We thank Pierre G&#246;nczy and the BioImaging &amp; Optics Platform (BIOP) at &#201;cole Polytechnique F&#233;d&#233;rale de Lausanne (EPFL), Lausanne, Switzerland, where the SIM images of the centrioles were acquired. Nikolai Klena and Davide Gambarotto are supported by the European Research Council (ERC) Starting Grant (StG) 715289 (ACCENT) and Maeva Le Guennec, Paul Guichard, and Virginie Hamel by the Swiss National Science Foundation (SNSF) PP00P3_157517. Susanne Borgers is supported by the University of Geneva.

<ol>
	<li>Bornens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+centrosome+in+cells+and+organisms.">The centrosome in cells and organisms.</a> Science. 335 (6067), 422-426 (2012).</li><li>Nigg, E. A., Holland, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Once+and+only+once:+mechanisms+of+centriole+duplication+and+their+deregulation+in+disease.">Once and only once: mechanisms of centriole duplication and their deregulation in disease.</a> Nature Reviews Molecular Cell Biology. 19 (5), 297-312 (2018).</li><li>Nigg, E. A., Raff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrioles,+centrosomes,+and+cilia+in+health+and+disease.">Centrioles, centrosomes, and cilia in health and disease.</a> Cell. 139 (4), 663-678 (2009).</li><li>G&#246;nczy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+molecular+architecture+of+centriole+assembly.">Towards a molecular architecture of centriole assembly.</a> Nature Reviews Molecular Cell Biology. 13 (7), 425-435 (2012).</li><li>Paintrand, M., Moudjou, M., Delacroix, H., Bornens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome+organization+and+centriole+architecture:+their+sensitivity+to+divalent+cations.">Centrosome organization and centriole architecture: their sensitivity to divalent cations.</a> Journal of Structural Biology. 108 (2), 107-128 (1992).</li><li>Winey, M., O'Toole, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centriole+structure.">Centriole structure.</a> Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 369 (1650), (2014).</li><li>Dippell, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+basal+bodies+in+paramecium.">The development of basal bodies in paramecium.</a> Proceedings of the National Academy of Sciences of the United States of America. 61 (2), 461 (1968).</li><li>Allen, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphogenesis+of+basal+bodies+and+accessory+structures+of+the+cortex+of+the+ciliated+protozoan+Tetrahymena+pyriformis.">The morphogenesis of basal bodies and accessory structures of the cortex of the ciliated protozoan Tetrahymena pyriformis.</a> The Journal of Cell Biology. 40 (3), 716-733 (1969).</li><li>Li, S., Fernandez, J. -. J., Marshall, W. F., Agard, D. A. <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+structure+of+basal+body+triplet+revealed+by+electron+cryo-tomography.">Three-dimensional structure of basal body triplet revealed by electron cryo-tomography.</a> The EMBO Journal. 31 (3), 552-562 (2012).</li><li>Guichard, P., Chr&#233;tien, D., Marco, S., Tassin, A. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procentriole+assembly+revealed+by+cryo-electron+tomography.">Procentriole assembly revealed by cryo-electron tomography.</a> The EMBO Journal. 29 (9), 1565-1572 (2010).</li><li>Guichard, 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=Native+architecture+of+the+centriole+proximal+region+reveals+features+underlying+its+9-fold+radial+symmetry.">Native architecture of the centriole proximal region reveals features underlying its 9-fold radial symmetry.</a> Current Biology. 23 (17), 1620-1628 (2013).</li><li>Hirono, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cartwheel+assembly.">Cartwheel assembly.</a> Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 369 (1650), (2014).</li><li>Bornens, M., Paintrand, M., Berges, J., Marty, M. C., Karsenti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+chemical+characterization+of+isolated+centrosomes.">Structural and chemical characterization of isolated centrosomes.</a> Cell Motility and the Cytoskeleton. 8 (3), 238-249 (1987).</li><li>Keller, L. C., Romijn, E. P., Zamora, I., Yates, J. R., Marshall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+isolated+chlamydomonas+centrioles+reveals+orthologs+of+ciliary-disease+genes.">Proteomic analysis of isolated chlamydomonas centrioles reveals orthologs of ciliary-disease genes.</a> Current Biology. 15 (12), 1090-1098 (2005).</li><li>Kilburn, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Tetrahymena+basal+body+protein+components+identify+basal+body+domain+structure.">New Tetrahymena basal body protein components identify basal body domain structure.</a> The Journal of Cell Biology. 178 (6), 905-912 (2007).</li><li>Bauer, M., Cubizolles, F., Schmidt, A., Nigg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Quantitative+analysis+of+human+centrosome+architecture+by+targeted+proteomics+and+fluorescence+imaging.">A Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging.</a> The EMBO Journal. 35 (19), 1-15 (2016).</li><li>Hamel, V., 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=Identification+of+Chlamydomonas+Central+Core+Centriolar+Proteins+Reveals+a+Role+for+Human+WDR90+in+Ciliogenesis.">Identification of Chlamydomonas Central Core Centriolar Proteins Reveals a Role for Human WDR90 in Ciliogenesis.</a> Current Biology. 27 (16), 2486-2498 (2017).</li><li>Cavalier-Smith, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=basal+body+and+flagellar+development+during+the+vegetative+cell+cycle+and+the+sexual+cycle+of+Chlamydomonas+reinhardii.">basal body and flagellar development during the vegetative cell cycle and the sexual cycle of Chlamydomonas reinhardii.</a> Journal of Cell Science. 16 (3), 529 (1974).</li><li>Geimer, S., Melkonian, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ultrastructure+of+the+Chlamydomonas+reinhardtii+basal+apparatus:+identification+of+an+early+marker+of+radial+asymmetry+inherent+in+the+basal+body.">The ultrastructure of the Chlamydomonas reinhardtii basal apparatus: identification of an early marker of radial asymmetry inherent in the basal body.</a> Journal of Cell Science. 117, 2663-2674 (2004).</li><li>Nakazawa, Y., Hiraki, M., Kamiya, R., Hirono, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SAS-6+is+a+cartwheel+protein+that+establishes+the+9-fold+symmetry+of+the+centriole.">SAS-6 is a cartwheel protein that establishes the 9-fold symmetry of the centriole.</a> Current Biology. 17 (24), 2169-2174 (2007).</li><li>Hamel, V., 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=Correlative+multicolor+3D+SIM+and+STORM+microscopy.">Correlative multicolor 3D SIM and STORM microscopy.</a> Biomedical Optics Express. 5 (10), 3326 (2014).</li><li>Guichard, 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=Cell-free+reconstitution+reveals+centriole+cartwheel+assembly+mechanisms.">Cell-free reconstitution reveals centriole cartwheel assembly mechanisms.</a> Nature Communications. 8, 14813 (2017).</li><li>Hiraki, M., Nakazawa, Y., Kamiya, R., Hirono, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bld10p+constitutes+the+cartwheel-spoke+tip+and+stabilizes+the+9-fold+symmetry+of+the+centriole.">Bld10p constitutes the cartwheel-spoke tip and stabilizes the 9-fold symmetry of the centriole.</a> Current Biology. 17 (20), 1778-1783 (2007).</li><li>Sahl, S. J., Hell, S. W., Jakobs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+nanoscopy+in+cell+biology.">Fluorescence nanoscopy in cell biology.</a> Nature Reviews Molecular Cell Biology. 18 (11), 685-701 (2017).</li><li>Hutner, S. H., Provasoli, L., Schatz, A., Haskins, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+Approaches+to+the+Study+of+the+Role+of+Metals+in+the+Metabolism+of+Microorganisms.">Some Approaches to the Study of the Role of Metals in the Metabolism of Microorganisms.</a> Proceedings of the American Philosophical Society. 94 (2), 152-170 (1950).</li><li>Keller, L. C., Marshall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+proteomic+analysis+of+chlamydomonas+centrioles.">Isolation and proteomic analysis of chlamydomonas centrioles.</a> Methods in Molecular Biology. 432 (1), 289-300 (2008).</li><li>Borlinghaus, R. T., Kappel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HyVolution-the+smart+path+to+confocal+super-resolution.">HyVolution-the smart path to confocal super-resolution.</a> Nature Methods. 13 (3), (2016).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>de la Rosa-Trev&#237;n, J. 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=Scipion:+A+software+framework+toward+integration,+reproducibility+and+validation+in+3D+electron+microscopy.">Scipion: A software framework toward integration, reproducibility and validation in 3D electron microscopy.</a> Journal of Structural Biology. 195 (1), 93-99 (2016).</li><li>Gartenmann, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combined+3D-SIM/SMLM+approach+allows+centriole+proteins+to+be+localized+with+a+precision+of+&#8764;4-5+nm.">A combined 3D-SIM/SMLM approach allows centriole proteins to be localized with a precision of &#8764;4-5 nm.</a> Current Biology. 27 (19), 1054-1055 (2017).</li><li>Jaenicke, L., Kuhne, W., Spessert, R., Wahle, U., Waffenschmidt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-wall+lytic+enzymes+(autolysins)+of+Chlamydomonas+reinhardtii+are+(hydroxy)proline-specific+proteases.">Cell-wall lytic enzymes (autolysins) of Chlamydomonas reinhardtii are (hydroxy)proline-specific proteases.</a> European Journal of Biochemistry. 170 (1-2), 485-491 (1987).</li><li>Salas, D., 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=Angular+reconstitution-based+3D+reconstructions+of+nanomolecular+structures+from+superresolution+light-microscopy+images.">Angular reconstitution-based 3D reconstructions of nanomolecular structures from superresolution light-microscopy images.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (35), 9273-9278 (2017).</li></ol>

The authors have nothing to disclose.

One of the challenges in biology is to decipher the precise localization of proteins in an architectural context. The centriole is an ideal structure to apply these methods, as its architecture has been studied using cryo-electron tomography, revealing interesting ultrastructural features along its length. However, due to its dimensions close to the resolution limit in optical microscopy, it is difficult to precisely localize a protein by immunofluorescence to a structural sub-region of the centriole using conventional microscopes30.
The resolution in optical microscopy is limited by the diffraction of light that gives, roughly, a lateral maximum resolution of 200 nm in optical microscopy24. However, this limit has been by-passed by one of the major breakthroughs of the last 20 years in optical microscopy: the invention of super-resolution methods. These approaches can image beyond the diffraction limits at different resolutions: 120 nm for SIM, about 50 nm for stimulated emission depletion (STED), and 20&#8211;40 nm for single-molecule localization microscopy (SMLM)24. With these new developments of super-resolution microscopy, the structural sub-regions of the centriole are attainable. However, in practice, it is still difficult to accurately determine the localization of a protein to a structural element for the main reason that the mature centrioles exist in only 2 copies per cell and have random orientations, which makes the interpretation of localization difficult. For this reason, a protocol was set up that allows researchers to image by super-resolution a large number of centrioles, increasing the chance to observe non-ambiguous orientations. Importantly, as this method relies on the use of isolated centrioles, we are providing a method to purify intact C. reinhardtii centrioles that contain mature centrioles and procentrioles.
Finally, owing to the range of centriole orientations that can be imaged with this protocol, single-particle analysis can be applied using electron microscopy software. This results in the generation of average classes of centrioles in a particular orientation. Importantly, these resulting 2-D images can then be used to assess the localization of a specific protein along the centriole. Indeed, this method can be applied to dual-color super-resolution images, and one color can be used to reveal the centriole skeleton (e.g., tubulin), while the other color can be attributed to a specific centriolar protein. By subtracting the averages obtained with one color or two colors, it becomes easier to register a protein along the centriole (proximal, central, or distal). Note that the two channels should be accurately aligned to prevent any misleading interpretations. Moreover, averages of top views will help decipher if a protein localizes inside the centriolar lumen, along the microtubule wall, or outside the centriole.
This method has the advantage to ascertain the localization of specific proteins that could be difficult to localize otherwise due to heterogeneous labeling. Note that other methods to map proteins within the centrioles have been described in correlative 3-D SIM/SMLM with, for instance, assessing the specific orientations of the centrioles by determining the elliptical profile of a marker forming a torus around the centriole by SIM imaging. Using this parameter, it is possible to localize protein with a precision of 4&#8211;5 nm30. Note also that the method described here uses isolated centrioles with intact procentrioles, a sign that the centriole architecture is most likely largely conserved. However, we cannot exclude that some architectural features are disturbed during purification, such as the centriole diameter varying with the concentration of divalent cations as amplified with the isolation of the human centrosome5.
One of the critical steps of the protocol presented here is to obtain sufficiently concentrated isolated centrioles in different orientations amenable to Fluo-SPA. To do so, first ensure the purity and the efficiency of the centriole isolation procedure. A low concentration of isolated centrioles will prevent proper imaging and further image processing. For this purpose, we are providing a method to enrich the number of centrioles per field of view. Depending on the number of centrioles in the fraction used, the volume loaded in the concentrator should be adjusted, with a maximum volume of 250 &#181;L.
Importantly, this method has been developed for a cell wall minus cw15- C. reinhardtii cells. In this strain, the fragility of the cell wall allows for a proper lysis of the cells and, thus, the liberation of its content. This protocol is not efficient for wild-type C. reinhardtii cells, as the cell wall prevents a proper lysis. Alternative strategies such as sonication or a pre-incubation of the cells with autolysin, an enzyme that can degrade the cell wall31, would have to be put in place to alter the cell wall prior to applying the isolation protocol presented here.
This set-up can be used with different types of microscopes, ranging from conventional confocal microscopes to high throughput dedicated super-resolution microscopes. Note that when doing SMLM, a special buffer is required for proper imaging and, thus, a chamber adapted for a 12 mm coverslip with the buffer on top of the coverslip should be used. Subsequent imaging will be performed with inverted microscopes. If the microscope set-up does not allow a 12 mm coverslip, the protocol presented here can be applied to 18 mm coverslips using a 30 mL round-bottom tube and a modified adaptor and concentrator. It is also important to note that the quality of the final reconstruction in SMLM will depend on the quality of the staining and of the primary antibody used, as well as the method of fixation.
In summary, we are providing a method that can be applied to image numerous centrioles followed by Fluo-SPA that will generate averages of centrioles in different orientations, thus helping to localize centriolar protein with precision. Importantly, this method can be applied more generally to isolated centrioles from other species, to other organelles, or to large macromolecular assemblies. Finally, the sample preparation approach presented here, combined with recent algorithm development for single-particle analysis of fluorescent SMLM data32, could open further improvement in the molecular cartography of large macromolecular assemblies.

