Dr. Pirta Hotulainen is acknowledged for her critical comments, invaluable for preparing this manuscript. Dr. Rimante Minkeviciene is acknowledged for her help in preparing the neuronal cultures used for the original experiments. All imaging was performed in the Biomedicum Imaging Unit. This work was supported by the Academy of Finland (D.M., SA 266351) and Doctoral Programme Brain &#38; Mind (A.A.)

Primary hippocampal neurons used in these experiments were harvested16 from embryonic day 17 Wistar rat embryos of either sex under the ethical guidelines of the University of Helsinki and Finnish law.
1. Sample preparation
<ol>
	<li>On high-fidelity glass coverslips, allow rat hippocampal neurons to grow in sparse culture conditions (~5,000-10,000 cells/cm2) for 14 days (14 days-in-vitro). 
	NOTE: Using sparse cultures reduces the chances of overlapping neurites, which helps quantify the MPS.</li>
	<li>Fix neurons using 4% paraformaldehyde for 12 min at room temperature. Wash the coverslip once in 0.2% BSA in PBS (BSA-PBS), then incubate for 10 min in a 1% solution of Triton-X in PBS at room temperature. Wash once in BSA-PBS.</li>
	<li>Dilute anti-ankyrin G antibodies (1:200) in BSA-PBS. Add antibody solution to the coverslip and incubate overnight at 4 &#176;C. Optionally, add chicken anti-MAP2 antibodies (see Table of Materials) to the antibody solution to demarcate the somato-dendritic domain.</li>
	<li>Wash cells once in BSA-PBS, followed by 0.1% Triton-X in PBS, then do a final wash in BSA-PBS.</li>
	<li>Dilute fluorophore-tagged anti-mouse secondary antibodies (1:200) (see Table of Materials) in BSA-PBS, add to the coverslip, and incubate for 1 h at room temperature. Wash the cells once in BSA-PBS, once in 0.1% Triton-X in PBS, then once in PBS.</li>
	<li>Prepare a 1 &#181;M solution of tagged phalloidin in PBS (see Table of Materials) and add it to the cells. Incubate for 2 h at room temperature. Wash cells once in PBS, once in 0.1% Triton-X in PBS, then once in PBS. 
	&#8203;NOTE: AlexaFluor488-tagged phalloidin was used here, but other tags will also work.</li>
	<li>For mounting the coverslip on a glass slide, apply a drop of mounting media on a glass slide, dip the coverslip in deionized water, and dab using a soft paper towel (to remove excess water).
	<ol>
		<li>Place the coverslip on the glass slide. Incubate at room temperature for 24 h. 
		&#8203;NOTE: No adverse effects on the sample were observed using hard-setting mounting media (refractive index 1.47 after curing).</li>
	</ol>
	</li>
</ol>
2. Imaging
<ol>
	<li>If possible, consult with the microscopy facility&#39;s personnel before imaging. Use an immersion oil calculator (see Table of Materials) to select the immersion oil suitable for the sample.</li>
	<li>Once the samples are ready, ensure the coverslips are clean and clear of any residue or excess mounting media. If necessary, use a cotton tip dipped in water or ethanol to clean. Place the sample in a 3D SIM-capable microscope (see Table of Materials) and find a cell to image.</li>
	<li>Adjust the power of the relevant laser lines and the exposure times to maximize the signal-to-noise ratio without significantly bleaching the sample. 
	NOTE: A good signal-to-noise ratio will show a clear grid-like appearance focused on the sample and lead to accurate high-resolution reconstructions. 488 nm and 640 nm laser lines were used to visualize F-actin and ankyrin G, respectively.</li>
