Name: A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions (Video) | JoVE Journal
Uploaded: 2017-07-11T14:00:00.000+00:00
Duration: 8 min 12 s
Description: Learn to analyze protein dynamics in filopodial protrusions using semi-automated image analysis software. This method enables parallel quantification of protrusion dynamics and spatially resolved protein concentration along filopodia.

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5 minutes

The authors acknowledge funding from the DFG (EXC-1003 to MG).

1. Cell Culture
<ol>
	<li>Culture HeLa or COS cells in Dulbecco's Modified Eagle Medium (DMEM) containing 4.5 g/L D-glucose, L-alanine-L-glutamine dipeptide, pyruvate, 10% fetal bovine serum, and 10 units/ml of penicillin/streptomycin. Culture neurons in culture-media without L-glutamine, glutamic acid or aspartic acid, supplemented with 0.5 mM L-alanine-L-glutamine dipeptide, serum-free neuronal supplements and 10 units/ml of penicillin/streptomycin.</li>
	<li>Once 40% confluency is reached, transfect cells with constructs of choice using a transfection reagent as per manufacturer's instructions. Keep transfected cells in incubator at 37 °C and 5% CO2 for 15-18 h.</li>
	<li>To reduce changes in pH and osmolarity (due to evaporation) during image acquisition, fill the cell culture chambers (prior to imaging) up to 90 % with 37 °C pre-warmed medium containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). To seal the lid, apply a thin layer of vacuum grease on the inside of the lid and gently press it on the culture chamber containing the transfected cells.</li>
</ol>
2. Image Acquisition
NOTE: The length of filopodia varies from 2-10 µm 13. Filopodia grow at an average velocity of 0.05-0.1 µm/s 13,14.
<ol>
	<li>Acquire images using a microscope with 60X or 100X objective and no pixel binning (here, a spinning disc confocal microscope). Use acquisition-rates greater than 1 Hertz (Hz) to track filopodial dynamics. To minimize out-of-focus artifacts, image filopodia close to the basal membrane (i.e. substrate surface).</li>
	<li>To assure smooth tracking, adjust the exposure time of the camera and laser intensity such that the signal to noise ratio (SNR) is greater than 4. Avoid saturation of individual channels (i.e. pixel values of 255 for 8-bit images and 65,535 for 16-bit images), as this will preclude subsequent image analysis.</li>
	<li>To avoid bleed-through, only use fluorescence tags compatible with laser lines and filters of the microscope (for details see reference 15).</li>
</ol>
3. Image Pre-processing
NOTE: Use ImageJ or other available software to pre-process images 16,17.
<ol>
	<li>If the sample is moving, correct for lateral drift using available software (e.g. https://github.com/NMSchneider/fixTranslation-Macro_for_ImageJ) prior to analysis. Exclude movies with axial drift (i.e. movement in z-direction).</li>
	<li>Correct background (i.e. grey values of areas outside of the cell), bleaching (i.e. continuous loss if fluorescence intensity due to damage to fluorescence proteins) and possible bleed-trough (i.e. signal from one fluorescence probe in the both channels) using available software (e.g. http://imagej.net/Category:Plugins and 16,17). 
	Note: Fluorescence intensities of individual channels are not altered by the software.</li>
	<li>To ensure subsequent image analysis, save movies corresponding to particular protein channels in grey scale as '.tiff' stacked file format in the work folder. 
	NOTE: The dimensions (i.e. size, length) of the stacks must be same for all channels.</li>
</ol>
4. Image Analysis – Step 1: Load Images
NOTE: The software described here was written in Matlab (referred to as programming software) and will run only with this program.
<ol>
	<li>Download the zipped folder containing all required files for image analysis from the following site: https://campus.uni-muenster.de/en/einrichtungen/impb0/nanoscale-forces-in-cells/software/. Unzip and copy files into the work folder.</li>