The centriole is an evolutionarily conserved organelle that lies at the core of the centrosome in animal cells and can act as a basal body (referred to as centrioles hereafter) to template cilia or flagella in many eukaryotes1,2. As such, centrioles are critical for fundamental cell biological processes ranging from spindle assembly to cell signaling. Therefore, defects in centriole assembly or function have been associated with several human pathologies including ciliopathies and cancers3.
Centrioles are nine-fold, symmetrical, microtubule triplet-based cylindrical structures that are, typically, ~450 nm long and ~250 nm wide4,5,6,7. Conventional electron microscopy and cryo-electron tomography of centrioles from different species have revealed that centrioles are polarized along their long axis with three distinct regions: a proximal region, a central core, and a distal region5,7,8,9,10,11. Importantly, each of these regions displays specific structural features. First, the lumen of the 100 nm-long proximal region contains the cartwheel structure connected to the microtubule triplet through the pinhead element12. Second, the 300&#8211;400 nm-long central region contains fibrous densities in the lumen and structural features along the inner face of the microtubules: the Y-shaped linker, the C-tubule tail, and the A-tubule stub9. Finally, the 50&#8211;100 nm distal region exhibits sub-distal and distal appendages that surround the distal part of the centriole5,13.
The last two decades have been marked by the discovery of an increasing number of centriolar proteins, leading to a current estimation of about 100 distinct proteins being part of the centriole14,15,16,17. Despite these advances, the precise localization of these proteins within the centriole remains elusive, particularly within structural sub-regions. Importantly, assigning a precise localization to structural regions of the centriole is crucial for a better understanding of their function. In this respect, C. reinhardtii centrioles have been instrumental in both aspects by first delimitating the different structural features along the cylinder9,18,19, which then allowed researchers to correlate the localization of a subset of proteins using fluorescent microscopy to a sub-structural region. This includes, for example, the proteins Bld12p and Bld10p, which localize in the proximal region, and in the cartwheel structure in particular20,21,22,23. The list of substructure localized proteins also includes POB15 and POC16, two novel proteins identified by mass spectrometry that decorate the inner central core region of C. reinhardtii centrioles17.
This paper provides a complete description of the method developed to isolate and image C. reinhardtii centrioles for subsequent super-resolution microscopy and single-particle averaging. To achieve this goal, it is important to delineate the technical limitations that need to be overcome. First, centriole purification can affect the overall architecture, with the cartwheel structure often being lost during the various steps of isolation9. Secondly, the dimensions of the centriole are very close to the diffraction limit in optical microscopy. Indeed, the lateral resolution that can be obtained in confocal microscopy is around 200 nm24, similar to the diameter of the centriole, and the resolution in the z-axis is about 2&#8211;3x lower, leading to an anisotropic volume. Thirdly, the heterogeneity of antibody labeling and centriole orientation could limit the interpretation needed to localize a protein in a specific centriolar sub-region. Finally, centrioles exist in only two copies per cell, making it difficult to acquire a large number of images and find an unambiguous centriole orientation. To circumvent these technical issues, we developed a method that relies on applying super-resolution microscopy on large numbers of isolated centrioles that adopt various orientations. We will first describe a protocol to purify C. reinhardtii centrioles that enables the purification of structurally intact centrioles and procentrioles containing the cartwheel. Then, we will describe a step-by-step protocol to concentrate the centrioles on coverslips for imaging by conventional or super-resolution fluorescent microscopy. This important step allows for increasing the number of centrioles imaged in multiple orientations. Finally, we will describe a procedure to perform single-particle averaging on data acquired on fluorescent microscopes that facilitates the detection of centrioles in different orientations. Altogether, this method can be applied to image centrioles from various species or other large macromolecular assemblies.

<table border="0" cellpadding="0" cellspacing="0" style="width:964px;" width="962">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:325px;">Mouse monoclonal anti-apha tubulin (clone DM1A)</td>
			<td style="width:155px;">Abcam</td>
			<td style="width:171px;">ab7291&#160;</td>
			<td style="width:313px;">dilution 1:300</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DNaseI</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">12 mm coverslips</td>
			<td>Roth</td>
			<td>YX03.1</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">18 mm coverslips</td>
			<td>Roth</td>
			<td>LH23.1</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">K2HPO4</td>
			<td>Fluka</td>
			<td>60355</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">KH2PO4</td>
			<td>Fluka</td>
			<td>60230</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tris base</td>
			<td>Biosolve Chimie SARL</td>
			<td>0020092391BS</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">acetic acid</td>
			<td>Carlo Erba Reagents</td>
			<td>524520</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NH4Cl</td>
			<td>Sigma</td>
			<td>A-4514</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CaCl2</td>
			<td>Sigma</td>
			<td>C-7902</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MgSO4</td>
			<td>Sigma</td>
			<td>63140-500G-F</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">steritop filter</td>
			<td>Millipore</td>
			<td>SCGPT05RE</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">sucrose</td>
			<td>Sigma</td>
			<td>S7903-1KG</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES</td>
			<td>AppliChem PanReac</td>
			<td>A3724,0250</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PIPES</td>
			<td>Sigma</td>
			<td>P6757-500G</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MgCl2</td>
			<td>ACROS ORGANICS</td>
			<td>197530010</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NP-40</td>
			<td>AppliChem PanReac</td>
			<td>A1694,0250</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Round-bottom (Kimble) tubes 15 mL</td>
			<td>Fisherscientific</td>
			<td>09-500-34</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Round-bottom (Kimble) tubes 35 mL</td>
			<td>Fisherscientific</td>
			<td>09-500-37</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">cover glass staining rack</td>
			<td>Thomas scientific</td>
			<td>8542E40</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">crystal polystyrene transmission lab box</td>
			<td>FISHERS</td>
			<td>11712944</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Methanol</td>
			<td>VWR</td>
			<td>20864.32</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BSA</td>
			<td>Roche</td>
			<td>10735086001</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Triton X100</td>
			<td>Roth</td>
			<td>3051.3</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">goat anti-mouse coupled to Alexa 488&#160;</td>
			<td>invitrogen</td>
			<td>A11029</td>
			<td style="width:313px;">dilution 1:1000</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">goat anti-rabbit coupled to Alexa 568</td>
			<td>invitrogen</td>
			<td>A11036</td>
			<td style="width:313px;">dilution 1:1000</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">mounting medium</td>
			<td>abcam</td>
			<td>ab188804</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tube, thinwall polypropylene</td>
			<td>Beckman Coulter</td>
			<td>326823</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Poly-D-Lysine 1 mg/mL</td>
			<td>SIGMA&#160;</td>
			<td>A-003-E</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Mouse monoclonal anti-Polyglutamylation modification mAb (GT335)</td>
			<td>Adipogen</td>
			<td>AG-20B-0020</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:325px;">glycerol mounting medium with DAPI and DABCO</td>
			<td style="width:155px;">Abcam</td>
			<td style="width:171px;">ab188804</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:325px;">50 mL conical tubes</td>
			<td style="width:155px;">Falcon</td>
			<td>14-432-22</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:325px;">Eppendorf 5810R centrifuge</td>
			<td style="width:155px;">Eppendorf</td>
			<td style="width:171px;">5811000622</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:325px;">Beckman JS-13.1 swinging bucket rotor</td>
			<td style="width:155px;">Beckman Coulter</td>
			<td>346963</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:325px;">Beckman SW 32Ti rotor</td>
			<td style="width:155px;">Beckman Coulter</td>
			<td>369694</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:325px;">parafilm</td>
			<td style="width:155px;">Bemis&#160;</td>
			<td>13-374-10</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:325px;">Leica TCS SP8 with Hyvolution mode</td>
			<td style="width:155px;">Leica&#160;</td>
			<td style="width:171px;"></td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:325px;">OSRAM L18W/954 LUMILUX&#160;</td>
			<td style="width:155px;">Luxe Daylight/ OSRAM</td>
			<td style="width:171px;"></td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:325px;">Whatman filter paper</td>
			<td style="width:155px;">Sigma&#160;</td>
			<td>WHA1001325</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:325px;">CrSAS-6/Bld12 antibody</td>
			<td style="width:155px;"></td>
			<td style="width:171px;"></td>
			<td style="width:313px;">dilution 1:300 (Hamel el al, 2014)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:325px;">Scipion</td>
			<td style="width:155px;">http://scipion.i2pc.es/</td>
			<td style="width:171px;"></td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:325px;">EM CCD camera (Andor iXON DU897)</td>
			<td style="width:155px;">Andor</td>
			<td style="width:171px;"></td>
			<td style="width:313px;"></td>
		</tr>
	</tbody>
</table>

isolation and fluorescence imaging for single-particle reconstruction of chlamydomonas centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell motility, or cell signaling. The green algae Chlamydomonas reinhardtii has proven to be an insightful model in the study of centriole architecture, function, and protein composition. Despite great advances toward understanding centriolar architecture, one of the current challenges is to determine the precise localization of centriolar components within structural regions of the centriole in order to better understand their role in centriole biogenesis. A major limitation lies in the resolution of fluorescence microscopy, which complicates the interpretation of protein localization in this organelle with dimensions close to the diffraction limit. To tackle this question, we are providing a method to purify and image a large number of C. reinhardtii centrioles with different orientations using super-resolution microscopy. This technique allows further processing of data through fluorescent single-particle averaging (Fluo-SPA) owing to the large number of centrioles acquired. Fluo-SPA generates averages of stained C. reinhardtii centrioles in different orientations, thus facilitating the localization of distinct proteins in centriolar sub-regions. Importantly, this method can be applied to image centrioles from other species or other large macromolecular assemblies.

C. reinhardtii Centriole Isolation: 
To isolate centrioles, cw15- C. reinhardtii cells were grown in liquid culture for several days under light and subsequently pelleted by centrifugation at 600 x g for 10 min. Pelleted cells were washed 1x with PBS and resuspended in a deflagellation buffer prior to deflagellation by performing a pH shock using 0.5 M acetic acid to a final pH of 4.5&#8211;4.7 for 2 min (Figure 1A). An addition of 1 N KOH was used to restore pH to 7.0. To separate detached flagella from cell bodies, deflagellated cells were first centrifuged to remove the bulk of the flagella. The pellet was then washed 2x with PBS and resuspended in 30&#160;mL of PBS prior to loading it slowly on a 25%-sucrose cushion (Figure 1B). The tube was spun at 600 x g for 15 min at 4 &#176;C to remove most of the detached flagella. After centrifugation, the cell bodies were spread in the sucrose cushion (Figure 1C) and recovered by removing the approximately 30 mL of supernatant (until the red arrow in Figure 1C). The resulting 20 mL of washed cells were then resuspended in 20 mL of cold PBS and centrifuged at 600 x g for 10 min. Next, the supernatant was discarded, and the cells were completely resuspended in 10 mL of PBS. The cells were transferred to a 250&#160;mL bottle and the lysis buffer was added to the resuspended cells at once. DNase was added to the lysis and incubated for 1 h at 4 &#176;C. After a centrifugation step to remove cell debris (see the white pellet in Figure 1D), the supernatant was collected and carefully loaded onto a 2 mL 60%-sucrose cushion prior to centrifugation at 10,000 x g for 30 min at 4 &#176;C. Note that for 100 mL of lysis, 8 tubes of 15 mL were used to perform this step, corresponding to 12.5 mL of lysis buffer loaded on 2 mL of sucrose per tube. After centrifugation, most of the supernatant was removed up to 1 mL above the cushion. The 1 mL of remaining supernatant was then collected with the 2 mL cushion and then mixed and pooled to obtain a final volume of 24 mL. The pool was then loaded on a 40%-, 50%-, 70%-sucrose gradient and spun at 68,320 x g for 1 h and 15 min at 4 &#176;C. Finally, the isolated centrioles were collected by making a hole in the bottom part of the centrifugation tube using a needle and the drops were collected in 12 x 500 &#181;L fractions. Due to the high density of the different sucrose, the drop formed very slowly at the beginning (70% sucrose) and then more rapidly (40% sucrose).
Immunofluorescence of Isolated Centrioles 
To assess the quality of the isolation procedure, 10 &#181;L of each collected gradient fraction was then centrifuged onto a coverslip using a coverslip support adaptor (Figure&#160;2A-2D). Importantly, to remove safely the coverslip, a custom hooked device was designed (Figure 2B). Next, the coverslips were analyzed by immunofluorescence. Antibodies against CrSAS-6(Bld12p) were used in this study to indicate the presence of the cartwheel structure and &#945;-tubulin (DMA1) to highlight the centriolar wall. By counting the number of centrioles that were positive for both CrSAS-6 and &#945;-tubulin per field of view and then calculating the total number of centrioles per fractions, it was possible to determine which fractions were enriched for isolated centrioles (Figure&#160;2F-2H). Interestingly, 6 fractions were enriched for centrioles (Figure&#160;2F and 2G, fractions #1&#8211;6), with a peak for fraction #3, while the last fractions were not, indicating that the purification worked. Note that in this particular experiment, 95% of the total centrioles were positive for CrSAS-6 and &#945;-tubulin in fraction #3. This indicates that most isolated centrioles retained their cartwheels. If no enrichment of centrioles is observed in the first fractions, the isolation procedure did not work and should be repeated. Note that some flagellar pieces can be observed mostly in the fractions devoid of centrioles.
