Exploring the intricacies of Toxoplasma gondii: Imaging mitochondrial membranes for insights into parasitic biology

Parasites are microorganisms that live in or on other organisms known as hosts. Their intimate relationships with their hosts range from mutualistic to parasitic, with some parasites causing harm and disease while others exert subtle influences on their hosts behavior or physiology. From microscopic protozoa to macroscopic helminths, parasites come in various forms and inhabit nearly every corner of our planet. As important as the relationship between the parasites and their hosts is, it is even more crucial to understand their inner life. Identifying inner structures and functions is essential for accurate diagnosis, treatment and drug development, knowing of risks associated with infection, complexity of ecosystem and survival mechanisms. 

Toxoplasma gondii, parasitic protozoan, is responsible for causing disease toxoplasmosis in humans or warm-blooded animals. Cats are usually the primary host where it can complete its life cycle while humans as intermediate hosts. In T. gondii, mitochondria is essential for parasite to survival and validate drug target. It contains two membrane, inner membrane and outer membrane that are mainly responsible for translocation of proteins and maintain the morphology during the lytic cycle. 

Here, we image both inner and outer membrane in T. gondii with single molecule localization microscopy in Abbelight SAFe MN 360 Ultimate 3D. We used spectral demixing STORM technique to carry out simultaneous multi color imaging. Only 640 laser is used for excitation in AF647 and CF680 fluorophores that labelled outer and inner membrane, respectively. Detected photons from each camera for same single molecule is allowed us to calculate intensity ratio which lead to the identification of each color in same time during the acquisition via Abbelight NeoLiveimaging software. Thanks to the aberration free and time saving approach for multicolor imaging, we are able to distinguish inner and outer membrane in nanoscale resolution. We believe, this study may allow to determine precise localization of mitochondrial proteins in reference to inner and outer membrane. 


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