Name: lens-free video microscopy for the dynamic and quantitative analysis of adherent cell culture
Uploaded: 2018-02-23T14:00:00
Description: Watch this Scientific Journal Video about Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture at JoVE.com

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1. cellekultur overvåking oppkjøp
<ol><li>Vokse HeLa celler i DMEM + glutamin (f.eksGlutaMAX) medium med 10% (v/v) fetal kalv inaktivert serum og 1% penicillin og streptomycin.</li>
	<li>Coat 6-vel glass bunnplater kultur med fibronectin (25 µg/mL) 1t. Så frø 2 x 104 celler per brønn.</li>
	<li>Under oppkjøpet, endre mediet hver tredje dag.</li>
	<li>Time-lapse oppkjøpet, bruke video linse-fri mikroskopet (kommersielt tilgjengelig). Merk: Dette er basert på det objektiv uten beregningsor...

<ol>
	<li>Zangle, T. A., Teitell, M. 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W., Seo, S., Erlinger, 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=High-throughput+lensfree+imaging+and+characterization+of+a+heterogeneous+cell+solution+on+a+chip.">High-throughput lensfree imaging and characterization of a heterogeneous cell solution on a chip.</a> Biotechnol Bioeng. 102 (3), 856-868 (2009).</li><li>Delacroix, R., 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=Cerebrospinal+fluid+lens-free+microscopy:+a+new+tool+for+the+laboratory+diagnosis+of+meningitis.">Cerebrospinal fluid lens-free microscopy: a new tool for the laboratory diagnosis of meningitis.</a> Sci Rep. 7, 39893 (2017).</li><li>Tinevez, J. -. Y., 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=TrackMate:+an+open+and+extensible+platform+for+single-particle+tracking.">TrackMate: an open and extensible platform for single-particle tracking.</a> Methods. 115, 80-90 (2016).</li><li>Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+cell+mass+and+growth+dynamics.">Optical imaging of cell mass and growth dynamics.</a> Am J Physiol Physiol. 295 (2), 538-544 (2008).</li><li>Liu, P. Y., 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=Cell+refractive+index+for+cell+biology+and+disease+diagnosis:+past,+present+and+future.">Cell refractive index for cell biology and disease diagnosis: past, present and future.</a> Lab Chip. 16, 634-644 (2016).</li><li>Rapoport, D. H., Becker, T., Mamlouk, A. M., Schicktanz, S., Kruse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+validation+algorithm+allows+for+automated+cell+tracking+and+the+extraction+of+biologically+meaningful+parameters.">A novel validation algorithm allows for automated cell tracking and the extraction of biologically meaningful parameters.</a> PLoS One. 6 (11), e27315 (2011).</li><li>Al-Kofahi, O., 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=Automated+cell+lineage+construction:+A+rapid+method+to+analyze+clonal+development+established+with+murine+neural+progenitor+cells.">Automated cell lineage construction: A rapid method to analyze clonal development established with murine neural progenitor cells.</a> Cell Cycle. 5 (3), 327-335 (2006).</li><li>Meijering, E., Dzyubachyk, O., Smal, I., van Cappellen, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+in+cell+and+developmental+biology.">Tracking in cell and developmental biology.</a> Semin Cell Dev Biol. 20 (8), 894-902 (2009).</li><li>Posakony, J. W., England, J. 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I dette papiret viser vi at linsen-fri video mikroskopi kan brukes i en inkubator for å fange kinetics av celler. For å beskrive generelle metodene forklarte vi hvordan en 2,7 dag time-lapse oppkjøpet av HeLa celler i kultur kan analyseres med standard celle-sporing algoritmer. Resultatet er et datasett med 2.2 x 106 celle målinger og 10,584 cell sykkelstier. Oppkjøpene ble utført på en kultur for Hela celler med en relativt stor avstand i celle til celle (celle tetthet < 500 celler/mm2) og c...

Kultivert pattedyrceller gjennom flere cellen sykluser og nøyaktig celle størrelsen og celle tørr er en utfordrende oppgave. Flere etikett uten optisk teknikker er stand til å utføre denne aktiviteten1,2: fase-shifting interferometry3, digital holografiske mikroskopi (DHM)4,5,6, 7, quadriwave lateral klipping interferometry8,9 og kvantitativ fase tomografi10,11. Disse metodene har ført til mange ny innsikt i forståelsen av cellen syklus av pattedyrceller. Men de er sjelden kombinert med automatisk cellen sporing algoritmer og deres gjennomstrømming fortsatt begrenset når du måler celle masse baner1 (N < 20 i henholdsvis3,4,5 , 6). derfor romanen optisk metode er nødvendig for å måle cellen masse baner med store statistikk (N > 1000).
I dette papiret demonstrere vi evnen linse-fri video mikroskopi samtidig bilde tusen celler direkte i inkubator, og deretter kvantifisere enkeltcelle beregninger langs tusenvis av individuelle cell sykkelstier. Objektivet-fri mikroskopi er en kvantitativ fase tenkelig teknikk som lar oppkjøpet av fase bilde av tett pakket cellene over en stor synsfelt (vanligvis flere titalls mm2, her 29,4 mm2)12,13 ,14,15. Flere beregninger på ett cellenivå bestemmes, f.ekscelle området, celle tørr masse, celle tykkelse, celle store lengden og celle størrelsesforholdet12,15, fra hvert bilde. Deretter bruker en celle-sporing algoritme, kan disse funksjonene tegnes for hver enkelt celle som en funksjon av eksperimentet tid14,15. Videre ved å registrere forekomsten av celledelinger i cellen sporene, er det mulig å trekke annen viktig informasjon som den første cellen tørr masse (bare etter celledeling), den siste tørr cellemasse (like før celledeling) og cellen syklus varighet, dvs, tiden mellom to etterfølgende divisjoner15. Alle disse målingene kan beregnes med veldig god statistikk (N > 1000) siden store synsfelt ville vanligvis tillater analyse av 200 til 10.000 celler i en enkelt linse-fri oppkjøpet.
For å forklare denne metodikken basert på objektivet-fri video mikroskopi, beskriver vi protokollen for å overvåke og kvantifisere en HeLa cellekultur 2,7 dager. Dataanalyse er en fire-trinns prosess basert på flere bølgelengde holografiske gjenoppbygging, celle-sporing, celle segmentering og celledeling algoritmer. Her er det vist at romlig oppløsning og relativt rask bildefrekvens (en acquisition hvert 10. minutt) med dette objektivet-fri video mikroskopi oppsettet er kompatibel med standard celle-sporing algoritmer. Full analyse av dataset resulterer i måling av 10,584 celle spor over hele cellen sykluser.
For å oppsummere, er linse-fri video mikroskopi et kraftig verktøy for å automatisk overvåke tusenvis av umerkede, usynkroniserte og uforandret celler per eksperimentet. hver celle spores over flere sykluser for cellen. Våre målinger dermed gir middelverdien av flere celle parametere, men enda viktigere, mellom cellen variasjon over en stor bestand av celler.

<table border="0" cellpadding="0" cellspacing="0" width="675">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Cytonote lens-free video microscope&#160;</td>
			<td style="width:208px;">Iprasense</td>
			<td style="width:156px;"></td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Horus acquisition software</td>
			<td>Iprasense</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well glass bottom culture plates</td>
			<td>MatTek corporation</td>
			<td>Part No: P06G-0-14-F&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM + GlutaMAX medium&#160;</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">heat-inactivated fetal calf serum&#160;</td>
			<td>Eurobio</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">penicillin and streptomycin&#160;</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin&#160;</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matlab, image processing toolbox&#160;</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

lens-free video microscopy for the dynamic and quantitative analysis of adherent cell culture

Her viser vi at linsen-fri video mikroskopi kan vi samtidig fange kinetics av celler direkte inne inkubator, og at det er mulig å overvåke og kvantifisere enkeltceller langs flere cellen sykluser. Vi beskriver hele protokollen som brukes til å overvåke og kvantifisere en HeLa cellekultur for 2,7 dager. Først celle kultur oppkjøpet utføres med en linse-fri video mikroskop, og deretter dataene er analysert etter en fire-trinns prosess: flere bølgelengde holografiske gjenoppbygging, celle-sporing, celle segmentering og celledeling gjenkjenning algoritmer. Resultatet viser vi at det er mulig å samle en datasett med mer enn 10.000 cell sykkelstier og mer enn 2 x 106 celle morfologiske målinger.

For holografiske gjenoppbyggingsprosessen, lys feltet er beskrevet av en skalar felt en (der <img alt="Equation 4" src="/files/ftp_upload/56580/56580eq4v3.jpg"> er komplekse verdien av på flyet på vei z fra eksempel og lateral posisjon <img alt="Equation 5" src="/files/ftp_upload/56580/56580eq5v3.jpg"> og bølgelengde λ). Lys overføring er modellert av Huygens-Fresnel teorien som gir kjerne Bjørnson <img alt="Equation 6" src="/files/ftp_upload/56580/56580eq...

Watch this Scientific Journal Video about Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture at JoVE.com

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Objektivet-fri video mikroskopi kan vi overvåke cellekulturer direkte i inkubator. Her beskriver vi full protokollen for å hente og analysere en 2,7 dagen oppkjøpet av kultivert HeLa celler, fører til et datasett 2.2 x 10 spor6 målinger av personlige celle morfologi og 10584 celle syklus.

Objektivet-gratis Video mikroskopi for dynamisk og kvantitativ analyse av tilhenger cellekultur

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

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