Tracking malaria movement in three dimensions
Dr. Jake Baum, Imperial College London; Yan-Hong Tan, The Walter and Eliza Hall of Institute of Medical Research, Australia; and colleagues.
The Plasmodium parasites responsible for malaria have a complex lifecycle that requires that the parasites be motile to target and infect cells in both mosquito and human hosts. Researchers led by Dr. Jake Baum from Imperial College London are using Imaris software to analyze 3-D Plasmodium movement with the goal of gaining information that could inform the development of drugs that target motility to stop the parasites, infection, and, therefore, malaria disease.
The investigators imaged 96-well plates of cultured Plasmodium ookinetes that expressed GFP and then used Imaris to track individual cells. Panel A shows a sample 3D ookinete track. The reconstructed 3D tracks in panel B are from a single well and show characteristic helical motion paths. In Panel C, reconstructed tracks from panel B are translocated to a common origin. They do not show any prominent bias in direction. Reprinted from Andrey Kan, et al., Cellular Microbiology 16(5):734–750.
Plasmodium parasites have three life cycle stages: sporozoites, merozoites, and ookinetes. Although motility is observed during all three life cycle stages, ookinetes, which are formed inside the midgut of infected mosquitoes, is the only life cycle stage during which the parasite shows motility outside of a host cell. For this reason, the researchers studied the biomechanics of Plasmodium ookinete motility and its role as a key bottleneck in its lifecycle. They also hoped to gain general insights into motility that occurs throughout the lifecycle.
3D cell tracking
Although scientists know that Plasmodium movement involves actin and myosin, the core biomechanics are not completely understood. The researchers turned to the automated time Spots objects tracking function of Imaris to accurately track and analyze single-cell ookinete motion in three-dimensions.
“The software was capable of analyzing very big file sizes produced by our imaging data analysis, something other packages were struggling with,” Dr. Baum says. “The resulting analytical data, including average speed, displacement, and distance proved to be very useful for our study, saving us a lot of time in calculating each data point compared to doing it manually.”
The investigators used a line-scanning confocal microscope to image 96-well plates of cultured Plasmodium ookinetes that expressed GFP. For time-lapse videos, they acquired 1 frame every 2 s for 10 minutes at 2.5 micron z depth intervals for a total depth of 40 microns. They then used the Spots tracking algorithm with Brownian motion for automated cell localization and tracking, setting the Spots diameter to 5 microns. The investigators randomly selected automatically reconstructed tracks and exported the resulting 3D coordinates into a Microsoft Excel file for analysis in MATLAB.
Ookinetes show helical movement
“One of the simplest, yet most striking bits of information we could glean from the Imaris analysis was that the malaria parasite ookinetes have a consistently left-handed helical movement, something striking that likely arises from their inherent chiral cell shape,” Dr. Baum says.
The researchers are now exploring the forces the parasite exerts on the extracellular milieu. They are using Imaris to construct 3D images of parasites moving through the matrix, but this time they embedded beads in the extracellular matrix. For this work, Imaris is helping them to create a useful visual representation of the data.
Research Paper: Andrey Kan, Yan-Hong Tan, Fiona Angrisano, Eric Hanssen, Kelly L. Rogers, Lachlan Whitehead, Vanessa P. Mollard, Anton Cozijnsen, Michael J. Delves, Simon Crawford, Robert E. Sinden, Geoffrey I. McFadden, Christopher Leckie, James Bailey, Jake Baum. Cellular Microbiology. May 2014; 16(5):734–750.