Nous tenons à remercier Jonathan Cooper Lab au Fred Hutchinson Cancer Research Center for la permission d&#39;utiliser leur cryostat. Ce travail a été soutenu par le NIH et la WM Keck Foundation. BM est un garçon Gordon et Betty Moore Foundation de la recherche Sciences de la Vie. Nous sommes reconnaissants à Daniel Gottschling pour avoir partagé son protocole de fixation avec nous.

1. Fixation Communautés de levure <ol><li> Cultiver les communautés de levure sur une membrane (par exemple, filtre à membrane Millipore MF; figure 2A) de filtre. Ce protocole correspond à une communauté typique de moins de 2x10 8 cellules sur un filtre à membrane 6 mm de diamètre. </li><li> Utilisez un morceau de papier filtre Whatman 541 pour couvrir le fond d&#39;un 1 cm de diamètre ainsi (figure 2B). Ce papier Whatman sera utilisé comme support pour transférer de la communauté dans les étapes ultérieures. Ajouter 1 ml de méthanol à 100% pour le bien, et laisser le puits dans -20 ° C pour la température de s&#39;équilibrer. </li><li> Mettre N 2 liquide dans un vase de Dewar. Geler la communauté dans l&#39;azote liquide par immersion de la communauté pendant au moins 15 secondes dans de l&#39;azote liquide 2 en utilisant une pince à épiler (figure 2C). </li><li> Transfert communauté congelé pour le bien de -20 ° C méthanol (figure 2D). Assurez-vous que la communauté est maintenu à l&#39;horizontale lorsque l&#39;immerger dans le méthanol.Attendre 20 min. Le MF filtre à membrane commence à se désintégrer dans le méthanol. </li><li> Transfert communauté en utilisant le support papier filtre Whatman 541 du méthanol et d&#39;un plat froid de Pétri conservées à -20 ° C pour l&#39;évaporation du méthanol (figure 2E). Lors du transfert, de tenir la communauté au-dessus du porte-filtre Whatman et attirent des gouttes supplémentaires de méthanol en utilisant un autre morceau de papier Whatman épaisse avant de mettre dans le plat froid de Petri. Cela permettra d&#39;assurer que les temps d&#39;évaporation est constante d&#39;un échantillon à l&#39;. </li><li> Attendez 4-6 heures pour l&#39;évaporation jusqu&#39;à ce que le porte-filtre Whatman paraît sèche. Évaporation insuffisante laisse méthanol résiduel qui interfère avec la coupe. Au cours de séchage des fissures et des déformations des causes dans les communautés. </li><li> Prélever l&#39;échantillon de -20 ° C congélateur. Coupez Whatman 541 papier filtre ne sont pas couverts par la communauté. </li><li> Faire un cube rectangulaire contenant une feuille d&#39;aluminium qui est suffisamment grande de telle sorte que l&#39;échantillon comporte au moins marge ~ 2 mm à partir dechaque côté (figure 2F). Placez la communauté sur la surface inférieure de la feuille d&#39;aluminium contenant; couvrir immédiatement l&#39;échantillon avec OCT pour 3 ~ 4 mm plus haut que communauté. Si importante, l&#39;orientation de l&#39;échantillon peut être ajustée dans cette étape par rapport aux côtés du récipient. Attendre 10 min. </li><li> Placer l&#39;échantillon OCT-embarqué sur une plaque métallique sur la glace sèche à geler. Stocker les communautés congelés à -20 ° C ou -80 ° C congélateur. </li></ol> 2. Sectionnement Communautés surgelés pour imagerie par fluorescence <ol><li> Régler la température de la chambre cryosection à -25 ° C et laisser les échantillons dans la chambre jusqu&#39;à ce que sa température atteigne la température de la chambre. </li><li> Monter l&#39;échantillon sur un cryo-fixation après application d&#39;une baisse de l&#39;OCT sur la montagne. Étaler uniformément octobre sur le côté de l&#39;échantillon avec exposés papier filtre Whatman (fond du récipient) de sorte que l&#39;épaisseur OCT est de 2-3 mm. Laissez-gel octobre intérieur de la chambre du cryostat avant soictioning. Assurer une marge PTOM de 2-3 mm sur tous les côtés de l&#39;échantillon permet de préserver l&#39;intégrité des sections. </li><li> Les côtés du bloc échantillon OCT peut être utilisée pour aligner l&#39;échantillon. Si l&#39;échantillon n&#39;est pas alignée par rapport à la lame, la coupe commence à partir d&#39;un coin ou un côté de l&#39;échantillon. Régler le montage de sorte que la lame coupe une parfaitement vertical (ou horizontal) de la surface du bloc à échantillons. </li><li> Sections typiques pour les communautés de levure sont ~ 14 um d&#39;épaisseur. Après avoir coupé une section, utilisez une lame de microscope conservé à température ambiante pour collecter le texte, par approchant lentement le curseur vers la section de coupe. La section va fondre et transférer à la lame de microscope. Jusqu&#39;à 18 articles peuvent être transférés sur une glissière. </li><li> Stocker les diapositives à des températures froides, de préférence à -20 ° C, avant d&#39;imagerie. Perte de signal est minime lorsqu&#39;il est conservé pendant une semaine à 4 ° C. Pour le signal de fluorescence maximale, l&#39;imagerie est de préférence effectuée immédiatement après la coupe. </li></ol>

