A. Ozcan ringrazia il sostegno del Premio Presidenziale per Early Career Scientists and Engineers (PECASE), Army Research Office (ARO) Young Investigator Award, National Science Foundation (NSF) premio alla carriera, Office of Naval Research (ONR) Young Investigator Award e National Institutes of Health (NIH) Innovator Award Nuovo direttore DP2OD006427 presso l&#39;Ufficio del Direttore, National Institutes of Health.

In questa sezione, si introducono i protocolli sperimentali per il nostro cellulare in base a grande campo microscopia a fluorescenza 13 e opto-fluidici piattaforma di citometria di imaging 14. Useremo perline fluorescenti e contrassegnati in modo fluorescente le cellule bianche del sangue per testare queste piattaforme di imaging. A. Preparazione del telefono cellulare Basato Wide-field microscopio a fluorescenza e Opto-fluidico citometro a flusso Imaging Il telefono cellulare in base a grande campo microscopio a fluorescenza o citometria a flusso è costituito da due parti principali: un telefono con fotocamera e un compatto opto-fluidico aggiuntivo allegato. 1. La fotocamera del telefono Mentre le tecniche presentate sono applicabili a qualsiasi telefono cellulare con fotocamera, abbiamo scelto Sony Erickson Aino come base per questi dispositivi. Questo cellulare ha un ~ 8 MegaPixel RGB sensore CMOS installato su di esso e un obiettivo incorporato che ha una lunghezza focale (f 1) di ~ 4,65 millimetri. </p&gt; 2. Opto-fluidico Attachment per Wide-field microscopia a fluorescenza L&#39;allegato ottico è stato progettato da Autodesk ed è stampato da un Elite Dimension stampante 3-D con materiale termoplastico ABSplus. In questo processo di stampa, materiali modello e il supporto vengono riscaldati in una testa di estrusione all&#39;interno della stampante e si depositano a strati su una base di modellazione. Quando questo passaggio è completato, il materiale di supporto può essere dissolto, lasciando un solido modello 3D del prototipo desiderato nostro disegno allegato ottico composto da LEDs (centro lunghezza d&#39;onda a 470 nm ~, Digikey), un filtro di plastica (# NT54-46, Edmund ottiche), un vassoio campioni, e una lente piano-convessa f 2 = 15 mm (# NT45-302, Edmund Optics). Tutti i led e filtri di plastica può essere facilmente modificato in base spettri dei fluorofori. I passaggi per assemblare questo opto-fluidico allegato comprendono: <ol><li> Posizionare la lente nell&#39;attacco Nella sua posizione specifica porta-lente. </li><li> Posizionare il filtro in plastica sul vassoio filtro e farlo scorrere in l&#39;attacco, o nastro adesivo il filtro in plastica di fronte al telefono cellulare fotocamera. </li><li> Inserire il vassoio LED in allegato. </li><li> Porre i vetrini campione di vetro nel cassetto campione. Far scorrere il vassoio dei campioni in attacco. Affronta i LED verso il campione. </li><li> Agganciare l&#39;attacco sul telefono cellulare, in modo tale che la lente in più è direttamente in contatto con il telefono cellulare fotocamera. </li><li> Utilizzare l&#39;interruttore per il sequestro conservativo per accendere il LED. </li><li> Immagine il campione di interesse con il telefono cellulare telecamera usando la sua &quot;modalità notturna&quot;. </li></ol> 3. Opto-fluidico di collegamento per Citometria Imaging fluorescente Quando vi è la necessità di schermare grandi volumi di campioni liquidi per la rilevazione di eventi rari, optofluidic dispositivo di citometria di flusso potrebbe essere preferito. Siamo in grado di modificare il nostro ampio settore del design microscopio a fluorescenza e di convertirlo in un citofluorimetro, wqui una base PDMS canale microfluidico vengono usati per fornire continuamente il campione di liquido attraverso il volume di imaging. Il collegamento ottico è progettato anche da Autodesk e stampato da Dimension Elite stampante 3-D. Si compone anche di LED (lunghezza d&#39;onda centrale a ~ 470 nm, Digikey), un filtro in plastica (# NT54-46, Edmund Optics), un vassoio di campione e una lente asferica (f = 4.5 mm) (prodotto # C230TME-A; Thorlab). I passaggi per assemblare questo opto-fluidico allegato comprendono: <ol><li> Posizionare la lente asferica in allegato. </li><li> Posizionare il filtro in plastica sul vassoio filtro e farlo scorrere in l&#39;attacco, o nastro adesivo il filtro in plastica di fronte alla lente della fotocamera del cellulare. </li><li> Far scorrere il canale microfluidica nello stesso opto-fluidico attacco. </li><li> Agganciare l&#39;attacco sul telefono cellulare in modo tale che la lente in più è direttamente in contatto con il telefono cellulare fotocamera. </li><li> Utilizzare l&#39;interruttore per il sequestro conservativo per accendere il LED. </li><li> Collegare il microfluidic canale per la pompa a siringa e consegnare il campione di liquido nel dispositivo microfluidico ad una portata costante. </li><li> Cattura un filmato delle cellule fluorescenti / particelle che attraversano il canale microfluidica utilizzando la modalità video del telefono cellulare fotocamera. </li></ol> B. Preparazione del campione 4. Preparazione di Micro-particelle fluorescenti Campioni <ol><li> Perline fluorescenti con diametro di 10 micron (perline rosse: prodotto # F8834 eccitazione / emissione 580nm/605nm, perline verdi: prodotti # F8836: eccitazione / emissione 505nm/515nm) vengono acquistati da Invitrogen (Carlsbad, CA). </li><li> Miscelare 10 ml di perline verdi fluorescenti, 10 microlitri di rosso perline fluorescenti con 40 ml di acqua deionizzata. </li><li> Place10 pl di questa miscela tallone su un vetrino utilizzando una micropipetta e mettere un altro vetrino sulla parte superiore di esso per realizzare una struttura a sandwich. </li><li> Inserisci questa struttura a sandwich nel cassetto campione e farla scorrere nel telefono cellulare attaccamento. </li&gt; </ol> 5. Preparazione del marcato in modo fluorescente globuli bianchi <ol><li> Prendere SYTO16 acido nucleico fluorescente etichettatura kit (# S7578, Life Technology) e tampone fosfato salino (PBS) fuori dal frigorifero e portarli a temperatura ambiente. </li><li> Trasferire 200 microlitri campione di sangue intero da provetta di raccolta del sangue con EDTA a 1,5 ml tubetto polistirolo (# 05-408-129, Fisher Scientific). </li><li> Aggiungere 1 ml di tampone di globuli rossi lisante (# R7757, Sigma-Aldrich) per il campione di 200 pl di sangue intero e mescolare accuratamente. </li><li> Dopo 5 min, centrifugare il campione lisato sangue e rimuovere la soluzione surnatante. </li><li> Risospendere il pellet di cellule bianche del sangue in 200 microlitri di buffer PBS e mescolare delicatamente. </li><li> Aggiungere 5 l 1 mM SYTO16 soluzione del campione di globuli bianchi. Avvolgere il campione con un foglio di alluminio e incubare in ambiente buio per ~ 30 min. </li><li> Centrifugare il campione di nuovo. Surnatante viene rimosso e l&#39;etichetta bianca del sangue pellet cellulare è ri-Sospese in PBS. </li><li> Microlitri 5-10 etichettato bianco cella campione sangue liquido ad un vetrino, e posizionare un foglio secondo coperchio sulla parte superiore del campione. </li><li> Inserire la diapositiva campione inserita nel vassoio di campione e l&#39;immagine utilizzando il telefono cellulare microscopio a fluorescenza. </li></ol> In alternativa <ol start="10"><li> Continuamente consegnare le cellule bianche del sangue fluorescente etichettati attraverso un canale microfluidica utilizzando una pompa siringa automatica, ma anche di acquisizione di un filmato fluorescente microscopica delle cellule che scorrono con il telefono cellulare fotocamera in modalità video. È necessario inoltre sottolineare che una pompa a siringa portatile alimentato a batteria o anche forza di gravità può essere utilizzato per guidare il flusso attraverso il canale microfluidico. </li></ol>