	<li>Set the upper and lower limits of the sample in the z-dimension and proceed to acquire a stack. 
	NOTE: z-step size of 125 nm was used here.</li>
	<li>For successful SIM reconstruction, ensure that the microscopy software (see Table of Materials) has an optical transfer file (OTF) suitable for the used dyes. 
	NOTE: These are typically created and maintained by specialized personnel. In the absence of a functional OTF, open-source tools are also available to perform reconstructions based on estimates17. In this work, up-to-date OTF files were used controlled by specialized personnel.</li>
	<li>Run the reconstruction algorithm on the stack to obtain a super-resolved reconstruction. Check the reconstructions for known artifacts and, if necessary, adjust the parameters to correct them. 
	NOTE: The default algorithm parameters typically give accurate results. Many of these artifacts involve some repeating patterns: striped lines along with multiple directions on one z-plane, &#39;ghosting&#39; (repeated features appearing in various z-planes), or a hexagonal &#39;honeycomb&#39; pattern emerging in some areas. These artifacts can often be fixed with better sample preparation, a better signal-to-noise ratio, adjusting the parameters of the reconstruction algorithm, or ensuring the refractive index of the chosen immersion oil is suitable for the mounting medium and sample. For a more detailed discussion of SIM artifacts and a freely-available tool to check for the presence of artifacts, see Ball et al.18</li>
	<li>Compare the SIM reconstruction to a wide-field image to observe improvements in resolution.</li>
	<li>When performing multi-color SIM, use the alignment algorithm to align the different channels correctly once a satisfactory reconstruction is ready. 
	&#8203;NOTE: The alignment algorithm uses an alignment reference file generated using a microsphere bead preparation as per the instruction of the microscope manufacturer.</li>
</ol>
3. Image analysis
<ol>
	<li>Actin rings in the MPS have a distinctive periodic appearance. Using image analysis software (see Table of Materials), create a maximum intensity projection image using all the focal planes where actin rings are visible.</li>
	<li>On the maximum intensity projection image, draw a perpendicular line across visible adjacent rings and record the fluorescence intensity along with the software&#39;s line &#39;Plot Profile&#39; function.</li>
	<li>To calculate the mean inter-peak distance, note the local maxima in the line profile and measure the distance between individual adjacent fluorescent intensity peaks. 
	NOTE: This can be easily calculated, for example, using the &#39;findpeaks&#39; function in MATLAB or the (open-source) GNU Octave platform.</li>
	<li>To evaluate the colocalization of different proteins with actin rings in the MPS, run a colocalization analysis procedure19,20,21 on SIM reconstructions of actin and the candidate protein. Manually draw a selection to define the AIS as a region of interest for the EzColocalization plugin in the software platform19 and run the analysis to calculate the Pearson&#39;s coefficient of correlation (PCC) of co-localization19,20,21. A PCC value close to 1 indicates strong colocalization. 
	NOTE: This protocol is summarized in Figure 1.</li>
</ol>

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Role of the axon initial segment in the regulation of protein transport and the maintenance of axonal identity.</a> Seminars in Cell &amp; Developmental Biology. 27, 44-51 (2014).</li><li>Brachet, A., 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=Ankyrin+G+restricts+ion+channel+diffusion+at+the+axonal+initial+segment+before+the+establishment+of+the+diffusion+barrier.">Ankyrin G restricts ion channel diffusion at the axonal initial segment before the establishment of the diffusion barrier.</a> Journal of Cell Biology. 191 (2), 383-395 (2010).</li><li>Nakada, 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=Accumulation+of+anchored+proteins+forms+membrane+diffusion+barriers+during+neuronal+polarization.">Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization.</a> Nature Cell Biology. 5 (7), 626-632 (2003).</li><li>Song, A. H., 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+selective+filter+for+cytoplasmic+transport+at+the+axon+initial+segment.">A selective filter for cytoplasmic transport at the axon initial segment.</a> Cell. 136 (6), 1148-1160 (2009).</li><li>Sun, X., 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=Selective+filtering+defect+at+the+axon+initial+segment+in+Alzheimer's+disease+mouse+models.">Selective filtering defect at the axon initial segment in Alzheimer's disease mouse models.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (39), 14271-14276 (2014).</li><li>Winckler, B., Forscher, P., Mellman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+diffusion+barrier+maintains+distribution+of+membrane+proteins+in+polarized+neurons.">A diffusion barrier maintains distribution of membrane proteins in polarized neurons.</a> Nature. 397 (6721), 698-701 (1999).</li><li>Kole, M. H., 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=Action+potential+generation+requires+a+high+sodium+channel+density+in+the+axon+initial+segment.">Action potential generation requires a high sodium channel density in the axon initial segment.</a> Nature Neuroscience. 11 (2), 178-186 (2008).</li><li>Xu, K., Zhong, G., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin,+spectrin,+and+associated+proteins+form+a+periodic+cytoskeletal+structure+in+axons.">Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons.</a> Science. 339 (6118), 452-456 (2013).</li><li>Leterrier, 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=Nanoscale+architecture+of+the+axon+initial+segment+reveals+an+organized+and+robust+scaffold.">Nanoscale architecture of the axon initial segment reveals an organized and robust scaffold.</a> Cell Reports. 13 (12), 2781-2793 (2015).</li><li>Leterrier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+axon+initial+segment:+An+updated+viewpoint.">The axon initial segment: An updated viewpoint.</a> The Journal of Neuroscience. 38 (9), 2135-2145 (2018).</li><li>Hamdan, H., 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=Mapping+axon+initial+segment+structure+and+function+by+multiplexed+proximity+biotinylation.">Mapping axon initial segment structure and function by multiplexed proximity biotinylation.</a> Nature Communications. 11 (1), 100 (2020).</li><li>Zhou, R., 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=Proteomic+and+functional+analyses+of+the+periodic+membrane+skeleton+in+neurons.">Proteomic and functional analyses of the periodic membrane skeleton in neurons.</a> bioRxiv. , (2020).</li><li>Valli, J., 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=Seeing+beyond+the+limit:+A+guide+to+choosing+the+right+super-resolution+microscopy+technique.">Seeing beyond the limit: A guide to choosing the right super-resolution microscopy technique.</a> Journal of Biological Chemistry. 297 (1), 100791 (2021).</li><li>Wang, T., 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=Radial+contractility+of+actomyosin+rings+facilitates+axonal+trafficking+and+structural+stability.">Radial contractility of actomyosin rings facilitates axonal trafficking and structural stability.</a> Journal of Cell Biology. 219 (5), 201902001 (2020).</li><li>Abouelezz, A., Stefen, H., Segerstr&#229;le, M., Micinski, D., Minkeviciene, R., Lahti, L., Hardeman, E., Gunning, P., Hoogenraad, C., Taira, T., Fath, T., Hotulainen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tropomyosin+Tpm3.1+is+required+to+maintain+the+structure+and+function+of+the+axon+initial+segment.">Tropomyosin Tpm3.1 is required to maintain the structure and function of the axon initial segment.</a> iScience. 23 (5), 101053 (2020).</li><li>Muller, M., Monkemoller, V., Hennig, S., Hubner, W., Huser, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-source+image+reconstruction+of+super-resolution+structured+illumination+microscopy+data+in+ImageJ.">Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ.</a> Nature Communications. 7, 10980 (2016).</li><li>Ball, G., 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=SIMcheck:+a+toolbox+for+successful+super-resolution+structured+illumination+microscopy.">SIMcheck: a toolbox for successful super-resolution structured illumination microscopy.</a> Scientific Reports. 5, 15915 (2015).</li><li>Stauffer, W., Sheng, H., Lim, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EzColocalization:+An+ImageJ+plugin+for+visualizing+and+measuring+colocalization+in+cells+and+organisms.">EzColocalization: An ImageJ plugin for visualizing and measuring colocalization in cells and organisms.</a> Scientific Reports. 8 (1), 15764 (2018).</li><li>Dunn, K. W., Kamocka, M. M., McDonald, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+evaluating+colocalization+in+biological+microscopy.">A practical guide to evaluating colocalization in biological microscopy.</a> American Journal of Physiology-Cell Physiology. 300 (4), 723-742 (2011).</li><li>Lahti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+analysis+supplement+to+the+research+article+Abouelezz,+Stefen,+Segerstr&#229;le,+Micinski,+Minkeviciene,+Lahti,+Hardeman,+Gunning,+Hoogenraad,+Taira,+Fath,+&amp;+Hotulainen.">Data analysis supplement to the research article Abouelezz, Stefen, Segerstr&#229;le, Micinski, Minkeviciene, Lahti, Hardeman, Gunning, Hoogenraad, Taira, Fath, &amp; Hotulainen.</a> Tropomyosin Tpm3.1 Is Required to Maintain the Structure and Function of the Axon Initial Segment. , (2021).</li><li>Abouelezz, A., Micinski, D., Lipponen, A., Hotulainen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-membranous+actin+rings+in+the+axon+initial+segment+are+resistant+to+the+action+of+latrunculin.">Sub-membranous actin rings in the axon initial segment are resistant to the action of latrunculin.</a> Journal of Biological Chemistry. 400 (9), 1141-1146 (2019).</li></ol>