	<li>Once installed, open the programming software and run 'filopodiaAnalysisM3.fig'. GUI Window #1 will open as shown in Figure 2A.</li>
	<li>Load the saved stacked '.tiff' files corresponding to a particular protein of interest in GUI Window #1 using the buttons &lt;1b&gt; for protein A, &lt;1c&gt; for protein B, &lt;1d&gt; for protein C. See Figure 2 and Table 1 for details. 
	NOTE: Protein A shown in GUI Window #1 acts as the reference channel for the final ratiometric analysis.</li>
	<li>Create a superimposed image of the cell from the protein channels by clicking &lt;1a&gt;.</li>
	<li>Click on &lt;2a&gt; to assign the first frame and &lt;2b&gt; for the last frame used for analysis.</li>
	<li>Optionally, crop the region of interest (ROI) containing the filopodium of interest using button &lt;2h&gt;, rotate the image using button &lt;2i&gt;, or delete unwanted regions using the free-hand drawing tool &lt;2j&gt;. 
	NOTE: To streamline image analysis, it is recommended to isolate the ROI (i.e. crop, rotate, delete) together with the other preprocessing steps (background subtraction, bleach corrections, etc.) using ImageJ.</li>
	<li>Move the slider &lt;2c&gt; for each frame for quality control and to check whether the filopodium remains clearly visible throughout the whole movie.</li>
</ol>
5. Image Analysis – Step 2: Generate Trace
<ol>
	<li>Click on button &lt;3a&gt; in the GUI Window #1 to open GUI Window #2 (see Figure 2B).</li>
	<li>Click on button &lt;4&gt; in the GUI Window #2 to generate the mask of the superimposed cell body (generated in GUI Window #1 after clicking &lt;1a&gt;). The program also generates the boundary of the mask, where the convex hull is implemented to get the vertex points.</li>
	<li>Click button &lt;5&gt;; a cursor will appear. Use the cursor to select the base (from where the distance of the filopodial tip is measured) followed by the tip of the filopodia in the frame where it first appears. To do this, move the slider in Window #2). 
	NOTE: To minimize errors in data output, place the base point vertically below the filopodial tip along the axis. Positioning the base point on other places (e.g. laterally shifted) may introduce a directional bias in fluorescence values within the cell body.</li>
	<li>Select the threshold length (above which the filopodia will bend) using &lt;6c&gt;. 
	Note: This length is defined as the distance between the base point (selected using &lt;5&gt;) and the boundary of the cell body (i.e. the region from where the filopodia starts growing). The distance is measured is in pixels.</li>
	<li>Specify the number of segments used to approximate the shape of the filopodia in box &lt;6a&gt;. 
	NOTE: The minimum number of segments depends on the maximal length reached by the filopodium, but also how the filopodium bends. Do not select a number of segments larger than the number of pixel between the base and the tip (i.e. the threshold length). Selecting more segments than the threshold length defined in step 5.4. (i.e. number of pixels between base and tip) will result in an overestimation of the length of the filopodium.</li>
	<li>Specify the scan width, which acts as horizontal scanner to place the nodes, in &lt;6b&gt;. 
	NOTE: These nodes are used by the program to fit the line joining the base and the tip with the body of the filopodium (i.e. to create the backbone). As a starting point, put a value in pixels equal to the maximum length multiplied by a factor of <img alt="1 over square root of 2 formula; mathematical expression relevant to probability theory." src="/files/ftp_upload/55653/55653eq1.jpg" data-altupdatedat="1788250015000" data-alt="Equation">.</li>
	<li>Specify the scan radius (in pixels) in box &lt;6d&gt;. Use a value that is approximately 50% larger than the observed inter-frame tip displacement. 
	NOTE: Using a very large scan radius might grab unwanted convex-hull points in frames where no real filopodial tip is present (e.g. out of plane movement or low SNR).</li>
	<li>Specify the bending angle in box &lt;6e&gt;. 