Next, to calculate the total number of centrioles per &#181;L, the number of centriole per field of view should be multiplied by the ratio as presented in Figure 2H. The resulting number should then be divided by the volume of the fraction used for the immunofluorescence to obtain the number of centrioles per &#181;L. In this particular isolation procedure, the most enriched fraction contained about 37 centrioles for an area of 0.00846 mm2 (with a field of view of 92 x 92 &#181;m2). The surface of the tube section was 7.5 mm in radius with a total surface of 176 mm2. The corresponding ratio was then 176/0.00846 = 20,803.8, so a total of 769,740 centrioles (37x20,803.8) in 10 &#181;L. Therefore, the number of centrioles in 1 &#181;L was 76,974.
Concentration of Isolated Centrioles on Coverslips: 
Increasing the number of centrioles per field increases the chance of detecting unambiguous centriole orientation, as well as increasing the chance of detecting similar orientations that can be used for further particle averaging procedures. As centrioles from the concentrated fractions are still sparse on the coverslip, we developed a centrifugation accessory to concentrate centrioles in the middle of the coverslip (Figure 3A) named a concentrator. Note that a .stl file with the precise measures for 3-D printing is provided with this manuscript.
First, one coverslip of 12 mm was mounted on the concentrator (Figure 3B). The adaptor was placed on top of the coverslip and the round-bottom tube was inverted and placed over the assembled adaptor and concentrator. The round-bottom tube was then gently inverted, thus allowing the loading of the sample (Protocol step 4.2). The centrioles were then centrifuged at 10,000 x g for 10 min at 4 &#176;C. After that, the centrioles were subjected to immunofluorescence and stained for CrSAS-6/Bld12p and &#945;-tubulin (Figure&#160;3C-3E). Importantly, without concentrator, about 30 centrioles were seen per field of view (Figure 3C), whereas 183 centrioles were seen per field of view when the concentrator was used (Figure&#160;3D-3F). Note that the centrioles covered only a disk of 4 mm in diameter in the middle of the coverslip. This result demonstrates that the concentration step works and allows a 6-fold enrichment of centrioles in a defined region of the coverslips, thus easing their detection and imaging.
Fluorescent Single-particle Averaging of Isolated C. reinhardtii Centrioles: 
Here, using SIM microscopy that can reach a resolution of about 120 nm, centrioles stained for monoglutamylated tubulin (GT335, Figure 4), a tubulin modification present in centriolar microtubules, were imaged24. C. reinhardtii centrioles are about 500 nm long, always in pairs, and often found with associated, newly duplicated probasal bodies (referred to as procentrioles hereafter) and striated microtubule-associated fibers19. Therefore, this final assembly was about 1 &#181;m large. For this reason, and in order to image the centrioles in their entirety, we recommend acquiring a Z-stack on the isolated centrioles.
Here, after the acquisition, a final image was generated by performing a maximum intensity projection using ImageJ28 (image/Stacks/Z project/max intensity projection, Figure 4B). From such images, a single-particle analysis using cryo-electron microscopy software was performed to make classes of centrioles with similar orientations, and then averaging was performed. To do so, the image color was first inverted so as to better visualize the objects (Figure 4C). Centrioles were picked manually in a box centered over each particle using the freely available Scipion software29 that integrates several electron microscopy software programs such as Xmipp3 (Figure 4D). Note that the size of the box has to be defined by the user. Here, boxes of 50 x 50 pixels for a pixel size of 31.84 nm were used. Next, all particles were extracted (Figure 4E) and aligned using Xmipp3 (Figure 4F). Next, a circular mask of 12 pixels in radius was applied to isolate each centriole from the centriolar-pair (Figure 4G). The particles were then classified, using Xmipp3, to generate several averages. Only homogenous class averages were kept, meaning that particles that diverge from the class averages were manually excluded. This step was repeated in order to generate a near-perfect average for each chosen orientation. After five iterations, two classes of averages were generated: a top view from 63 objects (Figure 4H) and a side view from 98 particles (Figure 4I) of monoglutamylated centrioles. The dimensions of the object were determined by measuring the distance between peaks of the intensity plot profile along the monoglutamylation signal.
Importantly, the length of the side-view class average is 260 nm with a diameter of 250 nm, comparable to the measured monoglutamylated tubulin signal that was shown to localize to a region of 286 &#177; 33 nm in length within the core of the centriole17.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58109/58109fig1.jpg" /> 
