Name: visualizing adhesion formation in cells by means of advanced spinning disk-total internal reflection fluorescence microscopy
Uploaded: 2019-01-21T14:00:00
Description: Watch this Scientific Journal Video about Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy at JoVE.com

We greatly thank the scientific community of the University Medical Center Hamburg-Eppendorf for supporting us with samples for evaluation. Namely, we thank Sabine Windhorst for NIH3T3 cells, Andrea Mordhorst for YFP-Vinculin and Maren Rudolph for RFP-Lifeact.

1. Preparation of cells
<ol>
	<li>Two days prior to the experiment, seed 3*105 HeLa or NIH3T3 cells in 2 mL of full growth medium per well of a 6-well cell-culture plate. Ensure that cells are handled in a laminar flow hood throughout this protocol.</li>
	<li>One day prior to the experiment, prepare the transfection reagents according to the manufacturer&#8217;s recommendations or an empirically determined protocol, e.g.:
	<ol>
		<li>Dilute 1 &#181;g of RFP-Lifeact and 1 &#181;g of YFP-Vinculin in a...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+performance+in+three-dimensional+fluorescence+microscopy.">Evaluating performance in three-dimensional fluorescence microscopy.</a> Journal of Microscopy. 228 (3), 390-405 (2007).</li><li>Trache, A., Lim, S. -. 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E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Transactions on Image Processing. 7 (1), 27-41 (1998).</li><li>Partridge, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+attachment+and+generation+of+mature+focal+adhesions+by+integrin-containing+filopodia+in+cell+spreading.">Initiation of attachment and generation of mature focal adhesions by integrin-containing filopodia in cell spreading.</a> Molecular Biology of the Cell. 17 (10), (2006).</li><li>Dubin-Thaler, B. J., Giannone, G., D&#246;bereiner, H. G., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanometer+analysis+of+cell+spreading+on+matrix-coated+surfaces+reveals+two+distinct+cell+states+and+STEPs.">Nanometer analysis of cell spreading on matrix-coated surfaces reveals two distinct cell states and STEPs.</a> Biophysical Journal. 86 (3), 1794-1806 (2004).</li><li>Choi, C. K., Vicente-Manzanares, M., Zareno, J., Whitmore, L. A., Mogilner, A., Horwitz, A. 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+and+&#945;-actinin+orchestrate+the+assembly+and+maturation+of+nascent+adhesions+in+a+myosin+II+motor-independent+manner.">Actin and &#945;-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor-independent manner.</a> Nature Cell Biology. 10 (9), 1039-1050 (2008).</li><li>Bachir, A. I., Zareno, J., Moissoglu, K., Plow, E. F., Gratton, E., Horwitz, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-associated+complexes+form+hierarchically+with+variable+stoichiometry+in+nascent+adhesions.">Integrin-associated complexes form hierarchically with variable stoichiometry in nascent adhesions.</a> Current Biology. 24 (16), 1845-1853 (2014).</li><li>Sun, Z., Guo, S. S., F&#228;ssler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-mediated+mechanotransduction.">Integrin-mediated mechanotransduction.</a> Journal of Cell Biology. 215 (4), 445-456 (2016).</li><li>Shao, L., Kner, P., Rego, E. H., Gustafsson, M. G. L. <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+3D+microscopy+of+live+whole+cells+using+structured+illumination.">Super-resolution 3D microscopy of live whole cells using structured illumination.</a> Nat. Methods. 8 (12), 1044-1046 (2011).</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 (6208), 1257998-1257998 (2014).</li><li>Fu, Y., Winter, P. W., Rojas, R., Wang, V., McAuliffe, M., Patterson, G. 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In this paper was presented the first successful implementation of SD and TIRF microscopy in a configuration suitable for performing live cell imaging experiments, i.e. high acquisition rates such as 2 SD-TIRF image stacks per minute at 3 different stage positions, corresponding to a total of 168 frames (circa 3 frames per second), were acquired. The few SD-TIRF microscopes that were described previously12,13, mainly lack of sufficiently high imaging spe...

