Time-lapse imaging di fluorescenza della crescita Root Arabidopsis con manipolazione accelerato dell&#39;ambiente root usando il RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Campione strategie di preparazione per la Spettrometria di Massa Imaging di 3D Models Cultura cellulare

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

Un chip microfluidico per ICPMS introduzione del campione

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

Divisione delle cellule dei mammiferi in matrici 3D tramite microscopia confocale quantitativa di riflessione

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Modelli avanzati di fegato 3D per test di genotossicità in vitro a seguito dell'esposizione a lungo termine ai nanomateriali

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

Misurazione continua del rumore biologico in Escherichia Coli utilizzando la microscopia time-lapse

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

Imaging in vivo spazio-temporale di sistemi di somministrazione di farmaci oculari mediante microscopia laser confocale a fibre ottiche

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

We report a stage-top, flexible environmental chamber for time-lapse imaging of live cells using upright stimulated Raman scattering microscopy with transmitted signal detection. Lipid droplets were imaged in SKOV3 cells treated with oleic acid for up to 24 h with a 3 min time interval.

Una camera flessibile per l'imaging di cellule vive time-lapse con microscopia a diffusione Raman stimolata

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many ...