Name: imaging intermediate filaments and microtubules with 2-dimensional direct stochastic optical reconstruction microscopy
Uploaded: 2018-03-06T13:30:00
Description: Watch this Scientific Journal Video about Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy at JoVE.com

Nous remercions Mickael Lelek, Orestis Faklaris et Nicolas Bourg de discussions fructueuses, Andrey Aristov et Elena Rensen pour aider avec la technique de Super-résolution et Shailaja Seetharaman pour une lecture attentive du manuscrit. Nous tenons à souligner le Citech UtechS BioImaging photoniques (Imagopole) d’Institut Pasteur (Paris, France) ainsi que le réseau d’infrastructures de France-BioImaging pris en charge par l’Office National Français des recherches (ANR-10-INSB-04 ; Investissements d’avenir) et la Région Ile-de-France (programme Domaine d'Intérêt Majeur-Malinf) pour l’utilisation du microscope Elyra. Ce travail a été soutenu par la la Ligue Contre le Cancer et le Français National Research Agency (ANR-16-CE13-0019).

1. couvre-objet en préparation (1 jour, 30 min)
<ol><li>Placez les lamelles de verre de 18 mm nº1.5H (170 µm +/-5µm) sur une grille en plastique.</li>
	<li>Mettre le panier dans un bécher de 100 mL rempli d’acétone et laisser tremper pendant 5 min.</li>
	<li>Transférer la crémaillère dans un bécher de 100 mL rempli d’éthanol absolu et laisser tremper pendant 5 min.</li>
	<li>Transférer la crémaillère dans un bécher de 100 mL nouveau rempli avec de l’éthanol absolu et placer le bécher dans un disp...