<ol>
	<li>Tolker-Nielsen, T., Molin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Organization+of+Microbial+Biofilm+Communities.">Spatial Organization of Microbial Biofilm Communities.</a> Microbial Ecology. 40 (2), 75-84 (2000).</li><li>Moller, S., 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=+In+Situ+Gene+Expression+in+Mixed-Culture+Biofilms:+Evidence+of+Metabolic+Interactions+between+Community+Members."> In Situ Gene Expression in Mixed-Culture Biofilms: Evidence of Metabolic Interactions between Community Members.</a> Appl. Environ. Microbiol. 64 (2), 721-732 (1998).</li><li>Lepp, P. W., 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=Methanogenic+Archaea+and+human+periodontal+disease.">Methanogenic Archaea and human periodontal disease.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (16), 6176-6181 (2004).</li><li>V&#225;chov&#225;, L., 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=Architecture+of+developing+multicellular+yeast+colony:+spatio-temporal+expression+of+Ato1p+ammonium+exporter.">Architecture of developing multicellular yeast colony: spatio-temporal expression of Ato1p ammonium exporter.</a> Environmental Microbiology. 11 (7), 1866-1877 (2009).</li><li>Min&#225;rikov&#225;, L., 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=Differentiated+Gene+Expression+in+Cells+within+Yeast+Colonies.">Differentiated Gene Expression in Cells within Yeast Colonies.</a> Experimental Cell Research. 271 (2), 296-304 (2001).</li><li>Shou, W., 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=Synthetic+cooperation+in+engineered+yeast+populations.">Synthetic cooperation in engineered yeast populations.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 1877-1882 (2007).</li><li>Momeni, B., 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=Cooperation+generates+a+unique+spatial+signature+in+microbial+communities.">Cooperation generates a unique spatial signature in microbial communities.</a> , (2012).</li><li>Kiernan, J., ed, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Histological and Histochemical Methods: Theory and Practice. , 4 (2008).</li><li>Piccirillo, S., 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=The+Rim101p/PacC+Pathway+and+Alkaline+pH+Regulate+Pattern+Formation+in+Yeast+Colonies.">The Rim101p/PacC Pathway and Alkaline pH Regulate Pattern Formation in Yeast Colonies.</a> Genetics. 184 (3), 707-716 (2010).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

La procédure présentée ici offre un moyen d&#39;inspecter la structure interne des communautés de levure. L&#39;étape de fixation méthanol rend la colonie de levure rigide 8 et préserve l&#39;intégrité de la colonie, mais il provoque aussi le point 8 de retrait qui doit être pris en compte dans l&#39;interprétation des résultats. Nous observons habituellement autour de 30% de rétrécissement dans la hauteur de colonies de levures, par rapport aux colonies directement intégrés dans un...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> Le méthanol 100% </td><td> JT Baker </td><td> 9070-01 </td><td></td></tr><tr><td> Tissue-Tek composé OCT </td><td> Andwin scientifique </td><td> 4583 </td><td></td></tr><tr><td> Whatman grade n ° 541 Papier filtre quantitatif </td><td> Whatman </td><td> 1541-047 </td><td></td></tr><tr><td> Millipore-MF filtre à membrane </td><td> Millipore </td><td> HAWP04700 </td><td></td></tr><tr><td> Cryostat </td><td> Leica </td><td> CM 1510 S </td><td></td></tr></tbody></table>

