This work was financed by a VIDI grant number H64.09.016 from The Netherlands Organization for Scientific Research (NWO) to CPF. CPF is grateful to Dr. Silvina A. Fratantoni for her critical comments and corrections on the final manuscript. GMRDL/EMMM are supported by the Dutch Technology Foundation STW (project 12151 and 11350), which is part of the NWO, and which is partly funded by the Ministry of Economic Affairs. We thank the Catherine Kitts and Peter Drent of Nikon Instruments Europe BV for assistance and support. HX was supported by the Royal Dutch Academy of Arts and Sciences (grant 11CDP10) and WT was supported by grant the Netherlands Organization for Scientific research (grant 820.02.006).

All experimental procedures involving animals were optimized to reduce animal suffering and were approved by the Commission for Animal Experimentation, University of Amsterdam, DEC protocol # DED204 and DED250.
1. Coverslip Preparation
<ol>
	<li>Cut down the coverslips to a size of 15 mm x 15 mm using a carbide or diamond scribe, so that they fit into the wells of a 12-well plate.</li>
	<li>While working in a fume hood, start coverslip coating by fully submerging coverslips in concentrated n...

<ol>
	<li>Gustafsson, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surpassing+the+lateral+resolution+limit+by+a+factor+of+two+using+structured+illumination+microscopy.">Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy.</a> J Microsc. 198, 82-87 (2000).</li><li>Yoshihara, Y., De Roo, M., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+spine+formation+and+stabilization.">Dendritic spine formation and stabilization.</a> Curr Opin Neurobiol. 19, 146-153 (2009).</li><li>Dailey, M. E., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamics+of+dendritic+structure+in+developing+hippocampal+slices.">The dynamics of dendritic structure in developing hippocampal slices.</a> J Neurosci. 16, 2983-2994 (1996).</li><li>Alvarez, V. A., Sabatini, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+and+physiological+plasticity+of+dendritic+spines.">Anatomical and physiological plasticity of dendritic spines.</a> Annu Rev Neurosci. 30, 79-97 (2007).</li><li>Jaworski, 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=Dynamic+microtubules+regulate+dendritic+spine+morphology+and+synaptic+plasticity.">Dynamic microtubules regulate dendritic spine morphology and synaptic plasticity.</a> Neuron. 61, 85-100 (2009).</li><li>Abbe, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beitr&#228;ge+zur+Theorie+des+Mikroskops+und+der+mikroskopischen+Wahrnehmung.">Beitr&#228;ge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung.</a> Archiv f&#252;r mikroskopische Anatomie. 9, 413-418 .</li><li>Gustafsson, M. G., Agard, D. A., Sedat, 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=I5M:+3D+widefield+light+microscopy+with+better+than+100+nm+axial+resolution.">I5M: 3D widefield light microscopy with better than 100 nm axial resolution.</a> J Microsc. 195, 10-16 (1999).</li><li>Heintzmann, R., Cremer, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laterally+modulated+excitation+microscopy:+improvement+of+resolution+by+using+a+diffraction+grating.">Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating.</a> Proc. SPIE 3568 Optical Biopsies and Microscopic Techniques III. , 185-196 (1999).</li><li>Karadagli&#402;&#225;, D., Wilson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+formation+in+structured+illumination+wide-field+fluorescence+microscopy.">Image formation in structured illumination wide-field fluorescence microscopy.</a> Micron. 39, 808-818 (2008).</li><li>Schermelleh, 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=Subdiffraction+multicolor+imaging+of+the+nuclear+periphery+with+3D+structured+illumination+microscopy.">Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy.</a> Science. 320, 1332-1336 (2008).</li><li>Gustafsson, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+structured-illumination+microscopy:+wide-field+fluorescence+imaging+with+theoretically+unlimited+resolution.">Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution.</a> Proc Natl Acad Sci U S A. 102, 13081-13086 (2005).</li><li>Nagerl, U. V., Willig, K. I., Hein, B., Hell, S. W., Bonhoeffer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+of+dendritic+spines+by+STED+microscopy.">Live-cell imaging of dendritic spines by STED microscopy.</a> Proc Natl Acad Sci U S A. 105, 18982-18987 (2008).</li><li>Kner, P., Chhun, B. B., Griffis, E. R., Winoto, L., Gustafsson, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+video+microscopy+of+live+cells+by+structured+illumination.">Super-resolution video microscopy of live cells by structured illumination.</a> Nat Methods. 6, 339-342 (2009).</li><li>James, C. 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=Aligned+microcontact+printing+of+micrometer-scale+poly-L-lysine+structures+for+controlled+growth+of+cultured+neurons+on+planar+microelectrode+arrays.">Aligned microcontact printing of micrometer-scale poly-L-lysine structures for controlled growth of cultured neurons on planar microelectrode arrays.</a> IEEE Trans Biomed Eng. 47, 17-21 (2000).</li><li>Dumitriu, D., Rodriguez, A., Morrison, 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=High-throughput,+detailed,+cell-specific+neuroanatomy+of+dendritic+spines+using+microinjection+and+confocal+microscopy.">High-throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy.</a> Nat Protoc. 6, 1391-1411 (2011).</li><li>Fitzsimons, C. 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=Knockdown+of+the+glucocorticoid+receptor+alters+functional+integration+of+newborn+neurons+in+the+adult+hippocampus+and+impairs+fear-motivated+behavior.">Knockdown of the glucocorticoid receptor alters functional integration of newborn neurons in the adult hippocampus and impairs fear-motivated behavior.</a> Mol Psychiatry. , (2012).</li><li>van Hooijdonk, L. W., 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=Lentivirus-mediated+transgene+delivery+to+the+hippocampus+reveals+sub-field+specific+differences+in+expression.">Lentivirus-mediated transgene delivery to the hippocampus reveals sub-field specific differences in expression.</a> BMC Neurosci. , (2009).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat Protoc. 1, 2406-2415 (2006).</li><li>Brewer, G. J., Torricelli, J. R., Evege, E. K., Price, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+survival+of+hippocampal+neurons+in+B27-supplemented+Neurobasal,+a+new+serum-free+medium+combination.">Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.</a> J Neurosci Res. 35, 567-576 (1993).</li><li>Izeddin, I., 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=Super-resolution+dynamic+imaging+of+dendritic+spines+using+a+low-affinity+photoconvertible+actin+probe.">Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe.</a> PLoS ONE. 6, (2011).</li></ol>

