Three-Dimensional Real-Time Imaging of Gliding Motility in Apicomplexan Parasites using 4D Dark Field Microscopy

Abstract

The speed and sensitivity of detection methods along with the efficiency of the process or signal representing the sample delimit optical imaging of living cells. To facilitate the imaging of living biological cells in real time in three dimensions, and in their native contrast, we have developed an imaging method composed of a dark field optical microscope coupled with a high-speed CMOS camera capable of capturing images at 5,000 fps at full field, equipped with a motorized assembly regulating the position of the objective. We have successfully applied the instrument to the imaging of 200nm gold particles and also to studying motility in the Apicomplexan parasite Neospora caninum. N. caninum is a close relative of the pathogenic apicomplexans Toxoplasma gondii (which causes disease in immunocompromised individuals) and Plasmodium falciparum (the causative agent of malaria), which exhibit the same characteristic dynamic motion. Apicomplexan parasites rely on apical constriction to lead the parasite in twirling, helical, and clockwise gliding motion for motility on solid surfaces and also to invade host cell membranes. The precise penetrative technique of these organisms has been little understood due to current restrictions in imaging, though it has been posited that more in-depth knowledge of the gliding motility can uncover the invasion mechanism. We are currently capable of successfully capturing 3D volumes of active N. caninum cells at 50-100Hz with a 5-20μm vertical transverse range. We employ a Fourier-based deconvolution algorithm to reduce noise and better visualize the 4D data, which is expected to facilitate the identification of features in N. caninum cells that are critical for its mechanism of invasion.