<ol>
	<li>Herrmann, H., Aebi, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermediate+Filaments:+Structure+and+Assembly.">Intermediate Filaments: Structure and Assembly.</a> Cold Spring Harb Perspect Biol. 8 (11), (2016).</li><li>Huber, F., Boire, A., Lopez, M. P., Koenderink, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+crosstalk:+when+three+different+personalities+team+up.">Cytoskeletal crosstalk: when three different personalities team up.</a> Curr Opin Cell Biol. 32, 39-47 (2015).</li><li>Leduc, C., Etienne-Manneville, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermediate+filaments+in+cell+migration+and+invasion:+the+unusual+suspects.">Intermediate filaments in cell migration and invasion: the unusual suspects.</a> Curr Opin Cell Biol. 32, 102-112 (2015).</li><li>Heilemann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subdiffraction-resolution+fluorescence+imaging+with+conventional+fluorescent+probes.">Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes.</a> Angew Chem Int Ed Engl. 47 (33), 6172-6176 (2008).</li><li>Gevaux, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nobel+Prize+in+Chemistry:+seeing+the+nanoscale.">Nobel Prize in Chemistry: seeing the nanoscale.</a> Nat Nanotechnol. 9 (11), 878 (2014).</li><li>Betzig, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313 (5793), 1642-1645 (2006).</li><li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nat Methods. 3 (10), 793-795 (2006).</li><li>Metcalf, D. J., Edwards, R., Kumarswami, N., Knight, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Test+samples+for+optimizing+STORM+super-resolution+microscopy.">Test samples for optimizing STORM super-resolution microscopy.</a> J Vis Exp. (79), (2013).</li><li>Dempsey, G. T., Vaughan, J. C., Chen, K. H., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+fluorophores+for+optimal+performance+in+localization-based+super-resolution+imaging.">Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging.</a> Nat Methods. 8 (12), 1027-1036 (2011).</li><li>Dempsey, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+user's+guide+to+localization-based+super-resolution+fluorescence+imaging.">A user's guide to localization-based super-resolution fluorescence imaging.</a> Methods Cell Biol. 114, 561-592 (2013).</li><li>van de Linde, 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=Direct+stochastic+optical+reconstruction+microscopy+with+standard+fluorescent+probes.">Direct stochastic optical reconstruction microscopy with standard fluorescent probes.</a> Nat Protoc. 6 (7), 991-1009 (2011).</li><li>Chazeau, A., Katrukha, E. A., Hoogenraad, C. C., Kapitein, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+neuronal+microtubule+organization+and+microtubule-associated+proteins+using+single+molecule+localization+microscopy.">Studying neuronal microtubule organization and microtubule-associated proteins using single molecule localization microscopy.</a> Methods Cell Biol. 131, 127-149 (2016).</li><li>Olivier, N., Keller, D., Rajan, V. S., Gonczy, P., Manley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+buffers+for+3D+STORM+microscopy.">Simple buffers for 3D STORM microscopy.</a> Biomed Opt Express. 4 (6), 885-899 (2013).</li><li>Eliasson, C., 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=Intermediate+filament+protein+partnership+in+astrocytes.">Intermediate filament protein partnership in astrocytes.</a> J Biol Chem. 274 (34), 23996-24006 (1999).</li><li>Leduc, C., Etienne-Manneville, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+microtubule-associated+motors+drives+intermediate+filament+network+polarization.">Regulation of microtubule-associated motors drives intermediate filament network polarization.</a> J Cell Biol. 216 (6), 1689-1703 (2017).</li><li>Gan, Z., 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=Vimentin+Intermediate+Filaments+Template+Microtubule+Networks+to+Enhance+Persistence+in+Cell+Polarity+and+Directed+Migration.">Vimentin Intermediate Filaments Template Microtubule Networks to Enhance Persistence in Cell Polarity and Directed Migration.</a> Cell Syst. 3 (5), 500-501 (2016).