YW and PK gratefully acknowledge funding support from the Singapore National Research Foundation, awarded to PK (NRF-NRFF-2011-04 and NRF2012NRF-CRP001-084). We also thank the MBI open lab and microscopy core facilities for infrastructure support.

1. Imaging Specimen Preparation
<ol>
	<li>Since background fluorescence signals interfere with fluorescence from fluorophore labels, clean the coverglasses by first rinsing them in de-ionized water (ddH2O) and then air-drying them using compressed air. Subsequently, perform plasma etching in a plasma cleaner for 15 sec, or longer if necessary.</li>
	<li>To enable drift correction and iPALM calibration, use #1.5 round (22-mm diameter) pre-cleaned coverglasses embedded with fluorescent nanoparticles as fiducia...

<ol>
	<li>Kanchanawong, P., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization-based+super-resolution+imaging+of+cellular+structures.">Localization-based super-resolution imaging of cellular structures.</a> Methods Mol Biol. 1046, 59-84 (2013).</li><li>Bertocchi, C., Goh, W. I., Zhang, Z., Kanchanawong, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+imaging+by+super+resolution+fluorescence+microscopy+and+its+emerging+applications+in+biomedical+research.">Nanoscale imaging by super resolution fluorescence microscopy and its emerging applications in biomedical research.</a> Crit Rev Biomed Eng. 41, 281-308 (2013).</li><li>Kanchanawong, P., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+light-based+imaging+of+three-dimensional+cellular+ultrastructure.">Advances in light-based imaging of three-dimensional cellular ultrastructure.</a> Curr Opin Cell Biol. 24, 125-133 (2012).</li><li>Betzig, E., 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=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313, 1642-1645 (2006).</li><li>Hess, S. T., Girirajan, T. P., Mason, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-high+resolution+imaging+by+fluorescence+photoactivation+localization+microscopy.">Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.</a> Biophys J. 91, 4258-4272 (2006).</li><li>Rust, M. J., Bates, M., 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=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nat Methods. 3, 793-795 (2006).</li><li>Heilemann, 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=Subdiffraction-resolution+fluorescence+imaging+with+conventional+fluorescent+probes.">Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes.</a> Angew Chem Int Edit. 47, 6172-6176 (2008).</li><li>Sharonov, A., Hochstrasser, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wide-field+subdiffraction+imaging+by+accumulated+binding+of+diffusing+probes.">Wide-field subdiffraction imaging by accumulated binding of diffusing probes.</a> P Natl Acad Sci USA. 103, 18911-18916 (2006).</li><li>F&#246;lling, J., Bossi, M., Bock, H., Medda, R., Wurm, C. A., Hein, B., Jakobs, S., Eggeling, C., Hell, S. W. <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+by+ground-state+depletion+and+single-molecule+return.">Fluorescence nanoscopy by ground-state depletion and single-molecule return.</a> Nat Methods. 5, 943-945 (2008).</li><li>Biteen, 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=Single-moldecule+active-control+microscopy+(SMACM)+with+photo-reactivable+EYFP+for+imaging+biophysical+processes+in+live+cells.">Single-moldecule active-control microscopy (SMACM) with photo-reactivable EYFP for imaging biophysical processes in live cells.</a> Nat Methods. 5, 947-949 (2008).</li><li>Shtengel, 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=Interferometric+fluorescent+super-resolution+microscopy+resolves+3D+cellular+ultrastructure.">Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure.</a> P Natl Acad Sci USA. 106, 3125-3130 (2009).</li><li>Huang, B., Wang, W., Bates, M., 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=Three-dimensional+super-resolution+imaging+by+stochastic+optical+reconstruction+microscopy.">Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy.</a> Science. 319, 810-813 (2008).</li><li>Dani, A., Huang, B., Bergan, J., Dulac, C., 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=Super+resolution+imaging+of+chemical+synapses+in+the+brain.">Super resolution imaging of chemical synapses in the brain.</a> Neuron. 68, 843-856 (2010).