The authors have nothing to disclose.

The protocol described here provides a method for visualizing and measuring MPS proteins using the super-resolution technique. As actin rings and other MPS components display a periodicity of ~190 nm9,10, conventional diffraction-limited imaging approaches cannot reveal the details of the MPS. Several microscopy setups may resolve diffraction-limited structures in super-resolution, and SIM represents a robust and uncomplicated option. Importantly, SIM is compatible with the most widely-used fluorophores, providing significant flexibility. Furthermore, SIM is effective in visualizing potentially dim structures in the MPS, such as actin rings in latrunculin-treated neurons22, as well as live neurons15.
An essential aspect for the success of this protocol is preserving the integrity of the MPS in the first place. The most critical steps to successful SIM imaging occur during sample preparation. For example, moderate fixation (4% PFA for 12 min at room temperature) and strong permeabilization (1% Triton-X for 10 min) provide the best results. It is crucial to keep in mind that harsh treatments that may be required for specific preparations may have a negative effect on the structural integrity of the MPS. Therefore, it is perhaps best to consider modifying such treatments to maximize the preservation of the MPS.
The other crucial factor to consider when preparing samples for imaging is to maximize the labeling density. Ideally, every molecule would be tagged and detected. For example, the anti-ankyrin G antibody used in this experiment is commonly used to label the AIS. It provides outstanding performance in conventional fluorescence microscopy, even when used at a dilution of 1:1000 and incubated for just 1 h at room temperature. However, to obtain good labeling density for super-resolution microscopy, it is highly effective to use 5-fold that concentration (1:200) and incubate the antibody overnight at 4 &#176;C. While the specific requirements for each antibody or labeling technique will vary and need to be determined experimentally, it is perhaps helpful to keep this in mind.
In addition, it is essential to note that achieving a high signal-to-noise ratio lends itself well to accurate and successful SIM reconstructions. A good rule of thumb is that the grid pattern should be visible in the individual images once the SIM modality is engaged. However, this is not always possible.
Finally, it is essential to note that SIM is among the weakest super-resolution techniques in resolving power14. Thus, while it is generally sufficient for revealing periodicity and overall organization of MPS proteins, it is significantly less capable of providing details about their interactions than stochastic optical reconstruction microscopy (STORM)10. Furthermore, the technique's utility described here is limited to the study of proteins for which a specific, well-performing antibody is available. However, this can partly be circumvented through exogenous expression of tagged proteins15.