	NOTE: The angle threshold is determined by the maximum angle the filopodium is bending during the whole analysis. Specifying the angle threshold helps the software to exclude tracking of unwanted structures growing from the side of the filopodia when the filopodia bends towards the cell body. The program works reliably for filopodia with tilting angles less than 45 degrees from the vertical axis.</li>
	<li>To start tracking, click the &lt;Track&amp;Analyze&gt; button in GUI Window #2. Click the 'History trace' box in GUI Window #2 to save the whole tracking protocol for future reference. 
	NOTE: After the tracking procedure is complete, the length of the filopodium in each frame is stored in the sheet named 'length_vel' of 'dynamics.xlsx' for future references. Likewise, all other tracking parameters are stored in the sheet named 'parameters' of 'dynamics.xlsx'.</li>
	<li>Optionally, if the filopodia is not automatically detected in all frames, use the following steps to manually correct for it.
	<ol>
		<li>Visit the respective frame using the slider in Window #2 and manually select the tip of the filopodium. 
		Note: Frames, where no convex hull points are detected will be represented by a blue region in the tracking window of GUI Window #2. It is mandatory to check the 'History trace' button before the tracking program was initiated for accessing the nodal coordinates in that frame.</li>
		<li>Select the reference points (base followed by tip) using &lt;6f&gt; in GUI Window #2. Specify the other parameters such as 'scan length', 'Accurate Measurement after', and 'maximum bending angle' for that frame as described in steps 5.4-5.8.</li>
		<li>Click button &lt;6i&gt; to save the new parameters (specific to that one frame). The steps are to be repeated for all frames indicated by the blue region.</li>
		<li>When finished, re-initialize the tracking procedure using &lt;7&gt;. 
		NOTE: This correction can also be used if the program suddenly switches from one filopodial to another within a movie. As an alternative, consider cropping movies that contain multiple filopodia.</li>
	</ol>
	</li>
</ol>
6. Image Analysis – Step 3: Spatio-temporal Protein Analysis
<ol>
	<li>For spatio-temporal analysis, select the box represented by &lt;8b&gt; followed by the protein channel of interest (&lt;8c&gt; and/or &lt;8d&gt; and/or &lt;8e&gt;). 
	Note: It is mandatory to select Protein A prior to ratiometric analysis (&lt;9a&gt;, &lt;9b&gt; and/or &lt;9c&gt;) since Protein A is used as reference.</li>
	<li>Click on &lt;8a&gt; to initiate protein tracking along the filopodial length using the trace generated in step 5.9.</li>
</ol>
7. Image Analysis – Step 4: Ratio-metric Protein Analysis
<ol>
	<li>Check box &lt;9b&gt; or &lt;9c&gt; and click on the button &lt;9a&gt; to get the spatio-temporal ratiometric plot. 
	NOTE: To avoid misrepresentation of relative protein concentration, the ratiometric image is not plotted as X/Y but as log(X/Y). For future use, the ratiometric plot is exported in '.png' and '.fig' format files, and raw plot data is stored within the 'dynamics.xlsx' file.</li>
</ol>
8. Image Analysis – Step 5: Filopodial Tip Analysis
<ol>
	<li>Check box &lt;8f&gt; and specify the tip length and threshold length from the base using &lt;8g&gt; and &lt;8h&gt;. Click on &lt;push button&gt; to save data to file 'dynamics.xlsx'. Click on &lt;Analyze Protein Intensities&gt; to generate the trace of protein intensities of the filopodial tip and to save for ratiometric analysis. 
	NOTE: Tip length determines the number of pixels at the tip used for the analysis. The software will return the average intensity value of the pixels at the tip over each frame. The threshold length determines the minimum distance from the base to tip above which the filopodium (filopodial tip) starts growing (and when the program starts tracking the intensity value of the tip). So the tip length must be smaller than the threshold length.</li>
	<li>Click on the desired ratio to be analyzed using &lt;9d&gt; or &lt;9e&gt;.</li>
	<li>Click on the compare button &lt;9a&gt; to generate the ratiometric data.</li>
</ol>