</li><li>Liu, 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=Talin+determines+the+nanoscale+architecture+of+focal+adhesions.">Talin determines the nanoscale architecture of focal adhesions.</a> P Natl Acad Sci USA. 112 (35), E4864-E4873 (2015).</li><li>Kanchanawong, 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=Nanoscale+architecture+of+integrin-based+cell+adhesions.">Nanoscale architecture of integrin-based cell adhesions.</a> Nature. 468, 580-584 (2010).</li><li>Wu, Y., Kanchanawong, P., Zaidel-Bar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin-delimited+adhesion-independent+clustering+of+e-cadherin+forms+the+nanoscale+building+blocks+of+adherens+junctions.">Actin-delimited adhesion-independent clustering of e-cadherin forms the nanoscale building blocks of adherens junctions.</a> Dev Cell. 32 (2), 139-154 (2015).</li><li>Szymborska, 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=Nuclear+Pore+Scaffold+Structure+Analyzed+by+Super-Resolution+Microscopy+and+Particle+Averaging.">Nuclear Pore Scaffold Structure Analyzed by Super-Resolution Microscopy and Particle Averaging.</a> Science. 341, 655-658 (2013).</li><li>Lawo, S., Hasegan, M., Gupta, G. D., Pelletier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subdiffraction+imaging+of+centrosomes+reveals+higher-order+organizational+features+of+pericentriolar+material.">Subdiffraction imaging of centrosomes reveals higher-order organizational features of pericentriolar material.</a> Nat Cell Biol. 14, 1148-1158 (2012).</li><li>Mennella, V., Agard, D. A., Huang, B., Pelletier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amorphous+no+more:+subdiffraction+view+of+the+pericentriolar+material+architecture.">Amorphous no more: subdiffraction view of the pericentriolar material architecture.</a> Trends Cell Biol. 24, 188-197 (2014).</li><li>Mennella, 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=Subdiffraction-resolution+fluorescence+microscopy+reveals+a+domain+of+the+centrosome+critical+for+pericentriolar+material+organization.">Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for pericentriolar material organization.</a> Nat Cell Biology. 14, 1159-1168 (2012).</li><li>Fletcher, D. A., Mullins, 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=Cell+mechanics+and+the+cytoskeleton.">Cell mechanics and the cytoskeleton.</a> Nature. 463, 485-492 (2010).</li><li>Salbreux, G., Charras, G., Paluch, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+cortex+mechanics+and+cellular+morphogenesis.">Actin cortex mechanics and cellular morphogenesis.</a> Trends Cell Biol. 22, 536-545 (2012).</li><li>Shtengel, 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=Imaging+cellular+ultrastructure+by+PALM,+iPALM,+and+correlative+iPALM-EM.">Imaging cellular ultrastructure by PALM, iPALM, and correlative iPALM-EM.</a> Method Cell Biol. 123, 273-294 (2014).</li><li>Juette, M. F., 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=Three-dimensional+sub-100+nm+resolution+fluorescence+microscopy+of+thick+samples.">Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples.</a> Nat Methods. 5, 527-529 (2008).</li><li>Lippincott-Schwartz, J., Patterson, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivatable+fluorescent+proteins+for+diffraction-limited+and+super-resolution+imaging.">Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging.</a> Trends Cell Biol. 19, 555-565 (2009).</li><li>Shannon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Communication+in+the+presence+of+noise.">Communication in the presence of noise.</a> Proc. IRE. 37, 10-21 (1949).</li><li>Good, N. E., 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=Hydrogen+ion+buffers+for+biological+research.">Hydrogen ion buffers for biological research.</a> Biochemistry. 5, 467-477 (1966).</li><li>Aitken, C. E., Marshall, R. A., Puglisi, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+oxygen+scavenging+system+for+improvement+of+dye+stability+in+single-molecule+fluorescence+experiments.">An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments.</a> Biophys J. 94, 1826-1835 (2008).</li><li>Shin, W. D., Goldman, R. D., Swedlow, J. R., Spector, D. 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> Live Cell Imaging: A Laboratory Manual. , (2010).