Our days, light microscopy techniques providing high/super resolution imaging in fixed and living specimen are developing rapidly. Super-resolution techniques such as stimulated emission depletion (STED), structured illumination microscopy (SIM) and photo-activation localization microscopy (PALM) or direct stochastic optical reconstruction microscopy (STORM), respectively, are commercially available and enable imaging of subcellular structures showing details almost on the molecular scale1,2,3,4,5,6. However, these approaches still have limited applicability for live imaging experiments in which large volumes need to be visualized with multiple frames per second acquisition speed. Varieties of highly dynamic processes regulated via the plasma membrane, e.g. endo-/exocytosis, adhesion, migration or signaling, occur with high speed within large cellular volumes. Recently, in order to fill up this gap, an integrated microscopy technique was proposed called spinning disk-TIRF (SD-TIRF)7. In detail, TIRF microscopy permits to specifically isolate and localize the plasma membrane8,9, while SD microscopy is one of the most sensitive and fast live imaging techniques for the visualization and tracking of subcellular organelles in the cytoplasm10,11. The combination of both imaging techniques in a single setup has already been realized in the past12,13, however, the microscope presented here (Figure 1) finally meets the criteria to perform live imaging SD-TIRF experiments of the aforementioned processes at 3 frames per second speed. Since this microscope is commercially available, the goal of this manuscript is to describe in details and provide open source tools and protocols for image acquisition, registration, and visualization associated with SD-TIRF microscopy.
The setup is based on an inverted microscope connected to two scan units via independent ports - the left port is linked to the SD unit and the back port to scanner unit for TIRF and photo-activation/-bleaching experiments. Up to 6 lasers (405/445/488/515/561/640 nm) can be used for excitation. For excitation and detection of the fluorescence signal, either a 100x/NA1.45 oil or 60x/NA1.49 oil TIRF objective, respectively, have been employed. The emitted light is split by a dichroic mirror (561 nm long-pass or 514 nm long-pass) and filtered by various band-pass filters (55 nm wide centered at 525 nm, 54 nm wide centered at 609 nm for green and red fluorescence, respectively) placed in front of the two EM-CCD cameras. Please note that more technical details about the setup are listed in Zobiak et al.7. In TIRF configuration, the SD unit is moved out of the light path within circa 0.5 s so that the same two cameras can be used for detection, allowing faster switching between the two imaging modalities compared to circa 1 s that was reported in the past13. This feature enables dual-channel simultaneous acquisition, thus 4 channels SD-TIRF imaging at previously unmatched speed and accuracy can be performed. Moreover, alignment between SD and TIRF images is unnecessary. Image alignment between the two cameras, however, has to be checked before starting the experiment and corrected if necessary. In the following protocol, a registration correction routine was implemented in a self-written ImageJ macro. Moreover, the macro was mainly designed to allow a simultaneous visualization of SD- and TIRF datasets despite their different dimensionality. The acquisition software itself did not provide these features.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Microscope and accessories</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >SD-TIRF microscope</td>
			<td >Visitron Systems</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Ti with perfect focus system</td>
			<td >Nikon</td>
			<td ></td>
			<td>Inverted microscope stand</td>
		</tr>
		<tr >
			<td >CSU-W1 T2</td>
			<td >Yokogawa</td>
			<td ></td>
			<td>Spinning disk unit in dual-camera configuration</td>
		</tr>
		<tr >
			<td >iLAS2&#160;</td>
			<td >Roper Scientific</td>
			<td ></td>
			<td>TIRF/FRAP scanner</td>
		</tr>
		<tr >
			<td >Evolve&#160;</td>
			<td >Photometrix</td>
			<td ></td>
			<td>EM-CCD cameras</td>
		</tr>
		<tr >
			<td >PiezoZ stage</td>
			<td >Ludl Electronic Products</td>
			<td ></td>
			<td>Motorized Z stage</td>
		</tr>
		<tr >
			<td >Bioprecision2 XY stage</td>
			<td >Ludl Electronic Products</td>
			<td ></td>
			<td>Motorized XY stage</td>
		</tr>
		<tr >
			<td >Stage top incubation chamber</td>
			<td >Okolab</td>
			<td >Bold Line</td>
			<td>Temperature, CO2 and humidity supply</td>
		</tr>
		<tr >
			<td >Cell culture</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >HeLa cervical cancer cells</td>
			<td >DSMZ</td>
			<td >ACC-57</td>
			<td></td>
		</tr>
		<tr >
			<td >NIH3T3 fibroblasts</td>
			<td >DSMZ</td>
			<td >ACC-59</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s phosphate buffered saline (PBS)</td>
			<td >Gibco</td>
			<td >14190144</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin-EDTA 0.05%</td>
			<td >Gibco</td>
			<td >25300054</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Modified Eagle Medium + GlutaMAX-I (DMEM)</td>
			<td >Gibco</td>
			<td >31966-021</td>
			<td></td>
		</tr>
		<tr >
			<td >OptiMEM</td>
			<td >Gibco</td>
			<td >31985070</td>
			<td>Reduced serum medium</td>
		</tr>
		<tr >
			<td >Fetal calf serum (FCS)</td>
			<td >Gibco</td>
			<td >10500064</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin/Streptomycin (PenStrep)</td>
			<td >Gibco</td>
			<td >15140148</td>
			<td></td>
		</tr>
		<tr >
			<td >Full growth medium (DMEM supplemented with 10% FCS and 1% PenStrep)</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >TurboFect</td>
			<td >ThermoFisher Scientific</td>
			<td >R0531</td>
			<td>Transfection reagent</td>
		</tr>
		<tr >
			<td >Ascorbic acid (AA)</td>
			<td >Sigma</td>
			<td >A544-25G</td>
			<td></td>
		</tr>
		<tr >
			<td >6-well cell culture plate</td>
			<td >Sarstedt</td>
			<td >83.392</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass bottom dishes</td>
			<td >MatTek</td>
			<td >P35G-1.5-10-C</td>
			<td>35mm, 0.17mm glass coverslip</td>
		</tr>
		<tr >
			<td >Fibronectin, bovine plasma</td>
			<td >ThermoFisher Scientific</td>
			<td >33010018</td>
			<td></td>
		</tr>
		<tr >
			<td >Neubauer improved chamber</td>
			<td >VWR</td>
			<td >631-0696</td>
			<td></td>
		</tr>
		<tr >
			<td >TetraSpeck beads</td>
			<td >ThermoFisher Scientific</td>
			<td >T7279</td>
			<td></td>
		</tr>
		<tr >
			<td >Plasmids</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >RFP-Lifeact</td>
			<td ></td>
			<td ></td>
			<td>Maren Rudolph, Institute of Medical Microbiology, University Medical Center Hamburg Eppendorf, Germany</td>
		</tr>
		<tr >
			<td >YFP-Vinculin</td>
			<td ></td>
			<td ></td>
			<td>Andrea Mordhorst, Institute of Medical Microbiology, University Medical Center Hamburg Eppendorf, Germany</td>
		</tr>
		<tr >
			<td >Software and plugins</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >VisiView</td>
			<td >Visitron Systems</td>
			<td ></td>
			<td>Version 3</td>
		</tr>
		<tr >
			<td >ImageJ</td>
			<td ></td>
			<td ></td>
			<td>Version 1.52c</td>
		</tr>
		<tr >
			<td >Turboreg plugin</td>
			<td ></td>
			<td ></td>
			<td>http://bigwww.epfl.ch/thevenaz/turboreg/</td>
		</tr>
		<tr >
			<td >Macro &#34;SD-TIRF_helper_JoVE.ijm&#34;</td>
			<td >this publication</td>
			<td ></td>
			<td>https://github.com/bzobiak/ImageJ</td>
		</tr>
		<tr >
			<td >Volocity</td>
			<td >PerkinElmer</td>
			<td ></td>
			<td >Version 6.2.2</td>
		</tr>
	</tbody>
</table>

visualizing adhesion formation in cells by means of advanced spinning disk-total internal reflection fluorescence microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

In order to show the potential of SD-TIRF imaging, an assay was developed that should reveal the spatio-temporal organization of cell-matrix adhesion complexes and their interaction with the cytoskeleton during cellular adhesion. Therefore, adherent HeLa or, alternatively, NIH3T3 cells were transfected with YFP-Vinculin and RFP-Lifeact for 18-24 h, trypsinized and seeded onto fibronectin-coated glass bottom dishes. These cell lines were chosen for their pronounced cytoskeleton and higher ...

Watch this Scientific Journal Video about Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy at JoVE.com

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

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Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations ...