Les auteurs tiennent à remercier Amanda Struckhoff pour l&#39;aide initiale avec l&#39;expérience. Ce travail a été soutenu par une subvention du NIH 5RO1CA115706.

<ol>
	<li>Lauffenburger, D. A., Horwitz, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration:+a+physically+integrated+molecular+process.">Cell migration: a physically integrated molecular process.</a> Cell. 84, 359-369 (1996).</li><li>Ridley, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration:+integrating+signals+from+front+to+back.">Cell migration: integrating signals from front to back.</a> Science. 302, 1704-1709 (2003).</li><li>Petrie, R. J., Doyle, A. D., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Random+versus+directionally+persistent+cell+migration.">Random versus directionally persistent cell migration.</a> Nat. Rev. Mol. Cell Biol. 10, 538-549 (2009).</li><li>Valone, F. H., Austen, K. F., Goetzl, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+random+migration+of+human+platelets.">Modulation of the random migration of human platelets.</a> J. Clin. Invest. 54, 1100-1106 (1974).</li><li>Biehlmaier, O., Hehl, J., Csucs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+speed+comparison+of+microscope+software+programs.">Acquisition speed comparison of microscope software programs.</a> Microsc. Res. Tech. 74, 539-545 (2011).</li><li>Struckhoff, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+regulation+of+ROCK+in+tumor+cells+controls+CXCR4-driven+adhesion+events.">Dynamic regulation of ROCK in tumor cells controls CXCR4-driven adhesion events.</a> J. Cell. Sci. 123, 401-412 (2010).</li><li>Abramoff, M. D., Magelhaes, 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> Biophoton. Internat. 11, 36-42 (2004).</li><li>Greenberg, J. H., Seppa, S., Seppa, H., Tyl Hewitt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+collagen+and+fibronectin+in+neural+crest+cell+adhesion+and+migration.">Role of collagen and fibronectin in neural crest cell adhesion and migration.</a> Dev. Biol. 87, 259-266 (1981).</li><li>Albini, A., Sporn, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tumour+microenvironment+as+a+target+for+chemoprevention.">The tumour microenvironment as a target for chemoprevention.</a> Nat. Rev. Cancer. 7, 139-147 (2007).</li><li>Yu, Y. P., Luo, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myopodin-mediated+suppression+of+prostate+cancer+cell+migration+involves+interaction+with+zyxin.">Myopodin-mediated suppression of prostate cancer cell migration involves interaction with zyxin.</a> Cancer. Res. 66, 7414-7419 (2006).</li><li>Teng, T. S., Lin, B., Manser, E., Ng, D. C., Cao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stat3+promotes+directional+cell+migration+by+regulating+Rac1+activity+via+its+activator+betaPIX.">Stat3 promotes directional cell migration by regulating Rac1 activity via its activator betaPIX.</a> J. Cell. Sci. 122, 4150-4159 (2009).</li><li>Wozniak, M. A., Kwong, L., Chodniewicz, D., Klemke, R. L., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R-Ras+controls+membrane+protrusion+and+cell+migration+through+the+spatial+regulation+of+Rac+and+Rho.">R-Ras controls membrane protrusion and cell migration through the spatial regulation of Rac and Rho.</a> Mol. Biol. Cell. 16, 84-96 (2005).</li><li>Orr, A. W., Elzie, C. A., Kucik, D. F., Murphy-Ullrich, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombospondin+signaling+through+the+calreticulin/LDL+receptor-related+protein+co-complex+stimulates+random+and+directed+cell+migration.">Thrombospondin signaling through the calreticulin/LDL receptor-related protein co-complex stimulates random and directed cell migration.</a> J. Cell Sci. 116, 2917-2927 (2003).</li><li>Smith, A., Bracke, M., Leitinger, B., Porter, J. C., Hogg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LFA-1-induced+T+cell+migration+on+ICAM-1+involves+regulation+of+MLCK-mediated+attachment+and+ROCK-dependent+detachment.">LFA-1-induced T cell migration on ICAM-1 involves regulation of MLCK-mediated attachment and ROCK-dependent detachment.</a> J. Cell Sci. 116, 3123-3133 (2003).</li></ol>

Nous n&#39;avons rien à communiquer.