The axon initial segment (AIS) is a short, uniquely specialized region of the proximal axon of vertebrate neurons1. The AIS comprises a transport filter and diffusion barrier essential in maintaining neuronal polarity by sorting somato-dendritic cargo2,3,4,5,6,7. In addition, the unique structure of the AIS allows it to accommodate clusters of voltage-gated ion channels that facilitate its function as the site of action potential initiation8. A highly stable structural complex underlies the unique functions of the AIS. Research within the last decade has revealed the presence of a membrane periodic skeleton (MPS) containing actin rings connected by spectrin and providing a scaffold for anchoring various AIS proteins9,10.
The distance between actin rings in the MPS (~190 nm)9,10 is under the resolution limit of conventional light microscopy. Early attempts to use electron microscopy to visualize the MPS were not successful, as the harsh preparation procedures involved failed to preserve the structure of the MPS. Thus, super-resolution microscopy techniques have proven invaluable in elucidating some of the structural details of the MPS11. However, the understanding of the AIS structural complex, the identity and functions of its components, and its spatiotemporal regulation are still incomplete. Recent proteomic studies succeeded in creating a sizeable list of proteins that localize to the AIS close to structural components of the AIS12,13. Still, details of their function and precise place in the AIS complex are lacking. Thus, super-resolution microscopy techniques serve as an essential tool to examine the accurate positions of these proteins relative to other MPS components and investigate their functions. Several light microscopy techniques can achieve resolutions higher than the diffraction limit of light, some even capable of localizing single molecules. However, many of these techniques typically require specialized fluorophores or imaging buffers, and image acquisition is often time-intensive14.
3D structured illumination microscopy (3D-SIM), owing to its ease of use and simple sample preparation requirements, requires no special reagents for imaging or sample preparation, works well with a wide array of fluorophores and samples, can be readily implemented in multiple colors, and is capable of live-cell imaging15. While the best possible resolution SIM offers (~120 nm) is low compared to many other super-resolution techniques, it is sufficient for many applications (for example, for resolving the components of the MPS in neurons). Thus, it is crucial to consider the requirement for specific applications to determine if SIM is a suitable choice or if an even higher resolution is necessary. Here, a protocol is described for using cultured hippocampal neurons and 3D-structured illumination microscopy (3D-SIM) to examine the position and organization of putative AIS proteins relative to actin rings in the MPS, as implemented in Abouelezz&#160;et al.16

<table><tbody><tr><td>24-well plates</td><td>Corning</td><td>3524</td><td></td></tr><tr><td>4% Paraformaldehyde</td><td></td><td></td><td></td></tr><tr><td>Alexa-488 Phalloidin</td><td>ThermoFisher</td><td>A12379</td><td></td></tr><tr><td>Alexa-647 anti-mouse</td><td>ThermoFisher</td><td>A31571</td><td></td></tr><tr><td>Anti-Ankyrin G antibody</td><td>UC Davis/NIH NeuroMab Facility, Clone 106/36</td><td>75-146</td><td></td></tr><tr><td>Anti-MAP2 antibody</td><td>Merck Millipore</td><td>AB5543</td><td></td></tr><tr><td>B-27</td><td>Invitrogen</td><td>17504044</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>BioWest</td><td>P6154</td><td></td></tr><tr><td>Deltavision OMX SR</td><td>GE Healthcare Life Sciences</td><td>N/A</td><td></td></tr><tr><td>Fiji software package</td><td>ImageJ</td><td></td><td></td></tr><tr><td>GNU Octave</td><td>GNU</td><td></td><td></td></tr><tr><td>High performance coverslips</td><td>Marienfeld</td><td>117530</td><td></td></tr><tr><td>Immersion Oil Calculator</td><td>Cytiva Life Sciences</td><td></td><td>https://tinyurl.com/ImmersionOilCalculator</td></tr><tr><td>L-Glutamine</td><td>VWR</td><td>ICNA1680149</td><td></td></tr><tr><td>MATLAB R2020a</td><td>Mathworks</td><td></td><td></td></tr><tr><td>Neurobasal media</td><td>Invitrogen</td><td>21103049</td><td></td></tr><tr><td>OMX SR</td><td>Delta Vision OMX</td><td></td><td></td></tr><tr><td>Primocin</td><td>InvivoGen</td><td>ant-pm-1</td><td></td></tr><tr><td>ProLong Gold mounting media</td><td>Invitrogen</td><td>P10144</td><td></td></tr><tr><td>softWoRx Deconvolution</td><td>Cytiva Life Sciences</td><td></td><td></td></tr><tr><td>Superfrost Slides</td><td>Epredia</td><td>ISO 8037/1</td><td></td></tr><tr><td>TetraSpeck microspheres 0.1 &amp;micro;m</td><td>ThermoFisher</td><td>T7279</td><td></td></tr><tr><td>Triton-X</td><td>Sigma</td><td>X100</td><td></td></tr></tbody></table>