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<li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+guide+to+choosing+fluorescent+proteins%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">A guide to choosing fluorescent proteins.</a> Nat Methods. 2 (12), 905-909 (2005).</li>
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</ol>

The authors have nothing to disclose.

Here we present a detailed protocol for tracking filopodial growth dynamics and analysis of relative protein concentrations in these dynamic structures via the convex-hull algorithm. Using the software, up to 3 channels can be compared pair-wise in a single run, whereby the relative concentrations of two channels (i.e. proteins) is determined throughout the extension/retraction cycle and stored as image and data files in separate folders. In addition to the routine operations, the software also provides a number...

Spatio-temporal control of actin regulatory proteins is associated with filopodium dynamics 1,2. Tracking spatially resolved protein concentration along the whole filopodial length through time is thus crucial to advance our understanding of the mechanisms underlying initiation, elongation, stabilization or collapse of these dynamic structures 3,4. Unlike protein analysis in the cytosol, where many cell shape changes occur at a larger scale, filopodia are dynamic micro structures that constantly buckle 5 and bend, thus precluding analysis using a simple approach such as a line-scan.
Different software solutions for tracking filopodial shape are available 6,7,8,9. Likewise, software for ratiometric tracking of protein dynamics within the cell body has been developed 10,11. To combine automated tracking of filopodial shape and spatio-temporal protein analysis, we recently developed an image analysis software based on the convex-hull algorithm 12. This novel analysis method, which is operated via a graphical user interface (GUI), combines for the first time, relative protein concentration along the filopodial length and growth velocity, thus allowing the accurate measurement of spatio-temporal protein distribution independent of movement of these dynamic structures 12.
The idea behind the software (source code is freely available, see below) is that one of the vertices of the convex hull will coincide with the tip of the filopodium (Figure 1A). By looking in the subsequent frame for the nearest vertex of the convex-hull, the moving tip can be tracked throughout the whole movie. Once the tip is detected in each frame, its position is used to draw an axis by joining the tip with a reference point at the base of the filopodium (Figure 1B). Finally, using equidistant nodal points, whose positions are determined by the median pixel with maximum intensity along the line orthogonal to the axis, are used to determine a backbone that follows the filopodial shape. Taking advantage of this adaptive backbone, a kymograph is generated to trace filopodial growth and protein concentrations for up to three channels along the filopodial length (Figure 1C).
<img alt="Filopodia analysis diagram with GUI steps and growth chart; includes fluorescence intensity scan." class="xfigimg" src="/files/ftp_upload/55653/55653fig1.jpg" data-altupdatedat="1788250015000" data-alt="Figure 1"> 
Figure 1: Working Principle of the Image Analysis Software. (A) The algorithm behind the software. In Step1 the user specifies the reference (base) and the vertex (the tip) of the filopodium. In Step 2-1 the backbone of the filopodium is obtained using the median pixel with maximum intensity value. In Step 3 the backbone is used for spatial protein intensity profile. In Step 2-2 the software automatically tracks the tip in the subsequent frame. The whole procedure iterates. (B) Snapshot of the algorithm with real filopodium introducing important elements such as the convex hull that is being used for tracking. (C) Overview of parameters that can be measured with the algorithm. This figure has been modified from reference 12. <a href="http://cloudflare.jove.com/files/ftp_upload/55653/55653fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The image analysis software is operated in Matlab (referred to as programming software) via a graphical user interface. To maximize flexibility and robustness for the particular experimental setting, the user can adjust a series of tracking parameters (e.g. permitted bending angle and inter-frame movement) and also make some corrections to the movies (e.g. cropping, rotation, removal of unwanted objects) (Figure 2A and Table 1).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>GUI</td>
			<td>No.</td>
			<td>Mandatory</td>
			<td colspan="5">Description</td>
			<td>Name (in GUI)</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>1a</td>
			<td>Y</td>
			<td colspan="5">Loading stacked .tiff file representing cell body (with box checked in) or create superimposed cell body from channels</td>
			<td>CellBody</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>1b</td>
			<td>Y</td>
