Immunofluorescence staining is the key technique for visualizing organization of endogenous cellular structures in single cells. Labeling and imaging of blood stage Plasmodium falciparum has always been challenging since it is a small intracellular parasite. A widely-used standard for parasite immunofluorescence is fixation in suspension with addition of minute amounts of glutaraldehyde to the paraformaldehyde-based solution. While this maintains red blood cell integrity, it has been postulated that antigenicity of the parasite proteins was, if at all, only slightly reduced. Here we show the deleterious effect that even these small quantities of glutaraldehyde can have on immunofluorescence staining quality and present an alternative cell seeding protocol that allows fixation with only paraformaldehyde. The highly improved signal intensity and staining efficiency enabled us to carry out RescueSTED nanoscopy on microtubules and nuclear pores and describe their organization in greater detail throughout the blood stage cycle.

免疫荧光染色是可视化单个细胞内源性细胞结构组织的关键技术。血液阶段恶性疟原虫的标记和成像由于它是一种小的细胞内寄生虫，因此一直具有挑战性。寄生虫免疫荧光的广泛使用的标准是在悬浮液中固定，并向基于多聚甲醛的溶液中添加微量的戊二醛。尽管这保持了红细胞的完整性，但据推测，如果有的话，寄生虫蛋白的抗原性只会稍微降低。在这里，我们显示了即使少量的戊二醛也可能对免疫荧光染色质量产生有害影响，并提出了另一种细胞接种方案，该方案只能用低聚甲醛固定。高度改善的信号强度和染色效率使我们能够对微管和核孔进行RescueSTED纳米检查，并在整个血液周期中更详细地描述它们的组织。