The authors have nothing to disclose.

In this article a working protocol to image dendritic spines from rat primary hippocampal neurons cultured in vitro using SIM is described. The primary hippocampal neuron culture method is an adaptation of the original method described by Kaech and Banker 18. The main differences are the use of Neurobasal/B27 culture medium, which eliminates the requirement of astroglial feeder cultures, and the addition of the mitotic inhibitor FUDR on day 3 which promotes neuronal survival while suppressing glial pr...

A dendritic spine is a small protrusion of the neuron membrane. This characteristic structure is specialized to typically receive input from a single synapse and represents the physical contact area between two neurons. Most functionally mature dendritic spines consist of a globular tip, termed head, and a thin neck that connects the head to the dendritic shaft. However, spines are not static and actively move and change their morphology continuously even in the adult brain 2. Within a 2 week period of time, rat primary hippocampal neuron cultures derived from late embryonic or early postnatal time develop complex dendritic arbors with numerous membrane protrusions that evolve from early filipodia to spine-like structures 3. Based on this dynamic behavior and other characteristics, dendritic spines are thought to provide an anatomical substrate for memory storage and synaptic transmission 4,5.
Given the critical role that dendritic spine size and shape have in synaptic function, it is important to measure their dimensions accurately. Spines vary from around 200 to 2,000 nanometers in length and can be readily identified by confocal laser-scanning fluorescence microscopy. However, spine dimensions other than length are usually below the conventional optical systems' resolution, theoretically fixed by diffraction around 200 nanometers 6. These resolving powers are insufficient for imaging finer details, such as the width of spine necks and heads. Much work has been dedicated to solve this problem and many relatively new super-resolution microscopy techniques have provided substantial progress. In particular, it is possible to achieve resolution beyond the classical limit without discarding any emission light by using laterally structured illumination microscopy (SIM) in a wide-field, non-confocal microscope 7-10. Using this technique in combination with non-linear microscopy techniques, it is theoretically possible to improve the lateral resolution of the optical microscope by an unlimited factor 11. However, in most experimental circumstances, SIM allows to surpass the resolution limit by a factor of two 1. Other super-resolution optical microscopy techniques such as Stimulated emission depletion (STED) microscopy 12 and photo-activation localization microscopy (PALM) 12 have been applied to imaging of dendritic spines. Localization-based methods such as PALM require very large numbers of raw images to achieve super-resolution and are therefore limited in speed. On the other hand, STED can achieve high imaging speed, although at relatively low photon counts and small fields of view, which may not be the case for SIM 13.
In this article the aim is to provide a working protocol to image dendritic spines from rat primary hippocampal neurons cultured in vitro using SIM. The protocol consists of two distinguishable phases: an initial one consisting of establishment, development, transfection and immunohistochemistry of rat primary hippocampal neuron cultures and a late phase dedicated to sample imaging.

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			<td>Fine forceps</td>
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			<td>Big forceps</td>
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			<td>Fine scissor</td>
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			<td>Big scissor</td>
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			<td>Blunt spatula</td>
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			<td>Dissecting microscope with illumination</td>
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			<td>Light microscope</td>
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			<td>37 &#176;C water bath</td>
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			<td>Laminar flow cell culture hood</td>
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			<td>High-temperature dry-oven</td>
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			<td>Bunsen burner</td>
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			<td>Cell culture incubator (5% CO2, 37 &#176;C)</td>
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			<td>Microcentrifuge</td>
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			<td>Orbital shaker</td>
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			<td>Nikon structured illumination microscope setup consisting of:</td>
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			<td>Nikon Eclipse Ti research inverted microscope with Perfect Focus System</td>
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			<td>Nikon CFI Apo TIRF 100x oil objective lens (N.A. 1.49)</td>
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			<td>4 Coherent Sapphire Lasers (458, 488, 514 and 561 nm exitation wavelength)</td>
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			<td>SIM Illuminator</td>
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			<td>Nikon Stage Controller</td>
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			<td>MCL Nano-Drive piezo controller</td>
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			<td>Nikon Intensilight C-HGFIE mercury lamp</td>
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			<td>SIM Microscope Enclosure temperature control</td>
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			<td>Andor EM-CCD Camera iXon DU897</td>
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			<td>PC with Microsoft Windows 7 Home Edition</td>
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			<td>Nikon&#8217;s NiS Elements 6.14 SIM software package</td>
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			<td>Nikon type A immersion oil</td>
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	</tbody>
</table>

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.

Described here is a standardized working protocol for imaging dendritic spines from rat primary hippocampal neurons in vitro using SIM. The protocol workflow and its crucial steps are shown in Figure 1. Overall, the protocol takes approximately 2 weeks of experimental work separated in a first phase of sample preparation, including culture, development and transfection of rat primary hippocampal neurons and immunohistochemistry, and second phase of sample imaging using SIM. The rat primary hippo...

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

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. ...

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15.7K Görüntüleme.  University of Amsterdam.  Bu makale Yapılandırılmış Aydınlatma Mikroskobu (SIM) kullanılarak in vitro hipokampalinden görüntü dendritik dikenler bir çalışma protokolünü açıklar. SIM kullanarak süper çözünürlüklü mikroskopi geliştirilmiş detaylar bireysel dendritik dikenler görüntüleme sağlayan, önemli ölçüde her üç boyutta ışık kırınım sınırı aşan görüntü çözünürlüğü sağlar. 