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Dr. Ozcan è il fondatore di una start-up che si propone di commercializzare l&#39;imaging di calcolo e strumenti di microscopia.

Abbiamo presentato i nostri risultati recenti sul telefono cellulare basato ampio campo microscopia a fluorescenza e opto-fluidici citometria a flusso di immagini utilizzando leggeri e compatti opto-fluidici allegati a telefoni cellulari fotocamere. Grazie a questa tecnologia piattaforma abbiamo ripreso oggetti fluorescenti incluse micro-particelle ed etichettati globuli bianchi in campioni di sangue intero. Pertanto, questo compatto ed economico cellulare set di strumenti basati imaging a fluorescenza può essere utile...

Microscopia e citometria a flusso sono ampiamente utilizzate tecniche di 1-12 nella ricerca biomedica e scientifica, nonché la diagnosi clinica per il conteggio e la caratterizzazione di vari tipi di cellule. Tuttavia, microscopi convenzionali e flusso-citometria strumenti sono relativamente complesse e costose, che limita il loro uso principalmente alla laboratori centrali consolidate. Recentemente abbiamo sviluppato un citometria compatto e leggero dispositivo di imaging a fluorescenza e microscopia integrato su un telefono cellulare, che mostra la promessa 13,14 a costi contenuti tradurre microscopia a fluorescenza, citometria a flusso e le relative micro tecniche di analisi di ambienti con risorse limitate per applicazioni di telemedicina vari settori: la salute globale. Nel optofluidic citometria a flusso di configurazione (vedere ad esempio la Figura 1C e 1D), una custom-designed polydimethysiloxane (PDMS) canale in base microfluidica è positioned davanti al telefono cellulare fotocamera-unità, dove emettitori di luce a diodi (LED) sono accoppiate di testa ai bordi del canale. Questo chip microfluidica, unito all&#39;interno campione liquido, forma un opto-fluidico guida d&#39;onda planare (composto ad esempio PDMS-liquido-PDMS) tale che la luce di eccitazione è guidata per pompare uniformemente i campioni etichettati fluorescenti all&#39;interno del micro-canali. L&#39;emissione di fluorescenza da questi oggetti etichettati, ad esempio, le cellule, è ulteriormente ripreso attraverso una lente aggiuntiva collocata subito dopo il telefono cellulare fotocamera unità ed è mappato sul cellulare Complementary Metal-Oxide-Semiconductor (CMOS) sensore di immagine. Poiché l&#39;emissione fluorescente viene raccolto perpendicolare al percorso di luce di eccitazione, un filtro di assorbimento di plastica poco costoso è sufficiente rimuovere la luce diffusa di eccitazione e può fornire un discreto campo scuro sfondo necessaria per l&#39;imaging fluorescente. Utilizzando un simile opto-fluidico disegno, possiamo anche l&#39;immagine degli oggetti fluorescenti in static modalità (vedi figura 1A e 1B), in cui sono le particelle fluorescenti sandwich tra due vetrini invece di fluire attraverso un canale microfluidico e l&#39;emissione fluorescente da queste particelle fluorescenti vengono catturati dal telefono cellulare sensore di immagine CMOS per conteggio di particelle e caratterizzazione. Sulla base delle diverse esigenze applicative, citometria a flusso o grande campo di microscopia a fluorescenza possono essere scelti. Per esempio, cellulare dispositivo citometria di flusso potrebbe essere particolarmente utile per lo screening di grandi volumi di campioni liquidi (ad esempio alcuni ml) per la rilevazione di cellule rare o patogeni. In questo manoscritto passiamo in rassegna alcuni dei nostri recenti risultati sull&#39;integrazione di microscopia a fluorescenza e di imaging strumenti di citometria a flusso su un telefono cellulare utilizzando compatte e convenienti opto-fluidici allegati. Questi cellulare a base di micro-analisi, citometria di imaging e le piattaforme di rilevamento in grado di fornire diverse opportunitàper la diagnostica di telemedicina e point-of-care, in particolare impatto la nostra lotta contro le sfide della salute globale nelle regioni delle risorse limitate del mondo.

<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Company</td>
			<td>Catalog Number </td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell-phone</td>
			<td>Sony</td>
			<td>Sony Ericsson Aino</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plano-convex lens</td>
			<td>Edmund Optics</td>
			<td># NT45-302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aspherical lens</td>
			<td>Thorlab</td>
			<td># C230TME-A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Edmund Optics</td>
			<td>#NT54-46</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Blue LED</td>
			<td>Digikey</td>
			<td>#365-1201-ND</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Battery</td>
			<td>Digikey</td>
			<td>#P032-ND</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polystyrene tube</td>
			<td>Fisher Scientific</td>
			<td>#05-408-129</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Red blood cell lysing buffer</td>
			<td>Sigma Aldrich</td>
			<td>R7757</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SYLGARD 184 SILICONE ELASTOMER KIT</td>
			<td>Dow Corning</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Red fluorescent beads (10 &mu;m)</td>
			<td>Life Technologies</td>
			<td>#F8834</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Green fluorescent beads (10 &mu;m)</td>
			<td>Life Technologies</td>
			<td>#F8836</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SYTO16 nucleic acid fluorescent labeling</td>
			<td>Life Technologies</td>
			<td># S7578</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone

Microscopia a fluorescenza e citometria a flusso sono ampiamente utilizzati strumenti nella ricerca biomedica e diagnosi clinica. Tuttavia questi dispositivi sono in generale relativamente ingombranti e costosi, rendendoli meno efficaci nelle impostazioni delle risorse limitate. Per far fronte a queste limitazioni potenzialmente, abbiamo recentemente dimostrato l&#39;integrazione di ampio campo di microscopia a fluorescenza e di imaging strumenti di citometria a flusso su telefoni cellulari utilizzando compatto, leggero, e costo-efficacia opto-fluidici allegati. Nel nostro disegno citometria di flusso, le cellule sono contrassegnati in modo fluorescente lavata attraverso un canale microfluidico che è posizionata sopra l&#39;esistente cellulare telecamera. Alimentati a batteria diodi emettitori di luce (LED) sono di testa accoppiata al lato di questo chip microfluidica, che agisce efficacemente come una guida d&#39;onda multimodale lastra, dove viene guidato la luce di eccitazione per eccitare uniformemente gli obiettivi fluorescenti. Il telefono cellulare fotocamera registra un film lasso di tempo di cellule fluorescenti fluisce attraversoil canale microfluidico, dove vengono elaborate le cornici digitali di questo film per contare il numero di cellule marcate nella soluzione bersaglio di interesse. Utilizzando un simile opto-fluidico disegno, possiamo anche un&#39;immagine queste cellule contrassegnati in modo fluorescente in modalità statica es intramezzando particelle fluorescenti tra due vetrini e catturare le loro immagini fluorescenti utilizzando il telefono cellulare fotocamera, che possono raggiungere una risoluzione spaziale di esempio ~ 10 micron su un grande campo di vista di ~ 81 mm 2. Questo telefono cellulare basato fluorescente citofluorimetria imaging e microscopia piattaforma potrebbe essere utile specialmente in impostazioni di risorse limitate, per esempio il conteggio delle cellule T CD4 + verso il monitoraggio dei pazienti HIV + o per il rilevamento di acqua da parassiti nell&#39;acqua potabile.

Con il nostro opto-fluidico di pompaggio / eccitazione schema (figure 1C e 1D), le cellule marcate fluorescente può essere continuamente consegnato nel canale microfluidica utilizzando una pompa a siringa, mentre il telefono cellulare fotocamera registra una time-lapse film fluorescente microscopica delle cellule che scorre. Questi film fluorescenti possono essere rapidamente analizzati utilizzando contorno rilevamento e algoritmi di monitoraggio 14,15 per determinare automa...

Watch this Scientific Journal Video about Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone at JoVE.com

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Esaminiamo i nostri risultati recenti sull&#39;integrazione di microscopia a fluorescenza e di imaging strumenti di citometria a flusso su un telefono cellulare utilizzando compatte e convenienti opto-fluidici allegati. Questi cellulare basato micro-dispositivi di analisi può essere utile per l&#39;analisi citometrica, ad esempio per eseguire operazioni di conteggio cellulari diversi e per high throughput screening di campioni di acqua, ad esempio in impostazioni delle risorse limitate.

Ampio campo di microscopia a fluorescenza e citometria di flusso Imaging fluorescente su un telefono cellulare

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

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23.5K Views.  University of California, Los Angeles. Esaminiamo i nostri risultati recenti sull'integrazione di microscopia a fluorescenza e di imaging strumenti di citometria a flusso su un telefono cellulare utilizzando compatte e convenienti opto-fluidici allegati. Questi cellulare basato micro-dispositivi di analisi può essere utile per l'analisi citometrica, ad esempio per eseguire operazioni di conteggio cellulari diversi e per high throughput screening di campioni di acqua, ad esempio in impostazioni delle r...