</li><li>Nieuwenhuizen, R. P., 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=Co-Orientation:+Quantifying+Simultaneous+Co-Localization+and+Orientational+Alignment+of+Filaments+in+Light+Microscopy.">Co-Orientation: Quantifying Simultaneous Co-Localization and Orientational Alignment of Filaments in Light Microscopy.</a> PLoS One. 10 (7), e0131756 (2015).</li><li>Yang, Z., Wang, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+fibrillary+acidic+protein:+from+intermediate+filament+assembly+and+gliosis+to+neurobiomarker.">Glial fibrillary acidic protein: from intermediate filament assembly and gliosis to neurobiomarker.</a> Trends Neurosci. 38 (6), 364-374 (2015).</li><li>Maslehaty, H., Cordovi, S., Hefti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symptomatic+spinal+metastases+of+intracranial+glioblastoma:+clinical+characteristics+and+pathomechanism+relating+to+GFAP+expression.">Symptomatic spinal metastases of intracranial glioblastoma: clinical characteristics and pathomechanism relating to GFAP expression.</a> J Neurooncol. 101 (2), 329-333 (2011).</li><li>Hol, E. M., Pekny, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+fibrillary+acidic+protein+(GFAP)+and+the+astrocyte+intermediate+filament+system+in+diseases+of+the+central+nervous+system.">Glial fibrillary acidic protein (GFAP) and the astrocyte intermediate filament system in diseases of the central nervous system.</a> Curr Opin Cell Biol. 32, 121-130 (2015).</li><li>Skalli, 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=Astrocytoma+grade+IV+(glioblastoma+multiforme)+displays+3+subtypes+with+unique+expression+profiles+of+intermediate+filament+proteins.">Astrocytoma grade IV (glioblastoma multiforme) displays 3 subtypes with unique expression profiles of intermediate filament proteins.</a> Hum Pathol. 44 (10), 2081-2088 (2013).</li><li>Endesfelder, U., Malkusch, S., Fricke, F., Heilemann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+estimate+the+average+localization+precision+of+a+single-molecule+localization+microscopy+experiment.">A simple method to estimate the average localization precision of a single-molecule localization microscopy experiment.</a> Histochem Cell Biol. 141 (6), 629-638 (2014).</li><li>Whelan, D. R., Bell, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+artifacts+in+single+molecule+localization+microscopy:+why+optimization+of+sample+preparation+protocols+matters.">Image artifacts in single molecule localization microscopy: why optimization of sample preparation protocols matters.</a> Sci Rep. 5, 7924 (2015).</li><li>Ovesny, M., Krizek, P., Borkovec, J., Svindrych, Z., Hagen, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ThunderSTORM:+a+comprehensive+ImageJ+plug-in+for+PALM+and+STORM+data+analysis+and+super-resolution+imaging.">ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging.</a> Bioinformatics. 30 (16), 2389-2390 (2014).</li><li>Olivier, N., Keller, D., Gonczy, P., Manley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+doubling+in+3D-STORM+imaging+through+improved+buffers.">Resolution doubling in 3D-STORM imaging through improved buffers.</a> PLoS One. 8 (7), e69004 (2013).</li><li>Nahidiazar, L., Agronskaia, A. V., Broertjes, J., van den Broek, B., Jalink, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+Imaging+Conditions+for+Demanding+Multi-Color+Super+Resolution+Localization+Microscopy.">Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy.</a> PLoS One. 11 (7), e0158884 (2016).</li><li>Huang, B., Wang, W., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+super-resolution+imaging+by+stochastic+optical+reconstruction+microscopy.">Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy.</a> Science. 319 (5864), 810-813 (2008).</li><li>Mikhaylova, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+bundled+microtubules+using+anti-tubulin+nanobodies.">Resolving bundled microtubules using anti-tubulin nanobodies.</a> Nat Commun. 6, 7933 (2015).</li><li>Nieuwenhuizen, R. P., 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=Measuring+image+resolution+in+optical+nanoscopy.">Measuring image resolution in optical nanoscopy.</a> Nat Methods. 10 (6), 557-562 (2013).</li><li>Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F., Jungmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+microscopy+with+DNA-PAINT.">Super-resolution microscopy with DNA-PAINT.</a> Nat Protoc. 12 (6), 1198-1228 (2017).</li></ol>