			<td colspan="5">Loading stacked image file corresponding to protein 1</td>
			<td>Protein 1</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>1c</td>
			<td>Y</td>
			<td colspan="5">Loading stacked image file corresponding to protein 2</td>
			<td>Protein 2</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>1d</td>
			<td>Y</td>
			<td colspan="5">Loading stacked image file corresponding to protein 3</td>
			<td>Protein 3</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>1e</td>
			<td>N</td>
			<td colspan="5">Resets everything to preloaded stacked image files</td>
			<td>Reset</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2a</td>
			<td>Y</td>
			<td colspan="5">Scroll bar to determine the initial frame for analysis in GUI window #2</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2b</td>
			<td>Y</td>
			<td colspan="5">Scroll bar to determine the final frame for analysis in GUI window #2</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2c</td>
			<td>Y</td>
			<td colspan="5">Scroll bar representing current frame</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2d</td>
			<td>N</td>
			<td colspan="5">The grey value of the pixels below which all pixels will be set to zero</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2e</td>
			<td>N</td>
			<td colspan="5">The grey value of the pixels above which all pixels will be set to maximum values</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2f</td>
			<td>N</td>
			<td colspan="5">Set the intensity values of the pixels specified by &lt;2e&gt; &amp; &lt;2f&gt;</td>
			<td>Set</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2g</td>
			<td>N</td>
			<td colspan="5">Play the intensity-adjusted movie</td>
			<td>Play</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2h</td>
			<td>N</td>
			<td colspan="5">Crop Image</td>
			<td>Crop</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2i</td>
			<td>N</td>
			<td colspan="5">Rotate Image</td>
			<td>Rotate</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>2j</td>
			<td>N</td>
			<td colspan="5">Delete regions in the whole stack</td>
			<td>Delete regions</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>3a</td>
			<td>Y</td>
			<td colspan="5">Click to open the ‘Analysis Window’ (GUI window #2)</td>
			<td>Tracking Window</td>
		</tr>
		<tr>
			<td>#1</td>
			<td>3b</td>
			<td>Y</td>
			<td colspan="5">Enter the size of a pixel in microns</td>
			<td>Enter Pixel size</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>4</td>
			<td>Y</td>
			<td colspan="5">Click to generate the boundary/edge image of the superimposed cell body</td>
			<td>Boundary</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>5</td>
			<td>Y</td>
			<td colspan="5">Click to select the base and tip of the filopodia</td>
			<td>Referernce</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6a</td>
			<td>Y</td>
			<td colspan="5">Enter the number of segments or nodes</td>
			<td>No of segments</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6b</td>
			<td>Y</td>
			<td colspan="5">Enter the scan length (perpendicular to axis)</td>
			<td>Scan Width</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6c</td>
			<td>Y</td>
			<td colspan="5">Enter the length above which filopodia starts bending</td>
			<td>Accurate Meas after</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6d</td>
			<td>Y</td>
			<td colspan="5">Enter the radius of the tip detection circle (i.e. area where the vertex can be localized in the next frame)</td>
			<td>Radius of tip detection</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6e</td>
			<td>Y</td>
			<td colspan="5">Enter the maximum angle the filopodium can bend from the vertical axis</td>
			<td>Angle Threshold</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6f</td>
			<td>N</td>
			<td colspan="5">Add reference points for base and tip for that specific frame</td>
			<td>Select reference</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6g</td>
			<td>N</td>
			<td colspan="5">Enter the length above which filopodia starts bending for that specific frame</td>
			<td>Accurate Meas after</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6h</td>
			<td>N</td>
			<td colspan="5">Enter the radius of the detection circle for that specific frame</td>
			<td>Radius of tip detection</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6i</td>
			<td>N</td>
			<td colspan="5">After entering all the parameters for the specific frame click to store the values to memory and file for further reference</td>
			<td>Add</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6j</td>
			<td>N</td>
			<td colspan="5">Click to delete the set manually parameters for that frame</td>
			<td>Delete</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6k</td>
			<td>N</td>
			<td colspan="5">Click to delete all parameters stored manually using the ‘optional features panel’ for all frames</td>
			<td>Reset</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>6l</td>
			<td>N</td>
			<td colspan="5">Check in before tracking to store all tracking results in memory for future reference</td>