measuring properties of the membrane periodic skeleton of the axon initial segment using 3d-structured illumination microscopy (3d-sim)

The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to the maintenance of neuronal polarity by sorting somato-dendritic cargo. A membrane periodic skeleton (MPS) comprising periodic actin rings provides a scaffold for anchoring various AIS proteins, including structural proteins and different ion channels. Although recent proteomic approaches have identified a considerable number of novel AIS components, details of the structure of the MPS and the roles of its individual components are lacking. The distance between individual actin rings in the MPS (~190 nm) necessitates the employment of super-resolution microscopy techniques to resolve the structural details of the MPS. This protocol describes a method for using cultured rat hippocampal neurons to examine the precise localization of an AIS protein in the MPS relative to sub-membranous actin rings using 3D-structured illumination microscopy (3D-SIM). In addition, an analytical approach to quantitively assess the periodicity of individual components and their position relative to actin rings is also described.

Using cultured rat hippocampal neurons and 3D-SIM, a protocol is described to visualize and measure actin rings and other components of the MPS in the AIS. Reconstructions of image stacks showed clear periodicity of actin rings (Figure 2A). In our hands, the mean inter-peak distance of actin rings in the MPS, visualized using Alexa 488-tagged phalloidin, was 190.36 &#177; 1.7 nm (mean &#177; SEM). This is in line with the previously reported average distance of ~190 nm between actin rings in the MPS. Similarly, an anti-ankyrin G antibody was used to visualize ankyrin G (Figure 2B). The colocalization of ankyrin G and F-actin was tested in the AIS using a colocalization analysis procedure to calculate the PCC of co-localization19,20,21. The PCC of colocalization of ankyrin G and F-actin fluorescence was 0.36 &#177; 0.03 (mean &#177; SEM, Figure 2B). As ankyrin G and actin rings bind &#946;IV-spectrin at different domains, they do not show significant colocalization. These data were adapted from Abouelezz et al.16
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63327/63327fig01.jpg" /> 
Figure 1: Schematic representation of the protocol for sample preparation for 3D-SIM imaging. Hippocampal neurons are harvested from rat embryos, dissociated, and allowed to grow on glass coverslips in culture for 14 days. Cells are then fixed and stained using tagged phalloidin and appropriate antibodies, then mounted on glass slides for 3D-SIM imaging. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63327/63327fig01large.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/63327/63327fig02.jpg" /> 
Figure 2: 3D-SIM reconstruction of the membrane periodic skeleton (MPS) in the axon initial segment (AIS). (A) F-actin (green) and ankyrin G (magenta) show a regular distribution in the AIS, visualized by 3D-SIM. Scale bar = 1 &#181;m. (B) Pearson&#39;s coefficients of correlation (PCC) of colocalization of ankyrin G with F-actin in the MPS in the AIS. The mean PCC of ankyrin G was 0.36 (black circle). Gray diamonds represent individual cells (n = 16), mid-line represents the median, error bars represent 25th and 75th percentiles. The data is adapted from Abouelezz et al.16 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63327/63327fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy 3D-SIM at JoVE.com

Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy 3D-SIM

The present protocol describes a method to visualize and measure actin rings and other components of the membrane periodic skeleton of the axon initial segment using cultured rat hippocampal neurons and 3D-structured illumination microscopy (3D-SIM).

Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy (3D-SIM)

The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to ...

measuring-properties-membrane-periodic-skeleton-axon-initial-segment

Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.0K Views.  Minerva Foundation Institute for Medical Research. Dit protocol maakt het mogelijk om het periodieke membraanskelet in het axon-initiële segment van gekweekte neuronen op te lossen. Door de lokalisatie van eiwitten te onderzoeken kan inzicht worden verkregen in de functie in het membraan periodiek skelet. Het belangrijkste voordeel van deze techniek is de robuustheid en het gebruiksgemak.Gestructureerde verlichtingsmicroscopie is in staat om het periodieke skelet van het membraan op te lossen en de monstervoorbereiding is eenvoudig en...

Video: Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy 3D-SIM

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

3D Reconstruction and Analysis of Thin Subcellular Neuronal Structures using Focused-Ion Beam Scanning Electron Microscopy Data

Recent advances in scanning electron microscope technologies now permit the rapid three-dimensional (3D) analysis of ultrathin subcellular processes. Here, a methodological pipeline is presented to identify, visualize, and analyze thin neuronal processes, such as those that project into the presynaptic boutons of other neurons (termed 'spinules'). Using freely available software packages, this protocol demonstrates how to use a decision tree to identify common neuronal subcellular structures using morphological criteria within focused ion beam scanning electron microscopy (FIB-SEM) image volumes, with particular attention on identifying a diversity of spinules projecting into presynaptic boutons. In particular, this protocol describes how to trace spinules within neuronal synapses to produce 3D reconstructions of these thin subcellular projections, their parent neurites, and postsynaptic partners. Additionally, the protocol includes a list of freely available open-source software programs for analyzing FIB-SEM data and offers tips (e.g., smoothing, lighting) toward improving 3D reconstructions for visualization and publication. This adaptable protocol offers an entry point into the rapid nanoscale analysis of subcellular structures within FIB-SEM image volumes.

A flexible methodological pipeline to identify, visualize, and quantify thin subcellular neuronal processes within focused ion beam scanning electron microscopy image volumes using user-friendly open-source software packages.

Recent advances in scanning electron microscope technologies now permit the rapid three-dimensional (3D) analysis of ultrathin subcellular processes. ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

Biology

Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

Membrane remodeling occurs constantly at the plasma membrane and within cellular organelles. To fully dissect the role of the environment (ionic conditions, protein and lipid compositions, membrane curvature) and the different partners associated with specific membrane reshaping processes, we undertake in vitro bottom-up approaches. In recent years, there has been keen interest in revealing the role of septin proteins associated with major diseases. Septins are essential and ubiquitous cytoskeletal proteins that interact with the plasma membrane. They are implicated in cell division, cell motility, neuro-morphogenesis, and spermiogenesis, among other functions. It is, therefore, important to understand how septins interact and organize at membranes to subsequently induce membrane deformations and how they can be sensitive to specific membrane curvatures. This article aims to decipher the interplay between the ultra-structure of septins at a molecular level and the membrane remodeling occurring at a micron scale. To this end, budding yeast, and mammalian septin complexes were recombinantly expressed and purified. A combination of in vitro assays was then used to analyze the self-assembly of septins at the membrane. Supported lipid bilayers (SLBs), giant unilamellar vesicles (GUVs), large unilamellar vesicles (LUVs), and wavy substrates were used to study the interplay between septin self-assembly, membrane reshaping, and membrane curvature.

Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

Septins are cytoskeletal proteins. They interact with lipid membranes and can sense but also generate membrane curvature at the micron scale. We describe in this protocol bottom-up in vitro methodologies for analyzing membrane deformations, curvature-sensitive septin binding, and septin filament ultrastructure.

Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy (3D-SIM)

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