Abstract
Malaria remains a major burden world-wide, but the disease-causing parasites from the genus Plasmodium are difficult to study in vitro. Owing to the small size of the parasites, subcellular imaging poses a major challenge and the use of super-resolution techniques has been hindered by the parasites’ sensitivity to light. This is particularly apparent during the blood-stage of the Plasmodium life cycle, which presents an important target for drug research. The iron-rich food vacuole of the parasite undergoes disintegration when illuminated with high-power lasers such as those required for high resolution in Stimulated Emission Depletion (STED) microscopy. This causes major damage to the sample precluding the use of this super-resolution technique. Here we present guided STED, a novel adaptive illumination (AI) STED approach, which takes advantage of the highly-reflective nature of the iron deposit in the cell to identify the most light-sensitive parts of the sample. Specifically in these parts, the high-power STED laser is deactivated automatically to prevent local damage. Guided STED nanoscopy finally allows super-resolution imaging of the whole Plasmodium life cycle, enabling multicolour imaging of blood-stage malaria parasites with resolutions down to 35 nm without sample destruction.
Highlights
Malaria remains a global threat, with 216 million cases world-wide and over 400,000 deaths in 2016 alone[1]
While no super-resolution information can be gained from non-fluorescent areas, unnecessary activation of the Stimulated Emission Depletion (STED) laser here still damages neighbouring fluorescent structures owing to the large size of the donut-shaped point spread function (PSF) of the STED laser
The use of super-resolution microscopy in the studies of malaria-causing parasites has been limited to early stages of the parasite life cycle[26] or to techniques that provide only moderate improvement in the resolution, such as SIM50
Summary
Malaria remains a global threat, with 216 million cases world-wide and over 400,000 deaths in 2016 alone[1]. The parasite’s proteins displayed on the red blood cell surface, such as the major virulence factor PfEMP1, facilitate cytoadherence of the infected cells to each other and to the blood endothelium[17,18,19,20,21] This sequesters infected cells from circulation and prevents their destruction in the spleen. During this time, the parasite grows and undergoes cell divisions producing approximately 20 new merozoites[22,23], which upon cell rapture are released into the blood stream to infect new red blood www.nature.com/scientificreports/. Hemozoin poses a major challenge during the imaging of malaria parasites It is highly reflective, making it very difficult to study by confocal microscopy, and it partly absorbs energy from high-power lasers, such as those used in STED. Energy absorption tends to decrease slightly with wavelength, but heating is still evident at 700 nm[42] and 820 nm[39] and severe around 670 nm[41]
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