			<td>History trace</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>7</td>
			<td>Y</td>
			<td colspan="5">Click to start tracking</td>
			<td>Track&amp;Analyze</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>8a</td>
			<td>N</td>
			<td colspan="5">Click to start tracking protein channel intensity</td>
			<td>Analyze Protein Intensities</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>8b</td>
			<td>N</td>
			<td colspan="5">Check in to track protein channel intensity along the filopodial length</td>
			<td>Whole filopodia</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>8c</td>
			<td>N</td>
			<td colspan="5">Check in for tracking the reference protein or protein A</td>
			<td>ProteinA</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>8d</td>
			<td>N</td>
			<td colspan="5">Check in for tracking the protein B</td>
			<td>ProteinB</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>8e</td>
			<td>N</td>
			<td colspan="5">Check in for tracking the protein C</td>
			<td>ProteinC</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>8f</td>
			<td>N</td>
			<td colspan="5">Check in to track average protein intensity in the tip</td>
			<td>Leading tip</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>8g</td>
			<td>N</td>
			<td colspan="5">Enter the length of the tip</td>
			<td>Tip Length</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>8h</td>
			<td>N</td>
			<td colspan="5">Enter the minimum distance from base above which the tip starts forming</td>
			<td>threshold</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>8i</td>
			<td>N</td>
			<td colspan="5">Click to save the leading tip analysis results to file</td>
			<td>Push Button</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>9a</td>
			<td>N</td>
			<td colspan="5">Click to initiate ratiometric protein analysis</td>
			<td>Compare</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>9b</td>
			<td>N</td>
			<td colspan="5">Check in to compare protein B with respect to A</td>
			<td>log10(B/A)</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>9c</td>
			<td>N</td>
			<td colspan="5">Check in to compare protein C with respect to A</td>
			<td>log10(C/A)</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>9d</td>
			<td>N</td>
			<td colspan="5">Check in to compare protein B with respect to A at the tip</td>
			<td>log10(B/A)</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>9e</td>
			<td>N</td>
			<td colspan="5">Check in to compare protein C with respect to A at the tip</td>
			<td>log10(C/A)</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>10a</td>
			<td>N</td>
			<td colspan="5">Choose other color-map (default: Jetplot)</td>
			<td>Color Map</td>
		</tr>
		<tr>
			<td>#2</td>
			<td>10b</td>
			<td>N</td>
			<td colspan="5">Edit the colormap</td>
			<td>Edit colormap</td>
		</tr>
	</tbody>
</table>
Table 1: Overview of All Functions Present in the GUI Windows #1 and #2.
Once this is accomplished, the program creates a convex hull and automatically tracks the tip throughout the movie. Parameters extracted from the movie, such as a ratiometric kymograph, growth velocity, and filopodial length are displayed and also stored in the work folder as images and as data files. Other parameters such as filopodial lifetime, growth rate and retraction rate can then be extracted and further analyzed from the stored data files (Figure 2B).
<img alt="Two GUI windows for scientific data analysis, showing image processing and graph plotting." class="xfigimg" src="/files/ftp_upload/55653/55653fig2.jpg" data-altupdatedat="1788250015000" data-alt="Figure 2"> 
Figure 2: Graphical User Interface for using the Image Analysis Software. (A) GUI Window #1 is used for loading and processing images. The program can load up to 3 protein channels, whereby 2 channels are compared pair-wise. The window comes with mandatory (blue) and optional features (green) for pre-processing the images prior to tracking (B) GUI Window #2 is used for tracking the filopodium as well as spatio-temporal and ratio-metric protein analysis. Again, optional features are marked in green. This figure has been modified from reference 12. <a href="http://cloudflare.jove.com/files/ftp_upload/55653/55653fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Here, we present a detailed protocol for sample preparation and software handling. We start with detailed instructions on culturing cells and acquiring movies optimized for image analysis. This section on data acquisition is followed by a detailed description for operating the image analysis software. Throughout the protocol, we introduce critical steps and optional features that should be considered when collecting and processing data. Finally, we analyze filopodia from different model systems with the image analysis software, before closing with a comparison of the described image analysis software with other programs available for filopodial quantification and a discussion on limitations and future direction.