Étapes cruciales dans le protocole
Nous présentons ici un protocole qui minimise les artefacts dans les images dSTORM des microtubules et IFs dans les cellules gliales. Artefacts peuvent être créées à chaque étape de la préparation de l’échantillon et l’imagerie : fixation, blocage, immunomarquage, dérive pendant l’acquisition, non-optimale clignotant conditions23. Nous énumérons ci-dessous les étapes plus critiques.
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Les filaments intermédiaires cytoplasmiques (IFs) sont 10 nm de diamètre homo - ou hétéropolymères d’un sous-ensemble spécifique de type cellulaire des protéines de l’IF. IFs participent dans une large gamme de fonctions cellulaires telles que les réponses de stress, la prolifération et la motilité cellulaire. Leur rôle essentiel est souligné par le fait que plus de 90 maladies humaines sont directement causées par des mutations dans les protéines de l’IF ; par exemple, les changements dans la composition de l’IF accompagne la croissance tumorale et la diffusion de1,2,3. Il est plus en plus évident que les trois systèmes de cytosquelette travaillent en collaboration pour contrôler les fonctions cellulaires tels que la polarisation de la cellule, division et migration2,3. Étant donné qu’un couplage étroit entre l’architecture spatiale et fonctions, il est crucial d’obtenir des aperçus de la structure de l’organisation de la fi avec les autres filaments et mieux comprendre la diaphonie du cytosquelette. Cet article fournit un protocole pour effectuer la Super-résolution technique dSTORM bicolores (stochastique optique Reconstruction examen microscopique direct)4 et comment il est utilisé pour étudier l’interaction entre le si et les microtubules en fixe, cultivés cellules en 2 dimensions (2D).
Plusieurs techniques de Super-résolution ont été développées au cours de la dernière décennie et là-bas découvertes furent à l’origine du prix Nobel de chimie en 20145. Parmi toutes ces techniques se trouve les seule molécule axées sur la localisation des méthodes comme PALM6 (microscopie de localisation Photo-Activated) qui utilise des protéines fluorescentes ayant, tempête de7 paires de fluorophores ou dSTORM4 avec des fluorophores classiques. Toutes ces méthodes sont basées sur le même principe qui consiste en (i) l’interrupteur de la plupart des journalistes fluorescentes dans un état « off » » l’état de (non fluorescent), (ii) l’activation stochastique d’une partie d'entre eux dans un fluorescent afin de localiser leurs position spatiale avec précision nanométrique et (iii) la répétition de ce processus afin d’activer autant de sous-ensembles de fluorophores que possible. Une image finale est reconstruite à l’aide de toutes les localisations des molécules activées, offrant une résolution latérale vers le bas pour ~ 20 à 40 nm. Plusieurs systèmes optiques commercialisés permettant PALM/STORM sont maintenant disponibles pour les biologistes pour expériences de routine. Ici, un tel système a été utilisé pour étudier l’association structurelle des microtubules et IFs. Parmi toutes les molécules simples axées sur la localisation méthodes, la technique de double-couleur dSTORM a été choisie, parce qu’il est bien adapté pour l’observation des régions lamellaires très minces (< 1 µm) des cellules adhérentes et peut apporter une amélioration significative de la résolution de l’image avec investissement minimal de temps et d’argent. En effet, dSTORM est une technique très pratique et polyvalente qui est compatible avec les colorants organiques standards couramment utilisées pour la coloration cellulaire et immunofluorescence.
Alors qu’une seule couleur dSTORM images sont relativement simples à obtenir en utilisant le fluorophore Alexa6478, dSTORM bicolore nécessite d’optimiser les conditions expérimentales afin que deux colorants peuvent clignoter correctement dans le tampon choisi, surtout si il est limité puissance du laser. Excellentes communications sont déjà disponibles sur les méthodes à mettre en place l’imagerie multicolore avec dSTORM9,10,11,12,13, et ces documents expliquent en détail les sources possibles de artefacts et la résolution de l’image dégradée ainsi que la façon de les surmonter. Dans cet article, les conditions expérimentales optimales en ce qui concerne la fixation des cellules, immunomarquage, préparation des échantillons, acquisition d’imagerie et la reconstruction de l’image sont décrits afin d’acquérir des images des réseaux denses de IF cytoplasmiques et microtubules dans cellules gliales avec dSTORM bicolores. En bref, la méthode d’extraction/fixation décrite à Chazeau et al.12 a été adaptée aux cellules gliales et utilisé avec une étape après fixation, concentration d’anticorps optimisée, un tampon de tempête avec 10 mM MEA décrit dans Dempsey9 et coll., qui a été jugées optimales pour l’expérimental mis en place et le type d’échantillon.
Les cellules gliales expriment principalement trois types de protéines IF : vimentine, nestin et GFAP (glial protéine fibrillaire acide). Ces trois protéines ont été montrés à polymériser conjointement dans les astrocytes14. Nous avons montré précédemment à l’aide de la microscopie d’illumination de Super-résolution structurée (SR SIM) que ces trois protéines IF se trouve dans le même filament unique de IF et qu’ils affichent semblables de distribution et de la dynamique dans les cellules gliales 15. En raison des similitudes entre les trois protéines IF, vimentine coloration a été utilisée comme reporter pour l’ensemble du réseau IF. À l’aide de dSTORM, nous avons réussi à résoudre Comment former des faisceaux IF le long des microtubules, qui n’était pas possible avec la diffraction limitée des techniques de microscopie15. Ces observations peuvent aider à comprendre comment la vimentine IFs peuvent microtubules modèle et réglementer leur trajectoire de croissance, favorisant le maintien d’un axe de polarité au cours de la migration de cellules16. Images de Super-résolution fourni des informations clées sur l’interaction mutuelle des deux sous-systèmes du cytosquelette et amené à mieux comprendre le lien entre l’association spatiale et la fonction locale qui pourrait être de type cellulaire spécifique17. En général, bicolores dSTORM peut servir à étudier la diaphonie entre les éléments du cytosquelette ou autres types d’organites, pourvu que l’immunomarquage bonnes conditions sont atteintes en termes de densité et de la spécificité. Cette technique sera utile pour mieux caractériser les cytosquelette des changements observés au cours de l’astrogliosis et dans le glioblastome, la tumeur plus malins et les plus courants du système nerveux central, où l’expression des protéines de l’IF est modifié 18 ,19,20,21.

