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.