<table><tbody><tr><td>DMEM</td><td>Life Technologies</td><td>31966-021</td><td></td></tr><tr><td>10% Fetal bovine serum</td><td>Biochrom AG</td><td>L11-044</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Life Technologies</td><td>11668-027</td><td></td></tr><tr><td>1% penicillin/streptomycin</td><td>Biochrom AG</td><td>12212</td><td></td></tr><tr><td>Neurobasal Medium</td><td>Life Technologies</td><td>21103-049</td><td></td></tr><tr><td>B27</td><td>Life Technologies</td><td>17504-044</td><td></td></tr><tr><td>HEPES (1M stock solution)</td><td>Life Technologies</td><td>15630</td><td></td></tr><tr><td>Citrine-N1</td><td>Addgene</td><td>54593</td><td></td></tr><tr><td>Labtech</td><td>Thermo</td><td>155411</td><td></td></tr><tr><td>Glutamax-I</td><td>Thermo</td><td>35050-061</td><td></td></tr><tr><td>Hela</td><td>Leibniz Institute DSMZ</td><td>ACC-57</td><td></td></tr><tr><td>COS 7</td><td>Leibniz Institute DSMZ</td><td>ACC-60</td><td></td></tr><tr><td>3T3 cells</td><td>Leibniz Institute DSMZ</td><td>ACC-59</td><td></td></tr><tr><td>Microscope</td><td>Nicon Eclipse</td><td></td><td></td></tr><tr><td>Camera</td><td>Andor</td><td>DU888 Ultra</td><td></td></tr><tr><td>Confocal Unit</td><td>Yokagawa</td><td>CSU-X1</td><td></td></tr><tr><td>Pyruvate</td><td>Gibco</td><td>31966-021</td><td></td></tr></tbody></table>

