This protocol enables quantification of the area, perimeter, and shape of intracellular structure from its two-dimensional image. This technique is quick and easy, requires only standard laboratory settings and a confocal microscope and uses opensource software. Demonstrating the procedure will be Anna Balcerak, a PhD student from my laboratory.
Begin by seeding four times 10 to the fifth cells in 0.5 milliliters of culture medium per well in a four-well collagen one coated chamber slide for a 24-hour culture in a 37 degree Celsius and five degree carbon dioxide incubator. The next morning, use an optical inverted microscope to verify the presence of at least 90%confluent monolayer and use a confocal microscope to obtain single Z-slice images. For focal adhesion analysis, open the images in ImageJ and select analyze, set scale, remove scale, and global to set the image scale in pixels.
To include the filename and the area of the region of interest in the measurement options, click analyze and set measurements and check the area and display label options. To subtract the background, select process and subtract background. Set the rolling ball radius to 50 pixels and check sliding paraboloid.
To determine the area of the smallest region of interest, outline the smallest single focal adhesion and click analyze and measure to measure the area. When at least 20 regions of interest have been selected and measured, calculate and save the mean of the obtained results. Set the upper boundary to 25%of a typical cell area.
To confer the image to binary, click image, adjust, and threshold, and check default, black and white, and dark background. To measure the number and areas of the regions of interest, select analyze and analyze particle and check pixel units, display results, clear results, and summarize. Transfer the slice, count, and total area average size data from the summary window to the data managing program of choice.
For focal adhesion quantification, open the focal adhesion ImageJ macro and enter the area of the smallest region of interest as the area of the smallest region of interest parameter. Set the threshold type value to manual or auto and save the changes. Then call the macro from ImageJ and select the image to be processed.
For manual cell shape analysis, open an image in an appropriate image processing software program and select the parameters to be measured. Use the freehand selection tool to manually delineate the cell borders as marked by the junction proteins of choice. The chosen parameters will be automatically calculated for each cell.
When all of the cells have been outlined, select edit, selection, and add to manager. Only complete entirely visible cells with uninterrupted borders should be selected. To make the measurement, mark all of the numbers appearing in the left box of the region of interest manager and click measure.
The results will appear in the results box and can be imported to the spreadsheet of choice. Automated cell analysis facilitates quantification of the large number of cells. For every new cell type, first run the macro to establish parameters.
When the macro has finished, select set cell size boundaries. Click the label of the smallest and largest cells and click measure. Set the value of the smallest cell and the biggest cell variables and save the changes.
Close all of the macro windows and select the image to be processed in grayscale. Then run the macro again. The macro will provide a table of the results that includes the cell shape index, aspect ratio, and regions of interest selections list data.
Here, representative images of focal adhesions counted with ImageJ including the final numbered outlines and the overlays of the focal adhesion outlines with the original image for both the control and knockdown cell lines can be observed. As illustrated in this analysis, the knockdown cells demonstrated a higher number of adhesions per cell as well as adhesions that are larger in size compared to control cell line cells. In these images, representative regions of untreated and chemotherapeutically treated cell monolayers are shown.
The outline cells were characterized according to their cell shape index values, for example in this analysis showing an increase in the last bin and a flattening of the main peaks for the drug-treated cells compared to untreated controls. A frequency distribution and cumulative distribution could then be generated for each cell culture. Grayscale imaging of the monolayers as demonstrated allowed the analysis and quantification of 512 cells from 12 fields of view revealing a relative frequency distribution of the cell shape index.
For this protocol to work, it is important to use images of the best possible quality. Proper cell seeding, staining, and imaging should ensure images of a sufficient experimental quality.
