To begin, place the strains of acanthamoeba under a bright field microscope. Visualize the amoeba at 4x magnification where the background appears light gray and the amoeba is dark. Adjust the microscope's lighting and focus to ensure the amoebae appear as solid dark circles.
Then configure the imaging software to record at regular intervals with a maximum gap of 30 seconds between images for accurate tracking. Track amoebas over multiple days, recording in segments of one hour for every 12 hours over five days. If the microscope and software permits, use the XY coordinates to record multiple wells or flow cell locations in a single session.
If necessary, stitch together multiple sections from a single well or flow cell for an expanded view at 4x magnification. Open the microscope file in the imaging software. The bio format's import options dialogue box is now seen.
Within the dialogue box, enable stack viewing, set view with to selection hyperstack. Then check use virtual stack in under memory management and set the color mode to default. Now open the bio format series options and select the required series.
Then press OK.Navigate to image, then select duplicate to duplicate only the image of a single well by choosing only one C channel and one Z channel. Work exclusively with the duplicated image for further manipulation. Click on image followed by type and 8 bit to convert the image.
Then choose process followed by subtract background. Set the rolling ball to 10. Check the light background option and select sliding paraboloid.
Now navigate to process followed by enhanced contrast. Adjust saturated pixel settings to 0.1%for individual trophozoites, or 0.3%for groups of cells and aggregates. Sequentially click on image, adjust, and threshold with default greater than black and white.
This will open the binary process settings. Choose default and check black background of binary masks. Adjust the threshold aggressively to minimize background noise while ensuring each amoeba is partially visible.
If the software inverted the colors to make the background white and amoeba black, go to edit and click on invert to switch it to a black background with white amoeba. Now sequentially click on process, followed by binary and close to connect slight gaps in the outer membrane of cells. Fill any remaining holes by clicking on process, binary, and fill holes.
Then use shape drawing tool and press edit followed by fill to remove any non-amoeba artifacts. If necessary, invert the image again to finalize the binary representation. To record the size of each amoeba, click on analyze, followed by analyze particles.
Then set the size to 10-infinity, the circularity between zero to one and the show to nothing. Check the only display results and summarize option to get the analysis without additional visuals. Save the result in CSV files, then click on file, followed by save as and TIFF to save the image in a TIFF format.
Edit the name as required.
