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, ...

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.

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