Figure 1:&#160;Purification of C. reinhardtii&#160;centrioles. (A) This is a schematic representation of each step leading to the isolation of C. reinhardtii centrioles. It includes representative steps of the protocol (B) before and (C) after centrifugation onto the 25%-sucrose gradient. The red arrow in panel C indicates the minimum volume to keep after centrifugation. (D) This panel shows the white pellet of lysed cells after centrifugation. The black arrow indicates the pellet. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58109/58109fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58109/58109fig2.jpg" /> 
Figure 2:&#160;Centrifugation set-up to perform immunofluorescence on isolated centrioles. (A) 10 &#181;L of each collected fraction is diluted first in 100 &#181;L of 10mM K-PIPES (pH 7.2). (B) This is a schematic representation of the centrifugation devices, encompassing a 15 mL round-bottom tube, a 12 mm coverslip, an adaptor for the coverslip, and a custom-made hooked device to recover the coverslip after centrifugation. Panels C and D show drawings explaining how to recover the coverslip after centrifugation. (C) Place the hooked tool into the hole present in the slotted edge of the adaptor and (D) gently pull. (E) These are pictures of the humid chamber needed to perform immunofluorescence imaging. The arrow indicates the 12 mm coverslip. (F) These are representative confocal images at 63X (zoom 2) of gradient fractions #1-8, collected during the purification procedure and stained for CrSAS-6 (red) and &#945;-tubulin (green). The insets correspond to the region indicated by a white box in the lower magnification views. Scale bar = 10 &#181;m. (G) This is a graph representing the number of centrioles positive for both CrSAS-6 and &#945;-tubulin per field of view in each fraction. Note the enrichment in fractions #1-6. The average number of centrioles per field in each fraction: #1 = 16.3 &#177; 4.7, #2 = 24.0 &#177; 4.6, #3 = 37.0 &#177; 17.0, #4 = 23.3 &#177; 11.4, #5 = 14.3 &#177; 2.1, #6 = 13.7 &#177; 9.3, #7 = 2.7 &#177; 1.2, #8 = 1.0 &#177; 0.0, #9 = 1.0 &#177; 0.0, #10 = 0.0 &#177; 0.0, #11 = 0.3 &#177; 0.6, #12 = 0.0 &#177; 0.0. For each fraction, 3 random fields were imaged. Note that, when counting the most enriched fraction #3, we found that 95% of the centrioles are positive for CrSAS-6 and &#945;-tubulin (n = 205 centrioles). (H) A ratio of the number of centrioles present in the measured area of the micrographs is used to calculate the total number of centrioles present in the area of the tube. The centriole number per &#181;L can be calculated from the 10 &#181;L originally centrifuged fractions. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58109/58109fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58109/58109fig3.jpg" /> 
Figure 3: Isolated centrioles are centrifuged using concentrators. (A) This panel shows the centrifugation devices needed to concentrate centrioles onto coverslips prior to immunofluorescence, including a 15 mL round-bottom tube, a concentrator, a 12 mm coverslip, and a support adaptor apparatus. (B) This panel shows the steps to assemble the centrifugation device. Panels C-E show confocal images of centrioles stained for CrSAS-6 (red) and &#945;-tubulin (green) (C) without or (D-E) with the concentrator. The insets correspond to the region indicated by a white box in the lower magnification views. The scale bar is 10 &#181;m. Note that centrioles are enriched in the middle of the coverslip (the dotted line represents the border of the concentrated area). (F) This graph represents the number of centrioles per field of view without and with the concentrator. Five random fields of view were analyzed. The average number of centrioles is, without concentrator, 29.8 &#177; 2.9, and with concentrator, 182.6 &#177; 11.5, P &#60;0.0001. The statistical significance was assessed by an unpaired t-test. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58109/58109fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58109/58109fig4.jpg" /> 
Figure 4:&#160;Single-particle averaging on isolated C. reinhardtii centrioles. (A) This panel shows Z stack images of centrioles stained with GT335 and acquired using a SIM microscope. (B) These panels show a maximal intensity Z projection of the stacked images. Scale bar = 1 &#181;m. (C) These panels show a representative image with an inverted contrast. The inset represents a zoom-in to visualize the centrioles better. (D) These panels show particle picking. The inset indicates how the particles were picked. (E) These are examples of 5 extracted particles. (F) This panel shows the particles after alignment. (G) This panel shows the particles after applying a mask. Panels H and I show two class averages: (H) a top view (63 particles) and (I) a side view (98 particles). The double arrows indicate the dimensions of the GT335 signal. Scale bar = 250 nm. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58109/58109fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1.&#160;<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58109/Adaptor_coverslip.stl" target="_blank">Please click here to download this file.</a>
Supplementary File 2.&#160;<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58109/concentrator.stl" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles at JoVE.com