<table>
	<tbody>
		<tr>
			<td>Minimum Essential Media (MEM)</td>
			<td>Gibco</td>
			<td>41090-028</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>non essential amino acid</td>
			<td>Gibco</td>
			<td>1140-050</td>
			<td>cell culture 1/100e</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>cell culture 1/100e</td>
		</tr>
		<tr>
			<td>F&#339;tal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>cell culture 1/10e</td>
		</tr>
		<tr>
			<td>0,05 % Trypsin-EDTA (1X)</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>PBS 10x</td>
			<td>fischer scientific</td>
			<td>11540486</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>coverslip 18 mm n&#176;1,5H</td>
			<td>Marienfield/Dominic dutsher</td>
			<td>900556</td>
			<td>coverslips</td>
		</tr>
		<tr>
			<td>paraformaldehyde (PFA) solution</td>
			<td>Sigma</td>
			<td>F8775</td>
			<td>cell fixation</td>
		</tr>
		<tr>
			<td>glutaraldehyde</td>
			<td>Sigma</td>
			<td>G5882</td>
			<td>cell fixation</td>
		</tr>
		<tr>
			<td>triton X100</td>
			<td>Sigma</td>
			<td>T9284</td>
			<td>non ionic surfactant. Used for cell fixation</td>
		</tr>
		<tr>
			<td>sodium borohydride (NaBH4)</td>
			<td>Sigma</td>
			<td>452904-25ML</td>
			<td>cell fixation</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td>sample passivation 3% in PBS</td>
		</tr>
		<tr>
			<td>Monoclonal Anti-Vimentin (V9) antibody produced in mouse</td>
			<td>Sigma</td>
			<td>V6630</td>
			<td>primary antibodies</td>
		</tr>
		<tr>
			<td>Rat anti alpha tubulin</td>
			<td>Biorad</td>
			<td>MCA77G</td>
			<td>primary antibodies</td>
		</tr>
		<tr>
			<td>Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 555</td>
			<td>Fisher scientific</td>
			<td>10635923</td>
			<td>secondary antibodies</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 647</td>
			<td>Fisher scientific</td>
			<td>10226162</td>
			<td>secondary antibodies</td>
		</tr>
		<tr>
			<td>TetraSpeck Microspheres, 0.1 &#181;m, fluorescent blue/green/orange/dark red</td>
			<td>Fisher scientific</td>
			<td>T7279</td>
			<td>fluorescent beads</td>
		</tr>
		<tr>
			<td>Chamlide magnetic chamber</td>
			<td>Live Cell Instrument</td>
			<td>CM-B18-1</td>
			<td>magnetic sample holder, maximum volume 1,2 mL</td>
		</tr>
		<tr>
			<td>Cysteamine (MEA)</td>
			<td>Sigma</td>
			<td>30070</td>
			<td>for the blinking buffer</td>
		</tr>
		<tr>
			<td>glucose oxidase from Aspergillus niger</td>
			<td>Sigma</td>
			<td>G0543</td>
			<td>for the blinking buffer</td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>sigma</td>
			<td></td>
			<td>for the blinking buffer</td>
		</tr>
		<tr>
			<td>catalase from bovine liver</td>
			<td>Sigma</td>
			<td>C9322</td>
			<td>for the blinking buffer</td>
		</tr>
		<tr>
			<td>Tris (trizma)</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td>for the blinking buffer</td>
		</tr>
		<tr>
			<td>Wash-N-Dry&#160;coverslip rack</td>
			<td>Sigma</td>
			<td>Z688568</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma</td>
			<td>P7793-1EA</td>
			<td>paraffin film</td>
		</tr>
		<tr>
			<td>ultrasonic cleaner</td>
			<td>Fisher scientific</td>
			<td>15-335-6</td>
			<td></td>
		</tr>
		<tr>
			<td>plasma cleaner</td>
			<td>Harrick plasma</td>
			<td>PDC-32G-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging intermediate filaments and microtubules with 2-dimensional direct stochastic optical reconstruction microscopy