</li><li>Aquino, 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=Two-color+nanoscopy+of+three-dimensional+volumes+by+4Pi+detection+of+stochastically+switched+fluorophores.">Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores.</a> Nat Methods. 8, 353-359 (2011).</li><li>Dempsey, G. T., Vaughan, J. C., Chen, K. H., Bates, M., 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=Evaluation+of+fluorophores+for+optimal+performance+in+localization-based+super-resolution+imaging.">Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging.</a> Nat Methods. 8, 1027-1036 (2011).</li><li>Pertsinidis, A., Zhang, Y., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subnanometre+single-molecule+localization,+registration+and+distance+measurements.">Subnanometre single-molecule localization, registration and distance measurements.</a> Nature. 466, 647-651 (2010).</li><li>Baddeley, D., Cannell, M. B., Soeller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+Localization+Microscopy+Data.">Visualization of Localization Microscopy Data.</a> Microsc Microanal. 16, 64-72 (2010).</li><li>El Beheiry, M., Dahan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ViSP:+representing+single-particle+localizations+in+three+dimensions.">ViSP: representing single-particle localizations in three dimensions.</a> Nat Methods. 10, 689-690 (2013).</li><li>Schnell, U., Dijk, F., Sjollema, K. A., Giepmans, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolabeling+artifacts+and+the+need+for+live-cell+imaging.">Immunolabeling artifacts and the need for live-cell imaging.</a> Nat Methods. 9, 152-158 (2012).</li><li>Shroff, H., Galbraith, C. G., Galbraith, J. A., Betzig, E. <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+photoactivated+localization+microscopy+of+nanoscale+adhesion+dynamics.">Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics.</a> Nat Methods. 5, 417-423 (2008).</li><li>Yamashiro, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+single-molecule+speckle+microscopy+reveals+modification+of+the+retrograde+actin+flow+by+focal+adhesions+at+nanometer+scales.">New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales.</a> Mol Biol Cell. 25, 1010-1024 (2014).</li><li>Shroff, 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=Dual-color+super+resolution+imaging+of+genetically+expressed+probes+within+individual+adhesion+complexes.">Dual-color super resolution imaging of genetically expressed probes within individual adhesion complexes.</a> P Natl Acad Sci USA. 104, 20308-20313 (2007).</li><li>Chen, B. 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=Lattice+light-sheet+microscopy:+imaging+molecules+to+embryos+at+high+spatiotemporal+resolution.">Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.</a> Science. 346, (2014).</li><li>Brown, T. 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=Super+resolution+fluorescence+imaging+of+mitochondrial+nucleoids+reveals+their+spatial+range,+limits,+and+membrane+interaction.">Super resolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction.</a> Mol Cell Biol. 31, 4994-5010 (2011).</li><li>Huang, F., 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=Video-rate+nanoscopy+using+sCMOS+camera-specific+single-molecule+localization+algorithms.">Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms.</a> Nat Methods. 10, 653-658 (2013).</li><li>Daostorm, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DAOSTORM:+an+algorithm+for+high-density+super-resolution+microscopy.">DAOSTORM: an algorithm for high-density super-resolution microscopy.</a> Nat methods. 8, 279 (2011).</li><li>Zhu, L., Zhang, W., Elnatan, D., Huang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Faster+STORM+using+compressed+sensing.">Faster STORM using compressed sensing.</a> Nat Methods. 9, 721-723 (2012).</li><li>Van Engelenburg, S. B., 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=Distribution+of+ESCRT+machinery+at+HIV+assembly+sites+reveals+virus+scaffolding+of+ESCRT+subunits.">Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits.</a> Science. 343, 653-656 (2014).</li><li>Vaughan, J. C., Jia, S., 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=Ultrabright+photoactivatable+fluorophores+created+by+reductive+caging.">Ultrabright photoactivatable fluorophores created by reductive caging.</a> Nat Methods. 9, 1181-1184 (2012).</li></ol>

