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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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. 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The authors have nothing to disclose.

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 ...

Spinning disk TIRF microscopy can be a useful tool for studying the role of protein during cytoskeletal network formation. This technique allowed three-dimensional imaging of fast cellular approaches that occur in the cell and at the same the precise localization of fluorescence molecule that are placed at the plasma membrane. This method can provide insight to research areas like microbiology, cell biology, and all those branches where it's important to study the interaction between the plasma membrane and the cellular environment.This method can be extended to those live imaging experiments where it's important to localize the structures in the plasma membrane and where you also want to maintain a high resolution of the remaining cellular volume. The crucial things one has to consider are the choice of the proper cell line to set up the TIRF illumination and the other imaging parameters and to find the focus of the transfected cells. The visual demonstration of this protocol would certainly help to avoid problems with the handling of the cells during image acquisition.Two days prior to the experiment, seed 3, 000 HELA or NIH 3T3 cells in two milliliters of full growth medium into each well of a six-well cell culture plate. The next day, prepare the transfection reagents. First, dilute one microgram of RFP Livact and one microgram of YFP Vinculin in a total of 200 microliters of reduced serum medium.Vortex the transfection reagent briefly. Then add four microliters of the mixture to 200 microliters of DNA. Vortex the reagents again and then incubate the transfection mix for 15 to 20 minutes at room temperature.After incubation, add the entire transfection mix drop-wise directly to the cells. Mix the wells by shaking the plate and then place back into the incubator. On the day of the experiment, prepare the sample for live imaging by first coating a 35 millimeter glass bottom dish with a 10 microgram per milliliter solution of Fibronectin in PBS.Leave the solution on the glass surface for 30 minutes at room temperature then remove it and let the dish air dry. Next, dilute a 0.1 micron diameter multi-fluorescent bead solution to a density of 1.8 billion particles per milliliter in distilled water. Add the solution to the Fibronectin-coated glass surface for 30 to 60 seconds.Then remove the solution and let the dish air dry. Precoating of the dishes with fluorescent beads is only necessary for two reasons, first if you want to find the TIRF plane before seeding the cells or to acquire a two-colored bead image for image registration. Now prepare an anti-oxidizing growth medium by adding 0.1 molar ascorbic acid solution at a final concentration of 0.1 millimolar in growth medium.Prewarm the media by placing it in a 37 degree Celsius water bath. Next, wash the cells with two milliliters of PBS and add 250 microliters of Trypsin EDTA to each well of the plate. Incubate the cells at 37 degrees Celsius and wait two to three minutes until the cells are fully detached.Resuspend the cells carefully in one milliliter of prewarmed anti-oxidizing growth medium and add them to an additional four milliliters of the medium in a 15 milliliter cell culture tube. Place the cell suspension with a slightly opened lid in an incubator set at 37 degrees Celsius buffered with 5%carbon dioxide. To begin, start the environmental control of the microscope to achieve stable cell culture conditions.Then place the glass bottom dish containing one milliliter of prewarmed anti-oxidizing growth medium into the sample holder of the preheated microscope. Next, in the imaging software, set up the acquisition settings at the microscope. Set the acquisition time interval to 30 seconds and the duration to between 60 and 90 minutes.Also, activate the autofocusing function of the hardware-based autofocus for every time point by setting the value to one. Now adjust the camera exposure to 200 milliseconds, the gain level to 500, and the laser power to 20%for every channel. High gain levels, low exposure time, and low laser power are recommended to reduce phototoxicity.Next, set the Z-stack for the spinning disk channels to 10 microns with 0.4 micron spacing, then set the bottom offset to zero so that the lowest plane will be the focus position of the hardware autofocus and deactivate the Z-stacks for the TIRF channels. Finally, activate the multi-point function stage positions. This will allow for up to three positions to be recorded over a 30-second interval.Now find the fluorescent beads with epifluorescent illumination, activate one TIRF channel and set the illumination angle to a value that denotes TIRF illumination. Next, activate the autofocus by pushing the button at the microscope panel and adjust the focus with the offset wheel. Once in focus, acquire a two-colored data set using TIRF 488 and TIRF 561 for subsequent bead-based image registration.Remove the cells from the incubator and mix the cell suspension by inverting the closed tube two to three times. Then apply one milliliter of the cells to the imaging dish. Quickly find double transfected cells with low level epifluorescent illumination.Once found, center the cells in the live camera preview using bright-field illumination and mark the position. Then find an additional one to two points of interest, save them to the positions list and begin data acquisition by clicking on the sequence button. In the beginning, the cells can easily detach due to the stage movement so it's better to set four to five positions and later on discard one or two.In order to generate a registration-free hyperstack in Fiji, first download and save the provided file SD-TIRF_helper_jove. ijm in the Fiji subfolder macros. Run the macro by clicking on the menu command starting with plugins then macros and finally run.If the color channels need registration correction, select the option and create a new bead-based registration reference known as a landmark file. Next, import the data with the bioformats importer and choose hyperstack as a viewing option. Load the image data set, select the spinning disk series in the first step and the TIRF series in the second step.At this point, Fiji will display the data sorted by channel and stage position. Apply the registration correction to the respective channels by loading the predetermined landmark file. Then select the desired color lookup table for every spinning disk and TIRF channel and merge them into a single multidimensional hyperstack.During the processing of the TIRF images, a number of zed planes is added to the bottom plane and this number matches with the number of the planes in the spinning disk data sets. That's very important for the visual representation of the final hyperstack. Using the setup described in this video, YFP and RFP-expressing cells were selected and their adhesion process observed during a 60-minute timelapse.As expected, cells were round shaped and only weakly adherent at the beginning with membrane protrusions sensing the environment and making contact with the substrate. Cell matrix contact strengthened quickly upon formation of so called nascent adhesions. Over the course of time, adhesion complexes enlarged and matured to focal adhesions.These elongated structures were predominantly apparent at the periphery of the cell. This resulted from forces that were exerted by actomyosin fibers which induced the strengthening of cell matrix adhesions as well as the bundling of actin fibers. The cell also flattened as a result of the actin network formation.The acquisition speed is dependent on the following parameters, the time interval, the exposure time, the number of zed planes and the number of positions. So it is very important to optimize these parameters for high acquisition rates. The implementation of the newest spinning disk technology will turn spinning disk TIRF microscopy into new super resolution microscopy technique that will allow imaging at high temporal rates.Spinning disk microscopy is a very powerful technique for studying processes that occur between the cell interior and the cell membrane. We have been shown that we can follow the dynamic of vesicle by acquiring very large image stacks within few seconds. Collimated laser light is hazardous for the eyes.Never look directly into the beam and use the safety precautions of the instrument to avoid direct light exposure.

visualizing-adhesion-formation-cells-means-advanced-spinning-disk

Research

JoVE Journal

Bioengineering

6.4K Görüntüleme.  University Medical Center Hamburg-Eppendorf. Spinning disk TIRF mikroskopisi sitoskelet alkanlığı sırasında proteinin rolünü incelemek için yararlı bir araç olabilir. Bu teknik, hücrede meydana gelen hızlı hücresel yaklaşımların üç boyutlu görüntülenmesi ve aynı zamanda plazma zarına yerleştirilen floresan molekülünün kesin lokalizasyonuna olanak sağlamıştır. Bu yöntem mikrobiyoloji, hücre biyolojisi ve plazma zarı ile hücresel çevre arasındaki etkileşimi incelemenin önemli olduğu tüm bu dallar ...