Le dosage en temps réel la migration aléatoire permet précis, sensible, l&#39;analyse des paramètres de migration cellulaires tels que la vitesse et le déplacement. Cette méthode n&#39;est pas limitée à la valeur finale de mesure de point, donc il est plus quantitative. Depuis, les cellules sont surveillés en milieu DMEM avec du sérum normal de; il réduit l&#39;effet délétère de plus d&#39;incubation dans le sérum des médias libres. Il a été montré que la migration des cellules dépend de la mise en f...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> DMEM </td><td> Thermo Scientific </td><td> SH30243.01 </td><td></td></tr><tr><td> FBS </td><td> Gemini Bio-Produits </td><td> 100-106 </td><td></td></tr><tr><td> Opti-Mem </td><td> Invitrogen </td><td> 31985-070 </td><td></td></tr><tr><td> Collagène </td><td> BD </td><td> 354231 </td><td></td></tr><tr><td> Microscope avec la chambre de cellules vivantes et automatisé contrôleur de platine XY </td><td> Olympe </td><td> Olympus 1X81 </td><td></td></tr><tr><td> Chambre de contrôle de l&#39;environnement </td><td> Neue </td><td> Chambre Neue Live Cell </td><td> La nôtre a une coutume construit plateau rectangulaire plaque en verre pour l&#39;optique appropriées par le haut de la chambre. La forme rectangulaire accueille plaques multi-puits utilisés dans bon nombre de nos expérits. </td></tr><tr><td> Automatique XY </td><td> Avant </td><td> Avant Proscan </td><td> Cela permet de multiplier retour précise positions XY sélectionnés au cours de la microscopie laps de temps. </td></tr><tr><td> Appareil photo </td><td></td><td> Hamamatsu EM appareil C9100 </td><td></td></tr><tr><td> Logiciel </td><td> Généralement achetés par l&#39;intermédiaire du fournisseur de microscope comme Olympus </td><td> 5 Réserver diapositives </td><td></td></tr></tbody></table>

quantitative analysis of random migration of cells using time-lapse video microscopy

La migration cellulaire est un processus dynamique, ce qui est important pour le développement embryonnaire, la réparation des tissus, la fonction du système immunitaire, et l&#39;invasion tumorale 1, 2. Lors de la migration directionnelle, les cellules se déplacent rapidement en réponse à un signal chimiotactique extracellulaire, ou en réponse à des signaux intrinsèques 3 prévus par le mécanisme de base motilité. La migration aléatoire se produit lorsque une cellule possède une faible directivité intrinsèque, permettant aux cellules d&#39;explorer leur environnement local. La migration cellulaire est un processus complexe, dans la cellule de réponse initial est soumise à la polarisation et s&#39;étend saillies dans le sens de la migration 2. Les méthodes traditionnelles de mesure de la migration, comme le test de migration Boyden chambre est une méthode simple pour mesurer la chimiotaxie in vitro, qui permet de mesurer la migration comme un résultat point de fin. Cependant, cette approche ne permet ni la mesure des paramètres de migration individuels, elle ne permet pas de visualisation de changements morphologiques que la cellule subit lors de la migration. Ici, nous présentons une méthode qui nous permet de contrôler les cellules migrent en temps réel en utilisant la vidéo - microscopie laps de temps. Depuis la migration et l&#39;invasion cellulaire sont caractéristiques du cancer, cette méthode sera applicable dans l&#39;étude de la migration des cellules cancéreuses et l&#39;invasion in vitro. La migration aléatoire des plaquettes a été considéré comme l&#39;un des paramètres de la fonction plaquettaire 4, d&#39;où cette méthode pourrait également être utile dans l&#39;étude des fonctions plaquettaires. Ce test présente l&#39;avantage d&#39;être rapide, fiable, reproductible, et ne nécessite pas d&#39;optimisation du nombre de cellules. Afin de maintenir des conditions physiologiquement appropriées pour les cellules, le microscope est équipé d&#39;alimentation en CO 2 et d&#39;un thermostat de température. Le mouvement cellulaire est contrôlé par la prise de vue à l&#39;aide d&#39;une caméra montée sur le microscope à intervalles réguliers. La migration cellulaire peut être calculée en mesurant la vitesse moyenne et unedéplacement mo yen, qui est calculée par un logiciel Slidebook.

Watch this Scientific Journal Video about Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy at JoVE.com

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cette méthode permet le suivi des cellules en temps réel et des mesures quantitatives de différents paramètres de migration cellulaires tels que la vitesse, le déplacement et la vitesse. Contrairement aux méthodes traditionnelles, cette approche temps réel n&#39;est pas basée sur des mesures quantitatives de point de terminaison de migration, mais plutôt il permet de surveiller et de calcul des paramètres différents en permanence.