cryosectioning yeast communities for examining fluorescence patterns

Les microbes vivent généralement dans les communautés. L&#39;organisation spatiale des cellules à l&#39;intérieur d&#39;une communauté est censé avoir un impact sur ​​la survie et la fonction de la communauté 1. Techniques optiques de sectionnement, y compris la microscopie confocale et biphotonique, se sont avérées utiles pour observer l&#39;organisation spatiale des communautés bactériennes et Archaea 2,3. Une combinaison de la microscopie confocale et sectionnement physique de colonies de levures a révélé organisation interne des cellules 4. Cependant, directe sectionnement optique en utilisant la microscopie confocale ou à deux photons a été seulement en mesure d&#39;atteindre quelques couches cellulaires profondes dans les colonies de levures. Cette limitation est probablement en raison de forte diffusion de la lumière à partir de 4 cellules de levure. Ici, nous présentons une méthode basée sur la fixation et découpe au cryostat à obtenir la distribution spatiale des cellules fluorescentes au sein des communautés de Saccharomyces cerevisiae. Nous utilisons le méthanol comme agent fixateurà préserver la répartition spatiale des cellules. Communautés fixes sont infiltrés par composé OCT, congelés et cryosectioned dans un cryostat. L&#39;imagerie par fluorescence des sections révèle l&#39;organisation interne des cellules fluorescentes sein de la communauté. Des exemples de communautés de levures consistant en souches exprimant rouges et verts protéines fluorescentes en évidence les potentialités du procédé découpe au cryostat pour révéler la distribution spatiale des cellules fluorescentes ainsi que celui de l&#39;expression des gènes dans les colonies de levure 2,3. Même si notre accent a été mis sur les communautés de Saccharomyces cerevisiae, la même méthode peut potentiellement être utilisée pour examiner les autres communautés microbiennes.

Images de fluorescence des coupes verticales des communautés de levure contenant rouges et verts marqués populations compétitifs 6 sont représentés à la figure 3. Ces communautés sont constituées de deux souches de levure prototrophes et leur concurrence pour les ressources partagées, y compris l&#39;espace, conduit à colonnes modèles 7, tel qu&#39;il apparaît dans les coupes verticales. Résolutions allant jusqu&#39;à une seule cellule peut être résolu en sections. ...

Watch this Scientific Journal Video about Cryosectioning Yeast Communities for Examining Fluorescence Patterns at JoVE.com

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Nous présentons un protocole de congélation et de découpe au cryostat communautés de levure pour observer les modèles internes de cellules fluorescentes. La méthode repose sur la fixation et le méthanol octobre-enrobage à préserver la répartition spatiale des cellules sans inactivation des protéines fluorescentes dans une communauté.

Découpe au cryostat Communautés de levure pour examen des caractéristiques de fluorescence

Microbes typically live  in communities. The spatial organization  of cells within a community is believed to impact the survival and function of the  ...

cryosectioning-yeast-communities-for-examining-fluorescence-patterns

Research

JoVE Journal

Biology

8.8K vues.  Fred Hutchinson Cancer Research Center. Nous présentons un protocole de congélation et de découpe au cryostat communautés de levure pour observer les modèles internes de cellules fluorescentes. La méthode repose sur la fixation et le méthanol octobre-enrobage à préserver la répartition spatiale des cellules sans inactivation des protéines fluorescentes dans une communauté.

Vidéo: Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Flash Freezing and Cryosectioning E12.5 Mouse Brain

Demonstrated in this video are the techniques for flash freezing and sectioning embryonic brain tissue from mouse. Useful tips for using the cryostat are given, including troubleshooting methods that can be used while cutting to ensure that the resultant tissues sections are free of cracks and other distortions.

Flash de congélation et Cryosectioning E12.5 cerveau de souris

Recherche

Biologie

Microbial Communities in Nature and Laboratory - Interview

Les communautés microbiennes dans la Nature et de laboratoire - Interview

Biology of Microbial Communities  - Interview

Biologie des communautés microbiennes - Interview

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 ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

L&#39;examen de la dynamique conformationnelle des protéines membranaires In situ Avec site-dirigé marquage fluorescent

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

Genomic IRM - une ressource publique pour l&#39;étude des schémas de séquence dans l&#39;ADN génomique

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Un test microscopie de fluorescence pour Mitophagy surveillance dans la levure<em&gt; Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Quantitatives de fluorescence Live Cell-microscopie Analyse de la levure à fission

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

L&#39;acquisition de fluorescence Time-lapse films de levure bourgeonnante et analyse de la dynamique de cellules uniques en utilisant des greffes

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

Cryosectioning Méthode de microdissection de murin côlon Muqueuse

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...