The authors have nothing to disclose.

The optical system of iPALM is based on a 4-&#960; dual-opposed objective design, as shown in Figure 1A. The setup is constructed using both custom-machined and commercial opto-mechanical parts, as described earlier23 and listed in Table 1. In addition to our setup, the Howard Hughes Medical Institute (HHMI) hosts a system that is accessible to the scientific community at the Advanced Imaging Center at the Janelia Research Campus. For the full mechanical drawi...

The visualization of complex cellular structures has long been integral to biological insights and discovery. Although fluorescence microscopy can image cells with high molecular specificity, its resolving power is limited by diffraction to ~ 200 nm in the image plane (x,y, or lateral dimension) and &#62; 500 nm along the optical axis (z, or axial dimension)1,2. Hence, the observation of ultrastructural features has historically been limited to electron microscopy (EM). Fortunately, the recent development of super resolution microscopy has circumvented this limit, enabling spatial resolution in the 10 - 100 nm range1-6. In particular, super resolution approaches based on single molecule localization, known by acronyms such as PALM (PhotoActivated Localization Microscopy)4, FPALM (Fluorescence PhotoActivated Localization Microscopy)5 (d)STORM (direct Stochastic Optical Reconstruction Microscopy)6,7, PAINT (Point Accumulation for Imaging Nanoscale Topography)8, GSDIM (Ground State Depletion Microscopy followed by individual molecular return)9, or SMACM (Single-Molecule Active-Control Microscopy)10, as well as their 3-dimensional (3D) implementations, such as interferometric PALM (iPALM)11 or 3D-STORM12, have been valuable in revealing novel insights into the nanoscale organization of numerous biological structures, including neuronal axons and synapses13, focal adhesions14,15, cell-cell junctions16, nuclear pores17, and centrosomes18-20, to name a few.
Another ultrastructural feature in cells for which super resolution microscopy is potentially useful is the actin cytoskeleton. The complex meshwork of filamentous (f)-actin in the cell cortex plays an essential role in the control of cellular shape and mechanical properties21. The organization of f-actin is actively and dynamically regulated though numerous regulatory proteins that strongly influence polymerization, crosslinking, turnover, stability, and network topology22. However, although the characterization of the f-actin meshwork architecture is important for mechanistic insights into a diverse range of cellular processes, the small size (~ 8 nm) of the f-actin filaments hampers their observation by conventional diffraction-limited light microscopy; thus, the visualization of actin fine structure has hitherto been exclusively performed by EM. Here, we describe protocols for visualizing the f-actin cytoskeleton in adherent mammalian cells, using the iPALM super resolution microscopy technique to take advantage of its very high precision capability in 3D11,23. Although the iPALM instrument is highly specialized, instruction on setting up such an instrument has been described recently23, while access to the iPALM microscope hosted by the Howard Hughes Medical Institute has also been made available to the research community with minimal cost. Additionally, the specimen preparation methods described herein are directly applicable to alternative 3D super resolution approaches, such as those based on astigmatic defocusing of the point spread function (PSF)12 or bi-plane detection24, which are more broadly available.
We note that a necessary ingredient for single-molecule localization-based super resolution microscopy in general is the photoswitchable fluorophore25, which allows the three critical requirements for single-molecule localization-based super resolution microscopy to be fulfilled: i) high single-molecule brightness and contrast relative to background signals; ii) sparse distribution of single molecules in a given image frame; and iii) high spatial density of labeling sufficient to capture the profile of the underlying structure (also known as Nyquist-Shannon sampling criterion)26. Thus, for satisfactory results, emphasis should be placed equally on both the proper preparation of specimens to optimize fluorophore photoswitching and to preserve the underlying ultrastructure, as well as on the instrumentation and acquisition aspects of the experiments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>optical table</td>
			<td>Newport, CA&#160;</td>
			<td>RS4000</td>
			<td>iPALM, installed on 4 Newport Stabilizer vibration isolators&#160;</td>
		</tr>
		<tr>
			<td>vibration isolator for optical table</td>
			<td>Newport, CA&#160;</td>
			<td>S-2000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>laser-642</td>
			<td>Newport, CA&#160;</td>
			<td>1185055</td>
			<td>output power=100mw</td>
		</tr>
		<tr>
			<td>laser-561</td>
			<td>Newport, CA&#160;</td>
			<td>1168931</td>
			<td>output power=200mw</td>
		</tr>
		<tr>
			<td>laser-488</td>
			<td>Newport, CA&#160;</td>
			<td>1137970</td>
			<td>output power=200mw</td>
		</tr>
		<tr>
			<td>laser-405</td>
			<td>Newport, CA&#160;</td>
			<td>1142279</td>
			<td>output power=100mw</td>
		</tr>
		<tr>
			<td>broadband dielectric mirrors&#160;</td>
			<td>Thorlabs, NJ</td>
			<td>BB1-E02</td>
			<td>laser combiner</td>
		</tr>
		<tr>
			<td>dichroic beamsplitter&#160;</td>
			<td>Semrock, NY</td>
			<td>LM01-427-25</td>
			<td></td>
		</tr>
		<tr>
			<td>acousto-optic tunable filter&#160;</td>
			<td>AA Opto-Electronic, France</td>
			<td>AOTFnC-VIS-TN</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear polarizer</td>
			<td>Newport, CA&#160;</td>
			<td>05LP-VIS-B</td>
			<td></td>
		</tr>
		<tr>
			<td>baseplate</td>
			<td>local workshop</td>
			<td>customized</td>
			<td></td>
		</tr>
		<tr>
			<td>turning mirror (22.5&#176;)</td>
			<td>Reynard Corpporation, CA</td>
			<td>customized</td>
			<td>22.5&#176; mirror</td>
		</tr>
		<tr>
			<td>motorized optic mounts&#160;</td>
			<td>New Focus, CA</td>
			<td>8816</td>
			<td></td>
		</tr>
		<tr>
			<td>motorized XYZ translation stage</td>
			<td>Thorlabs, NJ</td>
			<td>MT3/M-Z6</td>
			<td>sample holder</td>
		</tr>
		<tr>
			<td>T-Cube DC servo motor controller</td>
			<td>Thorlabs, NJ</td>
			<td>TDC001</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezo Phase Shifter</td>
			<td>Physik Instrumente, Germany</td>
			<td>S-303.CD</td>
			<td></td>
		</tr>
		<tr>
			<td>objective lens</td>
			<td>Nikon, Japan</td>
			<td>MRD01691</td>
			<td>objective. Apo TIRF 60X/1.49oil</td>
		</tr>
		<tr>
			<td>translation stage</td>
			<td>New Focus, CA</td>
			<td>9062-COM-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pico Motor Actuator</td>
			<td>New Focus, CA</td>
			<td>8301</td>
			<td></td>
		</tr>
		<tr>
			<td>rotary Solenoid/Shutter</td>
			<td>DACO Instruments, CT</td>
			<td>5423-458</td>
			<td></td>
		</tr>
		<tr>
			<td>3-way beam splitter</td>
			<td>Rocky Mountain Instruments, CO</td>
			<td>customized</td>
			<td>beamsplitter</td>
		</tr>
		<tr>
			<td>Piezo Z/Tip/Tilt scanner</td>
			<td>Physik Instrumente, Germany</td>
			<td>S-316.10</td>
			<td></td>
		</tr>
		<tr>
			<td>motorized five-axis tilt aligner&#160;</td>
			<td>New Focus, CA</td>
			<td>8081</td>
			<td></td>
		</tr>
		<tr>
			<td>Picmotor ethernet controller</td>
			<td>New Focus, CA</td>
			<td>8752</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezo controllers/amplifier/digital operation module</td>
			<td>Physik Instrumente, Germany</td>
			<td>E-509/E-503/E-517</td>
			<td></td>
		</tr>
		<tr>
			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-523/20</td>
			<td>filters</td>
		</tr>
		<tr>
			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-588/21</td>
			<td></td>
		</tr>
		<tr>
			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-607/30</td>
			<td></td>
		</tr>
		<tr>
			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-676/37</td>
			<td></td>
		</tr>
		<tr>
			<td>notch filter</td>
			<td>Semrock, NY</td>
			<td>NF01-405/488/561/635</td>
			<td></td>
		</tr>
		<tr>
			<td>motorized filter wheel with controllter</td>
			<td>Thorlabs, NJ</td>
			<td>FW103H</td>
			<td></td>
		</tr>
		<tr>
			<td>EMCCD</td>
			<td>Andor, UK&#160;</td>
			<td>DU-897U-CSO-#BV</td>
			<td>3 sets</td>
		</tr>
		<tr>
			<td>Desktop computers for controlling cameras and synchronization</td>
			<td>Dell</td>
			<td>Precision T3500</td>
			<td>PC, 4 sets</td>
		</tr>
		<tr>
			<td>coverslips with fiducial</td>
			<td>Hestzig, VA</td>
			<td>600-100AuF</td>
			<td>sample preparation. fiducial marks with various density and spectra&#160; available</td>
		</tr>
		<tr>
			<td>fibronectin&#160;</td>
			<td>Millipore, MT</td>
			<td>FC010</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Electron Microscopy Sciences, PA</td>
			<td>15710</td>
			<td>fixation. 16%</td>
		</tr>
		<tr>
			<td>glutaraldehyde&#160;</td>
			<td>Electron Microscopy Sciences, PA</td>
			<td>16220</td>
			<td>25%</td>
		</tr>
		<tr>
			<td>triton X-100</td>
			<td>Sigma aldrich, MO</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>HUVEC cells</td>
			<td>Life Technologies, CA</td>
			<td>C-015-10C</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 200</td>
			<td>Life Technologies, CA</td>
			<td>M-200-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Vessel Endothelial Factors</td>
			<td>Life Technologies, CA</td>
			<td>A14608-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td></td>
			<td>14190367</td>
			<td></td>
		</tr>
		<tr>
			<td>Pennicillin/Streptomycin</td>
			<td></td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Life Technologies, CA</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma aldrich, MO</td>
			<td>P1851</td>
			<td>PHEM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>1st base, Malaysia</td>
			<td>BIO-1825</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma aldrich, MO</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Millipore, MT</td>
			<td>5985</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Alexa Fluor 647 Phalloidin</td>
			<td>Invitrogen, CA</td>
			<td>A22287</td>
			<td>staining</td>
		</tr>
		<tr>
			<td>sodium borohydride (NaBH4)&#160;</td>
			<td>Sigma aldrich, MO</td>
			<td>480886</td>
			<td>quenching</td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>1st base, Malaysia</td>
			<td>BIO-1101</td>
			<td>imaging buffer</td>
		</tr>
		<tr>
			<td>glucose oxidase</td>
			<td>Sigma aldrich, MO</td>
			<td>G2133</td>
			<td></td>
		</tr>
		<tr>
			<td>catalase</td>
			<td>Sigma aldrich, MO</td>
			<td>C9322</td>
			<td></td>
		</tr>
		<tr>
			<td>cysteamine&#160;</td>
			<td>Sigma aldrich, MO</td>
			<td>30070</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Thorlabs, NJ</td>
			<td>G14250</td>
			<td></td>
		</tr>
		<tr>
			<td>vaseline</td>
			<td>Sigma aldrich, MO</td>
			<td>16415</td>
			<td>sample sealing</td>
		</tr>
		<tr>
			<td>lanolin</td>
			<td>Sigma aldrich, MO</td>
			<td>L7387</td>
			<td></td>
		</tr>
		<tr>
			<td>parafin wax</td>
			<td>Sigma aldrich, MO</td>
			<td>327204</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion oil&#160;</td>
			<td>Electron Microscopy Sciences, PA</td>
			<td>16915-04</td>
			<td>imaging. Cargille Type HF</td>
		</tr>
	</tbody>
</table>

three-dimensional super resolution microscopy of f-actin filaments by interferometric photoactivated localization microscopy (ipalm)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and &#62; 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Critical requirements for iPALM are the alignment, registration, and calibration of the optical systems. These are necessary to ensure proper interference within the 3-way beam splitter requisite for z-coordinate extraction. To enable continuous monitoring, constant point sources of fluorescence are necessary. This can be achieved using fluorescent Au or bi-metallic nanoparticles23 whose photoluminescence arise from localized surface plasmon resonance (LSPR). They act as a stab...

Watch this Scientific Journal Video about Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM at JoVE.com

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

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

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

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

The overall goal of this procedure is to visualize the actin cytoskeleton in adherent mammalian cells at the ultrastructural level, using a three-dimensional super resolution microscopy method called iPALM. iPALM imaging provides sub-trending nanometer spatial resolution in all three dimensions. This enables ultrastructures to be visualized using florescence probes, allowing high labeling specificity.The procedure presented in this video should be rapidly adaptable and instructive for visualizing other ultrastructural features in cells by single molecule localization based in super resolution microscopy. Begin this procedure by preparing the cells on cover glasses in a six well plate. To assemble the imaging sample, use fine forceps to gently remove the specimen cover glass from the buffer well.Then quickly and gently tap away excess buffer by touching the edge of the cover glass with folded, absorbent paper. Place the cover glass cell side facing up on a piece of clean lens paper. Rinse the sample by placing 30 to 50 microliters of the imaging buffer onto the sample.Remove excess buffer by tilting and tapping with folded absorbent paper. After repeating the rinsing step a few times, place 30 to 50 microliters of imaging buffer onto the sample. Blot dry the edge of the cover glass and place multiple very small dots of fast curing epoxy onto the dried area.Slowly lower another pre-cleaned number 1.5 cover glass onto the center of the cell containing 22 millimeter cover glass. Let the imaging buffer wet both cover glasses by capillary action. The small dots of fast curing epoxy should adhere to both cover glasses.Gently press upon the assembled sample using folded absorbent paper to spread the pressure evenly. Use sufficient pressure to make the sample cell thin and even, but not so much as to crush the cells. Gauge the proper thickness by observing the Newton's rings pattern.Make sure to perform this step gently to minimize air bubbles. If needed, practice with empty cover glasses several times beforehand. Next, seal the sample with melted Vaseline lanolin paraffin.Then rinse the sealed sample with deionized water. Blow dry the sample with compressed air to make ready for mounting onto the microscope. Move the spring-loaded top objective lens upward to allow for the removal of the sample holder.Place the sealed specimen onto the sample holder and secure with several small rare-earth magnets. Apply immersion oil on both sides of the imaging sample. Place the sample holder back into the optical path and gently lower the top objective lens.Turn on the excitation lasers. Also turn on the EMCCD cameras in frame transfer mode. Next, rotate in the proper emission filters.Activate a mechanical shutter to block the top beam path and open the bottom beam path. Bring the bottom objective lens into focus by translation in small increments using the piezo actuator. Once the fiducial is in focus, open the top beam path while blocking the bottom beam path and bring the top objective lens into focus in a similar manner.Monitor the width of the fiducial on the computer display for optimal focus. For proper centration, open both the top and bottom beam paths. Manually adjust the top objective lens while the bottom objective is held constant using a pair of microfine set screws until the fiducial images are overlapped as closely as possible, ideally within one pixel.Subsequently, perform fine adjustments so that the fiducial images overlap within one tenth of an EMCCD pixel. Adjust the top two axis piezo mounted mirrors via the control software while holding both objective lenses and the bottom reflection mirrors constant. Compare the centers of the fiducials of the top and bottom objective views via the computer display to guide the process.With the lasers on, the cameras continuously streaming, and both the top and bottom beam paths open, oscillate the sample holder Z piezo using a sinusoidal voltage waveform generated by the control software for a continuous Z-axis oscillation over a magnitude of 400 nanometers. Manually translate the motorized beam splitter assembly up or down until the intensity of the fiducial oscillates. Due to the desired single photon interference effect, this signifies the close matching of the optical path lengths.A peak to valley ratio of greater than 10 can be achieved in optimal cases. To ensure that both the amplitude and phase at each surface are as uniform as possible across the field, adjust the bottom mirror of the beam splitter assembly in small steps to fine tune the gap and the tilt angles. Perform beam splitter fine alignment by translating the sample in eight nanometer Z steps over 800 nanometers.Monitor the fiducial intensity among cameras one through three. Adjust the height, position, and tilt of the bottom mirror within the beam splitter assembly in small steps such that the oscillation phase of camera one relative to camera two is maximized, ideally at 120 degrees. Once the initial alignment is complete, translate the sample to scan for a suitable field of view that contains both cells to image and multiple fiducials nearby.Place an enclosure around the system to block stray light and ambient perturbations. Once the imaging area is found, translate the sample again and record the calibration curve for use in subsequent Z coordinate extractions. Using the command acquire calibration scans versus sample piezo position in the main interface.Once a desired area is found and the calibration curve is obtained, enter the appropriate file names into the software. Open both the top and bottom beam paths, then increase the excitation power of the 642 nanometer laser to the maximum. For Alexa Fluor 647, an initial period of Fluor-4 switching off may be needed.Expose with a constant 642 nanometer excitation for five minutes, or longer if necessary, until single molecule blinking is observed. The software allows an automatic increase of 405 nanometer photoactivation during the course of the acquisition. Large numbers of acquisition frames are usually required for filamentous features to be clearly visible.When ready, commence the acquisition of raw image sets using the command start iPALM acquisition in the main interface. During the acquisition, tune the photo activation level by adjusting the intensity of the 405 nanometer laser to maintain the proper blinking density as needed. When the sample cover slip is assembled properly, Newton's rings can be observed by naked eyes under ambient light or standard fluorescent lighting.Extraction of the axial positions of fluorescence emitters requires simultaneous multi-phase interference in iPALM imaging, which is calibrated by the intensity changes of a given fiducial in three EMCCDs. The intensities are then normalized and fit to determine the phase differences. Here is the reconstruction iPALM image with each localization coordinate rendered by a 2D Gaussian with the width corresponding to localization uncertainty.The Z coordinate is colored according to the color bar. Areas of dense and sparse filamentous features can be seen. Shown here are reconstruction iPALM images of actin cytoskeleton of HUVEC cells labeled by Alexa Fluor 647 phalloidin.The image color indicates the Z coordinates according to the color bar. The transverse cross section histogram is generated from the XY localization coordinates along the long axis of the boxed area. Gaussian fitting shows full-width at half maximum of 43.88 nanometers associated with the transverse cross section.This histogram is of the Z position of localization coordinates in the boxed area. Gaussian fitting shows full-width at half maximum of 17.50 nanometers. Once mastered, this technique can be done in 90 minutes if it is performed properly.After watching this vide, you should have a understanding of how to visualize the actin cytoskeleton at the ultrastructural using iPALM through simple preparation and mounting, optical alignment, raw data acquisition, and the subsequent analysis for reconstructing super resolution images. After its development, this technique paved the way for researches in the field of cell biology to explore the nanoscale architecture of the actin cytoskeleton, as well as cell adhesion structures such as the focal adhesions.

three-dimensional-super-resolution-microscopy-f-actin-filaments

Research

JoVE Journal

Biology

10.6K vistas.  South University of Science and Technology of China, Shenzhen. The overall goal of this procedure is to visualize the actin cytoskeleton in adherent mammalian cells at the ultrastructural level, using a three-dimensional super resolution microscopy method called iPALM. iPALM imaging provides sub-trending nanometer spatial resolution in all three dimensions. This enables ultrastructures to be visualized using florescence probes, allowing high labeling specificity.The procedure presented in this video should be rapidly adaptable and instructive for ...