Analyse quantitative des migrations au hasard des cellules en utilisant la microscopie vidéo time-lapse

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

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16.7K vues.  LSU School of Medicine. Cette méthode permet le suivi des cellules en temps réel et des mesures quantitatives de différents paramètres de migration cellulaires tels que la vitesse, le déplacement et la vitesse. Contrairement aux méthodes traditionnelles, cette approche temps réel n'est pas basée sur des mesures quantitatives de point de terminaison de migration, mais plutôt il permet de surveiller et de calcul des paramètres différents en permanence.

Vidéo: Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Monitoring Actin Disassembly with Time-lapse Microscopy

Surveillance actine démontage Time-lapse microscopie

Recherche

Biologie

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Imagerie time-lapse de la mitose après transfection de siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Time-lapse de microscopie du début de l&#39;embryogenèse dans les Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

L&#39;utilisation de la microscopie à Time Lapse Visualisez anoxie induite Suspended Animation en<em&gt; C. elegans</em&gt; Embryons

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Analyse quantitative de l&#39;autophagie en utilisant avancée Fluorescence Microscopy 3D

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Visualisation de développement cranio-facial dans les SOX10: Kaede poisson zèbre transgénique ligne à l&#39;aide Time-lapse de microscopie confocale

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time points. Time intervals can be set through a computer interface that controls the microscope-integrated camera. This kind of microscopy requires both the ability to acquire very rapid events and the signal generated by the observed cellular structure during these events. After the images have been collected, a movie of the entire experiment is assembled to show the dynamic of the molecular events of interest. Time-lapse video microscopy has a broad range of applications in the biomedical research field and is a powerful and unique tool for following the dynamics of the cellular events in real time. Through this technique, we can assess cellular events such as migration, division, signal transduction, growth, and death. Moreover, using fluorescent molecular probes we are able to mark specific molecules, such as DNA, RNA or proteins and follow them through their molecular pathways and functions. Time-lapse video microscopy has multiple advantages, the major one being the ability to collect data at the single-cell level, that make it a unique technology for investigation in the field of cell biology. However, time-lapse video microscopy has limitations that can interfere with the acquisition of high quality images. Images can be compromised by both external factors; temperature fluctuations, vibrations, humidity and internal factors; pH, cell motility. Herein, we describe a protocol for the dynamic acquisition of a specific protein, Parkin, fused with the enhanced yellow fluorescent protein (EYFP) in order to track the selective removal of damaged mitochondria, using a time-lapse video microscopy approach.

Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

Time-Lapse Video Microscopy pour l&#39;évaluation de EYFP-Parkin Agrégation comme un marqueur pour Cellular Mitophagy

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time ...

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

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

La microscopie vidéo sans lentille pour la dynamique et l’analyse Quantitative de la Culture de cellules adhérentes

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

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

Étiquetage spécifique des nucléoïdes mitochondriaux pour la microscopie d’illumination structurée time-lapse

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...

Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy

Bdellovibrio bacteriovorus is a small gram-negative, obligate predatory bacterium that kills other gram-negative bacteria, including harmful pathogens. Therefore, it is considered a living antibiotic. To apply B. bacteriovorus as a living antibiotic, it is first necessary to understand the major stages of its complex life cycle, particularly its proliferation inside prey. So far, it has been challenging to monitor successive stages of the predatory life cycle in real-time. Presented here is a comprehensive protocol for real-time imaging of the complete life cycle of B. bacteriovorus, especially during its growth inside the host. For this purpose, a system consisting of an agarose pad is used in combination with cell-imaging dishes, in which the predatory cells can move freely beneath the agarose pad while immobilized prey cells are able to form bdelloplasts. The application of a strain producing a fluorescently tagged &#946;-subunit of DNA polymerase III further allows chromosome replication to be monitored during the reproduction phase of the B. bacteriovorus life cycle.

Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy

Presented here is a protocol that describes monitoring of the complete life cycle of predatory bacterium Bdellovibrio bacteriovorus using time-lapse fluorescence microscopy in combination with an agarose pad and cell-imaging dishes.

Imagerie à cellules vivantes du cycle de vie du prédateur bactérien Bdellovibrio bactériovore à l’aide de la microscopie par fluorescence time-lapse

Bdellovibrio bacteriovorus is a small gram-negative, obligate predatory bacterium that kills other gram-negative bacteria, including harmful pathogens. ...