Video: Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

Le nanoparticelle fluorescenti per la misurazione della concentrazione di ioni in sistemi biologici

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

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Bioingegneria

Lensless Fluorescent Microscopy on a Chip

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for high-throughput screening applications. This emerging technology platform has the potential to eliminate the need for bulky and/or costly optical components through the help of novel theories and digital reconstruction algorithms. Along the same lines, here we demonstrate an on-chip fluorescent microscopy modality that can achieve e.g., &lt;4&mu;m spatial resolution over an ultra-wide field-of-view (FOV) of &gt;0.6-8 cm2 without the use of any lenses, mechanical-scanning or thin-film based interference filters. In this technique, fluorescent excitation is achieved through a prism or hemispherical-glass interface illuminated by an incoherent source. After interacting with the entire object volume, this excitation light is rejected by total-internal-reflection (TIR) process that is occurring at the bottom of the sample micro-fluidic chip. The fluorescent emission from the excited objects is then collected by a fiber-optic faceplate or a taper and is delivered to an optoelectronic sensor array such as a charge-coupled-device (CCD). By using a compressive-sampling based decoding algorithm, the acquired lensfree raw fluorescent images of the sample can be rapidly processed to yield e.g., &lt;4&mu;m resolution over an FOV of &gt;0.6-8 cm2. Moreover, vertically stacked micro-channels that are separated by e.g., 50-100 &mu;m can also be successfully imaged using the same lensfree on-chip microscopy platform, which further increases the overall throughput of this modality. This compact on-chip fluorescent imaging platform, with a rapid compressive decoder behind it, could be rather valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

A lensless on-chip fluorescent microscopy platform is demonstrated that can image fluorescent objects over an ultra-wide field-of-view of e.g., &#62;0.6-8 cm2 with &#60;4&#956;m resolution using a compressive sampling based decoding algorithm. Such a compact and wide-field fluorescent on-chip imaging modality could be valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

Senza lenti microscopia a fluorescenza su un chip

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for ...

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. 
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

Protocolli Preparazione del campione, Imaging, e d&#39;analisi per Knife-edge Microscopia Scansione

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Analizzando internalizzazione cellulare di nanoparticelle e batteri da Multi-spettrale Citometria a Flusso Imaging

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Localizzazione e quantificazione relativa dei nanotubi di carbonio in cellule con immagini multispettrali Citometria a Flusso

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

Un nuovo metodo per la localizzazione Reporter perline fluorescenti vicino alla superficie coltura cellulare per la trazione microscopia a forza

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

Visualizzazione di formazione di aderenze in cellule per mezzo di avanzati filatura microscopia di fluorescenza di riflessione interna totale disco

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

Un metodo automatizzato per eseguire l'analisi in vitro micronucleoutilizzando la citometria del flusso di imaging multispettrale

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies for the detection of CTCs include flow cytometry, microfluidic devices, and real-time polymerase chain reaction (RT-PCR). Despite recent advances, methods for the detection of early ovarian cancer metastasis still lack the sensitivity and specificity required for clinical translation. Here, a novel method is presented for the detection of ovarian circulating tumor cells by photoacoustic flow cytometry (PAFC) utilizing a custom three dimensional (3D) printed system, including a flow chamber and syringe pump. This method utilizes folic acid-capped copper sulfide nanoparticles (FA-CuS NPs) to target SKOV-3 ovarian cancer cells by PAFC. This work demonstrates the affinity of these contrast agents for ovarian cancer cells. The results show NP characterization, PAFC detection, and NP uptake by fluorescence microscopy, thus demonstrating the potential of this novel system to detect ovarian CTCs at physiologically relevant concentrations.

A protocol is presented to detect circulating ovarian tumor cells utilizing a custom-made photoacoustic flow system and targeted folic acid-capped copper sulfide nanoparticles.

Rilevamento del cancro ovarico utilizzando la citometria a flusso fotoacustico

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies ...

Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome formulation, liposomes may be preferentially taken up by different cell types in the body. This may influence the efficacy of the therapeutic particle as progression of different diseases is tissue- and cell-type-specific. In this protocol, we present one method for synthesizing and fluorescently labeling liposomes using DSPC, cholesterol, and PEG-2000 DSPE and the lipid dye DiD as a fluorescent label. This protocol also presents an approach for delivering liposomes in vivo and assessing cell-specific uptake of liposomes using flow cytometry. This approach can be used to determine the types of cells that take up liposomes and quantify the distribution and proportion of liposome-uptake across cell types and tissues. While not mentioned in this protocol, additional assays such as immunofluorescence and single-cell fluorescence imaging on a cytometer will strengthen any findings or conclusions made as they permit assessment of intracellular staining. Protocols may also need to be adapted depending on the tissue(s) of interest.

The goal of this protocol is to synthesize fluorescently-labeled liposomes and use flow cytometry to identify in vivo localization of liposomes at a cellular level.

Preparazione, somministrazione e valutazione dell'assorbimento cellulare tessuto-specifico in vivo di liposomi marcati con coloranti fluorescenti

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome ...