Name: three-dimensional super resolution microscopy of f-actin filaments by interferometric photoactivated localization microscopy (ipalm)
Uploaded: 2016-12-01T12:30:00
Description: Watch this Scientific Journal Video about Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM at JoVE.com

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>
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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 ...

Research

JoVE Journal

Biology

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...