a graphical user interface for software-assisted tracking of protein concentration in dynamic cellular protrusions

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

Using COS cells transfected with a marker for filamentous actin (f-tractin18, red) and a cytosolic reference (green), we found actin-rich filopodial protrusions (Figure 3A, top panel). Time series showed that filopodia rapidly extend and retract (Figure 3A, middle panel). Using the image analysis software, we then traced individual filopodia. Comparison of filopodial length measured by hand vs. the image a...

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

The overall goal of this image analysis software is the parallel analysis of protrusion dynamics and relative protein concentration along the entire filopodial length. This method can really help us understand key questions in cell biology, especially the individual proteins involved in extension and retraction of filopodia. The main advantage of the technique is that the adaptive shape tracking algorithm provided, allows quantitative assessment of protrusion dynamics and spatially resolved protein localisation.To begin, culture neurons, HeLa or COS cells, in supplemented medium as detailed in the text protocol. Once 40%confluency is reached, transvect the cells with the constructs of choice, using a transvection reagent as per manufacturer's instructions. Keep the transvected cells in an incubator at 37 degree celsius and 5%CO2 for 15 to 18 hours.Following incubation, fill the cell culture chambers up to 90%with 37 degree celsius pre-warmed medium, containing 20 milli molar HEPES, to reduce changes in pH and osmolarity due to evaporation during image acquisition. To seal the lid, apply a thin layer of vacuum grease on the inside of the lid and gently press it on the culture chamber containing the transvected cells. Possibly the most important part for image analysis is the image quality.One thing we have to take care is the signal to noise ratio, which has to be more than four and we can ensure that, by adjusting the exposure time of the camera and the laser intensity and force would tracking the frame rate has to be more than one hertz. Acquire the images using a microscope with a 60x or 100x objective and no pixel bending. Use acquisition rates not less than one hertz to track filopodial dynamics.To minimize out of focus artifacts, image filopodial close to the basil membrane. To ensure smooth tracking, adjust the exposure time of the camera and laser intensity such that the signal to noise ratio is greater than four. Once this is complete, start the image acquisition.Following image pre-processing, as described in the text protocol, load the images by downloading the zipped folder containing all required files for image analysis. Unzip and copy files into the work folder. Once installed, start the graphical user interface by opening the file named, filopodiaAnalysisM3.fig.Load the saved stack TIFF files for individual proteins in the graphical user interface or GUI. Using the buttons 1B for protein A, 1C for protein B and optionally, 1D for protein C.Create a superimposed image of the cell from the protein channels by clicking 1A. Then, click on 2A, to assign the first frame and click on 2B, to assign the last frame for the analysis.Optionally, crop the region of interest or ROI, containing the filopodium of interest using the 2H button. Rotate the image using the 2I button, or delete unwanted regions by using the free handed drawing tool, 2J. Move the slider, 2C for each frame for quality control and to check whether the filopodium remains clearly visible throughout the whole movie.To generate the trace, first, click on the 3A button in the GUI window one, to open GUI window two. Click on the four button in GUI window two, to generate the mass of the superimposed cell body that was generated in GUI window one, after clicking 1A. We recommend spending some time on optimizing parameters such as, the number of segments, the scan width and the scan radius for your experimental data set.Move the slider in window number two, to check where the filopodial tip first appears. Then, click button five and the cursor will appear. Use the cursor to select the base from where the distance of the filopodial tip is measured.Followed by the tip of the filopodia in the frame where it first appears. Next, select the threshold length above which the filopodia will bend, using 6C. Then, specify the number of segments used to approximate the shape of the filopodia in box 6A.Specify the scan width, which acts as a horizontal scanner, to place the nodes in 6B. Specify the scan radius in box 6D, use a value that is approximately 50%larger than the observed inter-frame tip displacement. Then, specify the bending angle in box 6E.Check the box's history trace in GUI window two, to save the whole tracking protocol for future reference and then click the track and analyze button to start tracking. For spatial temporal analysis, select the box represented by 8B, followed by the protein channel of interest. Click on 8A, to initiate protein tracking along the filopodial length, using the previously generated trace.For ratiometric protein analysis, check box 9B or 9C and click on the button, 9A to get the spatial temporal ratiometric plot. To perform the filopodial tip analysis, check box 8F and specify the tip length and threshold length from the base, using 8G and 8H. Click on the push button, to save the data to an excel file named, dynamics.xlsx.Then, click on analyze protein intensities to generate the trace of protein intensities of the filopodial tip and to save for ratiometric analysis. Click on the desired ratio to be analyzed, using 9D or 9E. Finally, click on the compare button, 9A to generate the ratiometric data.The analysis of COS cells transvected with a marker for filamentous actin in red, and a cytosolic reference in green reveal actin-rich filopodial protrusions. A time series shows actin-rich filopodial protrusions rapidly extend and retract. Using the image analysis software, the individual filopodia were traced.Image analysis software reliably track filopodial extension, in addition to growth and retraction rates of an individual filopodia, chemigraphs also depict concentration of actin normalized to the cytosolic reference. If used properly, this technique allows quantitative analysis of protrusion dynamics and spatially resolved protein concentration along the filopodia. When attempting the procedure, it is important to remember the acquisition speed and to keep the signal to noise ratio larger than four, while not saturating individual images.

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Research

JoVE Journal

Biology

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Here, we present a protocol for single particle tracking image analysis that allows quantitative evaluation of diffusion coefficients, types of motion and cluster sizes of single particles detected by fluorescence microscopy.

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

This article presents a method for spatiotemporal analysis of mobile, single-molecule Förster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

This protocol aims to measure the dynamic parameters (protrusions, retractions, ruffles) of protrusions at the edge of spreading cells.

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy

This protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided alongside directions to prevent potential pitfalls.

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

This article provides a detailed description of how to create samples for single-protein tracking in solid-supported lipid bilayers. It also explains a straightforward fluorescence microscope with single-molecule sensitivity and a fast frame rate. Finally, we outline the procedure for extracting single-protein trajectories.

Tracking Single Proteins in Lipid Bilayers Using Fluorescence Microscopy

Learn to analyze protein dynamics in filopodial protrusions using semi-automated image analysis software. This method enables parallel quantification of protrusion dynamics and spatially resolved protein concentration along filopodia.

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

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