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

We thank Mickael Lelek, Orestis Faklaris and Nicolas Bourg for fruitful discussion, Andrey Aristov and Elena Rensen for help with the super-resolution technique and Shailaja Seetharaman for careful reading of the manuscript. We gratefully acknowledge the UtechS Photonic BioImaging (Imagopole) Citech of Institut Pasteur (Paris, France) as well as the France-BioImaging infrastructure network supported by the French National Research Agency (ANR-10-INSB-04; Investments for the Future), and the R&#233;gion Ile-de-France (program Domaine d'Int&#233;r&#234;t Majeur-Malinf) for the use of the Elyra microscope. This work was supported by the the Ligue Contre le Cancer and the French National Research Agency (ANR-16-CE13-0019).

1. Coverslips preparation (Day 1, 30 min)
<ol>
	<li>Place the 18-mm n&#186;1.5H (170 &#181;m +/- 5&#181;m) glass coverslips on a plastic rack.</li>
	<li>Put the rack in a 100-mL beaker filled with acetone and soak for 5 min.</li>
	<li>Transfer the rack to a 100-mL beaker filled with absolute ethanol and soak for 5 min.</li>
	<li>Transfer the rack to a new 100 mL beaker filled with absolute ethanol and place the beaker in an ultrasonic cleaner device (see Table of materials) at room temperature. Press &#34;on&#34; and w...

<ol>
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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. 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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. 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Critical steps in the protocol
We present here a protocol which minimizes artifacts in the dSTORM images of microtubules and IFs in glial cells. Artifacts can be created at every step of the sample preparation and imaging: fixation, blocking, immunolabeling, drift during acquisition, non-optimal blinking conditions23. We list below the most critical steps.
Cleaning the coverslips is an important step to limit the non-specific...

Cytoplasmic intermediate filaments (IFs) are 10 nm-diameter homo- or heteropolymers of a cell type specific subset of IF proteins. IFs participate in a large range of cellular functions such as cell motility, proliferation and stress responses. Their key role is highlighted by the fact that more than 90 human diseases are directly caused by mutations in IF proteins; for instance, changes in IF composition accompanies tumour growth and spreading1,2,3. There is growing evidence that the three cytoskeleton systems work in collaboration to control cellular functions such as cell polarization, division and migration2,3. Since there is a tight coupling between spatial architecture and functions, it is crucial to get insights into the structural organization of the IF with the other filaments and better understand the cytoskeletal cross-talk. this article provides a protocol to perform the super-resolution technique dual-color dSTORM (direct STochastic Optical Reconstruction Microscopy)4 and how it is used to investigate the interaction between IF and microtubules in fixed, cultured cells in 2 dimensions (2D).
Several super-resolution techniques have been developed over the past decade, and there discoveries were at the origin of the Nobel Prize in Chemistry in 20145. Among all these techniques lies the single molecule localization-based methods like PALM6 (Photo-Activated Localization Microscopy) which uses photoswitchable fluorescent proteins, STORM7 with pairs of fluorophores or dSTORM4 with conventional fluorophores. All these methods are based on the same principle which consists of (i) the switch of most of the fluorescent reporters into an &#34;off&#34; state&#34; (non-fluorescent), (ii) the stochastic activation of a subset of them into a fluorescent state in order to localize their spatial position with nanometer accuracy, and (iii) the repetition of this process in order to activate as many subsets of fluorophores as possible. A final image is reconstructed using all the localizations of the activated molecules, providing a lateral resolution down to ~20-40 nm. Several commercialized optical systems allowing PALM/STORM are now available for biologists for routine experiments. Here, one such system was used to study the structural association of microtubules and IFs. Among all the single-molecule localization-based methods, the dual-color dSTORM technique was selected, because it is well suited to observe very thin lamellar regions (&#60;1 &#181;m) of adherent cells and can provide significant improvement of image resolution with minimal time and money investment. Indeed, dSTORM is a very convenient and versatile technique that is compatible with the standard organic dyes routinely used for cellular staining and immunofluorescence.
While one color dSTORM images are relatively simple to obtain using the fluorophore Alexa6478, dual color dSTORM requires to optimize the experimental conditions so that two dyes can blink properly in the chosen buffer, especially when there is limited laser power. Excellent papers are already available on the methods to set up multi-color imaging with dSTORM9,10,11,12,13, and these papers explain in detail the possible sources of artifacts and degraded image resolution as well as how to overcome them. In this article, the optimal experimental conditions in terms of cells fixation, immunostaining, sample preparation, imaging acquisition and image reconstruction are described in order to acquire images of dense cytoplasmic IF networks and microtubules in glial cells with dual-color dSTORM. Briefly, the extraction/fixation method described in Chazeau et al12 was adapted to glial cells and used with a post-fixation step, optimized antibodies concentration, a STORM buffer with 10 mM MEA described in Dempsey9 et al. which was found to be optimal for the experimental set up and sample type.
Glial cells mainly express three types of IF proteins: vimentin, nestin and GFAP (glial acidic fibrillary protein). These three proteins were shown to co-polymerize in astrocytes14. We previously showed using super-resolution structured illumination microscopy (SR SIM) that these three IF proteins can be found in the same single IF filament and that they display similar distribution and dynamics in glial cells 15. Due to the similarities between the three IF proteins, vimentin staining was used as a reporter for the whole IF network. Using dSTORM, we managed to resolve how IF form bundles along microtubules, which was not possible with diffraction limited microscopy techniques15. These observations may help to understand how vimentin IFs can template microtubules and regulate their growth trajectory, promoting the maintenance of a polarity axis during cell migration16. Super-resolution images provided key information on the mutual interaction of the two cytoskeletal subsystems, and brought insight into the link between spatial association and local function which could be cell-type specific17. In general, dual-color dSTORM can be used to study the crosstalk between cytoskeletal elements or other types of organelles, provided that good immunostaining conditions are reached in terms of density and specificity. This technique will be useful to better characterize the cytoskeleton changes observed during astrogliosis and in glioblastoma, the most common and most malignant tumor of the central nervous system, where the expression of IF proteins is altered 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

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

A microscope equipped with 50 mW 405 nm and 100 mW 488, 561 and 642 nm solid-state lasers, an EMCCD 512x512 camera, an alpha Plan Apo 100X/1.46 objective and Band Pass 570-650 / Long Pass 655 emission filters was used for the representative results presented below.
Figure 1A gives an example of molecule density and signal to noise ratio that should be used during raw image acquisition. Good quality ...

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

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

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

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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 ...

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 ...

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 ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

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 ...

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 ...

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

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 ...

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 ...

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

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) ...

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

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 ...

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

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 ...