Navigating imaging artifacts: challenges with visualizing intrinsically fluorescent antimalarials

Elucidating the target of an antiparasitic small molecule can aid in the drug development process. However, various technical challenges related to parasite biology prevent timely target identification. Microscopy methods determining the co-localization of a drug with subcellular markers is a powerful approach that provides evidence of its localization within the cell. This technique can help pinpoint the primary site of action of novel therapeutics, which can assist in elucidating their mode of action. Intrinsically fluorescent drugs allow for subcellular localization without extensive medicinal chemistry. We imaged the intrinsically fluorescent drugs PRC1590 and quinine with digestive vacuole stains to determine these compounds' localization in the malaria parasite, Plasmodium falciparum. We observed that imaging intrinsically fluorescent compounds often produces imaging artifacts due to spectral overlap and autofluorescence of P. falciparum, resulting in false positive co-localization findings. Our work outlines critical methodological considerations when studying subcellular localization of intrinsically fluorescent antimalarials.