Le cytosquelette, composé de microfilaments d’actine et les microtubules filaments intermédiaires (si), joue un rôle clé dans le contrôle de la forme des cellules, la polarité et la motilité. L’Organisation des réseaux actine et les microtubules a été particulièrement étudiée, mais celui d’IFs n’est pas encore pleinement caractérisée. IFs ont un diamètre moyen de 10 nm et forme un réseau s’étendant dans tout le cytoplasme de la cellule. Ils sont physiquement associés d’actine et les microtubules grâce à des moteurs moléculaires et linkers du cytosquelette. Cette association étroite est au cœur des mécanismes de réglementation qui assurent la régulation coordonnée des trois réseaux du cytosquelette requis pour la plupart des fonctions cellulaires. Il est donc indispensable de visualiser IFs seuls et aussi avec chacun des autres réseaux du cytosquelette. Cependant, les réseaux de IF sont extrêmement denses dans la plupart des types de cellules, en particulier dans les cellules gliales, qui rend sa résolution très difficile à réaliser avec la microscopie en fluorescence standard (latéral résolution de ~ 250 nm). Microscopie de Reconstruction stochastique optique directe (dSTORM) est une technique qui permet un gain en résolution latérale d’un ordre de grandeur. Ici, nous montrons que la résolution latérale dSTORM est suffisante pour résoudre l’organisation dense des réseaux IF et, en particulier, des faisceaux de IF entourant les microtubules. Serré de ces associations sont susceptible de participer à la régulation coordonnée de ces deux réseaux et mai, expliquer comment modèle IFs vimentine et stabiliser l’Organisation des microtubules ainsi que pourraient influencer microtubule dépendant du trafic vésiculaire. Plus généralement, nous montrons comment l’observation des deux composants du cytosquelette avec bicolores dSTORM technique apporte nouvel aperçu de leur interaction mutuelle.

Un microscope équipé de caméra de 512 x 512, un alpha Plan Apo 100 X 50 mW 405 nm et les 100 mW 488, 561 et 642 nm à semi-conducteurs lasers, une EMCCD / 1,46 objectif et Band passent 570-650 / passent longtemps 655 filtres d’émission a été utilisé pour les résultats représentatifs présentés ci-dessous.
Figure 1 a donne un exemple de la densité de la molécule et de signal / bruit qui...

Watch this Scientific Journal Video about Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy at JoVE.com

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

Imagerie des Filaments intermédiaires et Microtubules avec directes 2 dimensions de Reconstruction stochastique optiques de microscopie

L’objectif global de cette méthodologie est de donner les conditions expérimentales optimales de préparation des échantillons pour l’acquisition d’images et de la reconstruction afin d’effectuer des images dSTORM 2D bicolore des microtubules et des filaments intermédiaires dans les cellules fixes

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

The overall goal of this methodology is to create the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual-colored D-STORM imaging of microtubules and intermediate filaments in fixed cells. This method can help characterize the physical interactions between dense and complex cytoskeletal networks such as intermediate filaments and microtubules. The main advantage of this technique is that it allows the observation of cellular structures, which are not resolved with conventional fluorescence microscopy.Visual demonstration of this method is critical as the control of sample preparation and acquisition of robe-linking data is crucial in order to limit the generation of artifacts. After growing U373 cells according to the text protocol, fix the cells by first using PBS to wash them once. Then, remove the PBS and add one milliliter per well of preheated extraction fixation solution.Incubate the cells at room temperature for 60 seconds. Then remove the solution and add one milliliter per well of freshly prepared and preheated 4%PFA diluted in cytoskeleton buffer. Place the 12-well plate back into the incubator at 37 degrees Celsius for 10 minutes.Then remove the PFA and add three milliliters of PBS per well. Use PBS to wash the cells twice. Then remove the PBS and add freshly prepared 10 millimolars sodium borohydride diluted in PBS in order to quench the autofluorescence from the glutaraldehyde.Incubate the cells at room temperature for seven minutes and transfer the recycled sodium borohydride into a specific bin. Use PBS to wash the wells three times for five minutes each. Then remove the PSB and add 5%BSA in PBS as a blocking solution.After immunostaining samples and preparing solutions in buffers according to the text protocol, mount the coverslip containing the labeled cells on a magnetic sample holder. And add the imaging buffer to completely fill the chamber. Apply the lid and make sure that there are no air bubbles between the imaging buffer and the lid.Using absolute ethanol, clean the bottom of the sample and verify that there are no leaks. Then if necessary, use grease to properly seal the sample hold. To carry out imaging, switch on the microscope and the acquisition software.Set up the acquisition parameters according to the text protocol, using the 100-fold NA 1.46 objective, the 1.6-fold Optivar lens, and the TIRF UHP mode. Create a 647 track to image the vimentin filaments labeled with Alexa Fluor 647. Select the 405 and 642 nanometer laser lines and the LP 655 emission filter.Then, create a 555 track to image the microtubules labeled with Alexa Fluor 555. Select the 561, 488, and 405 nanometer laser lines to image the fluorophores and the BP 570 to 650 emission filter. Next, add oil to the objective and place the sample on the lens.Then, select the locate tab and open the shutter of the fluorescence lamp. Find the focus and use the eyepiece to look for the cells to image. Then use the joystick to position the cells in the center of the field.Click on the acquisition tab to return to acquisition mode. Then select the 647 track and set the 642 laser power to 0.2%and the 405 laser power to its minimal value. Set the exposure time to 50 milliseconds and the gain to 50.Then choose continuous acquisition mode to observe the cell on the screen. Make sure that there is at least one fluorescent bead in the field of view. Use an autoscale mode for the display and adjust the focus.Next, take a wide field epifluorescence image of the vimentin network by clicking on snap and save the image. To check TIRF illumination, click on TIRF, press continuous, adjust the acquisition parameters in order to have a homogenous field of illumination. Then press snap and save the image.Check the 555 track. Uncheck the 647 track, and select the 555 track. Take an epifluorescence image of the microtubule network and save it.Then take a TIRF image and save it. Now, check the 647 track. Uncheck the 555 track, and select the 647 track.Set the gain to zero and the exposure time to 10 milliseconds. Then press continuous. Set the 642 nanometer laser power to 100%in order to pump most of the Alexa 647 dyes to the dark state.Once the fluorophores start blinking, increase the exposure to 15 milliseconds and the gain to 300. Use the range indicator mode to verify that single molecule signals do not saturate the camera as indicated by the red color. In that case, decrease the gain until the saturation disappears.The beads will remain saturated at the beginning of the acquisition. Press stop to discontinue observation of the cell. Expand the online processing tool and click online processing PALM to visualize the STORM image during acquisition.Use nine for the peak mask size and six for the peak intensity to noise. Now, press start acquisition. During acquisition, refocus on the cell if necessary.Adjust the exposure time and eventually switch on the 405 nanometer laser line starting at the 0.001%to keep a medium density of fluorophores with a minimum distance of one micrometer between single molecules. Make sure that the peak of localization precision on the histogram below the reconstructed image is centered around or below 10 nanometers. Press stop to end the movie when more than one million localizations have been reached.Save the raw data from the STORM acquisition with the CZI format. Then in the channels tool, check track 555, uncheck track 647, and select track 555. Next, press the continuous button.Set the exposure time to 30 milliseconds and the gain to zero. Then set both 488 and 561 lasers to 100%to pump Alexa 555 dyes to the dark state. Use the HILO mode of illumination.Once the fluorophores are depleted, press stop. Set the gain to 250 and decrease the 488 laser to 50%Use the range indicator to check that single molecules do not saturate the camera. If that is the case, decrease the gain.Now, press start acquisition. During acquisition, increase the gain gradually to reach the limit of saturation. Increase step-by-step the 405 nanometer laser power to activate the molecules and reach a medium density in the field of view.Decrease the 488 nanometer laser power to 30%when the 405 laser is about 15%Then decrease gradually the 488 while increasing the 405 laser power in order to keep the blinking of the molecules as fast as possible and with a medium density. The 488 laser line coupled with the 561 excitation laser at maximal intensity increases both the on and off rates of the fluorophores, whereas the 405 laser line only increases the on rate. Stop the acquisition when 500, 000 localizations or more have been reached or when no more molecules are blinking.Then save the raw data from the STORM acquisition with the CZI format. To carry out image reconstruction using the PAL-Drift tool, choose the model-based correction type with automatic segments with a maximum size of eight. Press apply multiple times in a row until the drift is corrected.This model-based correction applies an algorithm based on cross-correlation analysis, which corrects the drift. Using the PAL filter tools, improve the appearance of the STORM image by selecting only the molecules which were best localized. For example, limit the localization precision to 30 nanometers and the PSF to 120 to 180 nanometers.With the PAL-Render tool, use a pixel resolution of five or 10 nanometers and parameters according to the text protocol. Select render auto dynamic range HR with a 95%value and render auto dynamic range SWF with a 90%value. Then select render best quality.In the PALM menu of the processing tab, select PALM convert. Press select and then apply. Finally, save the created image with the format LSM and not CZI.This figure shows a typical example of a D-STORM image of vimentin filaments immuno-labeled with Alexa 647. The increase in resolution can clearly be observed upon comparison with the image acquired with a standard, wide-field microscope. The fluorescence intensity profiles presented in this graph show that super resolution microscopy imaging allows the resolution of vimentin bundles.The D-STORM resolution is sufficient to count the number of filaments present in the bundles. Shown here is a typical example of the dual-colored D-STORM imaging of vimentin and microtubule networks immuno-labeled with Alexa 647 and Alexa 555, respectively. Good vimentin images should have at least 5, 000 localizations per micrometer squared and 2, 500 per micrometer squared for microtubules.Once mastered, this technique can be done in three days if it performed properly. While attempting this procedure, it is important to adapt the methods of fixation and parametrization to the protein that would be imaged. For example, fixation with only glutaraldehyde gives poor results for intermediate filaments, but is best for microtubule fixation.It is important to remember to use freshly prepared samples and solutions, especially MEA, whose pH has to be carefully adjusted. After its development, this technique paved the way for researchers in the field of cell biology to explore the morphology of nanoscopic cellular structures. After watching this video, you should have a good understanding of how to perform sample preparation, image acquisition, and reconstruction of 2D dual-colored D-STORM images of intermediate filaments and microtubules.Don't forget that working with some solutions, especially PFA, NABH4 can be extremely hazardous and precautions such as wearing gloves, working under the chemical hood, and using specific bins should always been taken while performing this procedure.

imaging-intermediate-filaments-microtubules-with-2-dimensional-direct

Research

JoVE Journal

Biology

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Étiquetage des cellules souches avec des colorants fluorescents pour les non-invasive de détection avec l&#39;imagerie optique

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Recherche

Biologie

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Entretien et nettoyage du microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

Principales composantes d&#39;un microscope optique

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Contraste de phase et les interférences de contraste différentiel (DIC) Microscopie

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

L&#39;exocytose imagerie dans les cellules bipolaires rétine par microscopie TIRF

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

Imagerie directe du calcium ER avec Targeted-estérase colorant induit Chargement (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

Imagerie et de reconstruction 3D de structures vasculaires cérébraux embryonnaires poisson zèbre

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo suivi 4-Dimensional de hématopoïétiques cellules souches et progénitrices adultes dans la voûte du crâne de souris de moelle osseuse

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and &#62; 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

Trois dimensions Super Resolution Microscopie de Filaments F-actine par interférométrique photoactive Localisation Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

Drosophila Préparation et imagerie longitudinale de la fonction cardiaque in vivo en utilisant cohérence optique Microscopie (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...