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

We have developed a strategy to purify and image a large number of centrioles in different orientations amenable for super-resolution microscopy and single-particle averaging.

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell ...

isolation-fluorescence-imaging-for-single-particle-reconstruction

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.4K Views.  University of Geneva. We have developed a strategy to purify and image a large number of centrioles in different orientations amenable for super-resolution microscopy and single-particle averaging.

Video: Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and genetics because of its simple life cycle, ease of growth and manipulation for genetic analysis, genomic resources, and transformability of the nucleus and both organelles. Mating strains is a common practice when genetic approaches are used in Chlamydomonass, to create vegetative diploids for analysis of dominance, or following tetrad dissection to ascertain nuclear vs. organellar inheritance, to test allelism, to analyze epistasy, or to generate populations for the purpose of map-based cloning. Additionally, genetic crosses are routinely used to combine organellar genotypes with particular nuclear genotypes. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad separation. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas. Key parameters and trouble spots are explained. Finally, resources for further information and alternative methods are provided.

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Time-lapse Imaging of Mitosis After siRNA Transfection

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

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

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

Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large macromolecular complexes. Compared to X-ray crystallography, it has some unique advantages. First, single particle EM reconstruction does not need to crystallize the protein sample, which is the bottleneck in X-ray crystallography, especially for large macromolecular complexes. Secondly, it does not need large amounts of protein samples. Compared with milligrams of proteins necessary for crystallization, single particle EM reconstruction only needs several micro-liters of protein solution at nano-molar concentrations, using the negative staining EM method. However, despite a few macromolecular assemblies with high symmetry, single particle EM is limited at relatively low resolution (lower than 1 nm resolution) for many specimens especially those without symmetry. This technique is also limited by the size of the molecules under study, i.e. 100 kDa for negatively stained specimens and 300 kDa for frozen-hydrated specimens in general.
For a new sample of unknown structure, we generally use a heavy metal solution to embed the molecules by negative staining. The specimen is then examined in a transmission electron microscope to take two-dimensional (2D) micrographs of the molecules. Ideally, the protein molecules have a homogeneous 3D structure but exhibit different orientations in the micrographs. These micrographs are digitized and processed in computers as "single particles". Using two-dimensional alignment and classification techniques, homogenous molecules in the same views are clustered into classes. Their averages enhance the signal of the molecule's 2D shapes. After we assign the particles with the proper relative orientation (Euler angles), we will be able to reconstruct the 2D particle images into a 3D virtual volume.
In single particle 3D reconstruction, an essential step is to correctly assign the proper orientation of each single particle. There are several methods to assign the view for each particle, including the angular reconstitution1 and random conical tilt (RCT) method2. In this protocol, we describe our practice in getting the 3D reconstruction of yeast exosome complex using negative staining EM and RCT. It should be noted that our protocol of electron microscopy and image processing follows the basic principle of RCT but is not the only way to perform the method. We first describe how to embed the protein sample into a layer of Uranyl-Formate with a thickness comparable to the protein size, using a holey carbon grid covered with a layer of continuous thin carbon film. Then the specimen is inserted into a transmission electron microscope to collect untilted (0-degree) and tilted (55-degree) pairs of micrographs that will be used later for processing and obtaining an initial 3D model of the yeast exosome. To this end, we perform RCT and then refine the initial 3D model by using the projection matching refinement method3.

This article describes a standard method to get a three-dimensional (3D) reconstruction of biological macromolecules using negative staining electron microscopy (EM). In this protocol, we explain how to get the 3D structure of the Saccharomyces cerevisiae exosome complex at medium resolution using the random conical tilt reconstruction method (RCT).

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large ...

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission1 by delivering neurotransmitters and neuropeptides onto neurons reproducibly2. Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation3 has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo4,5. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels.
A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain6. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current2,7, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be &tilde; 1 &mu;m or less to minimize diffusional 'leak', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette.
Fabrication of micropipettes is facilitated with sophisticated electronic pullers8. Micropipettes are pulled from glass capillary tubes containing a filament that 'wicks' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below). 
The present protocol demonstrates microiontophoresis of acetylcholine (ACh+ Cl-) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40BAC-GCaMP2 transgenic mice9 enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.

Microiontophoresis entails movement of ions from a micropipette in response to a difference in electrical potential between the inside and outside of the micropipette. Biologically active molecules are thereby delivered in proportion to electrical current. We illustrate acetylcholine microiontophoresis in conjunction with micromanipulation to study endothelium-dependent vasodilation in the microcirculation.

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental  preparation. ...

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.

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

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

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Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Time-lapse Imaging of Mitosis After siRNA Transfection

Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

DNA Electroporation, Isolation and Imaging of Myofibers