In vitro and in vivo documentation of quantum dots labeled Trypanosoma cruzi–Rhodnius prolixus interaction using confocal microscopy

Abstract

Semiconductor quantum dots (QDs) are highly fluorescent nanocrystals markers that allow long photobleaching and do not destroy the parasites. In this paper, we used fluorescent core shell quantum dots to perform studies of live parasite-vector interaction processes without any observable effect on the vitality of parasites. These nanocrystals were synthesized in aqueous medium and physiological pH, which is very important for monitoring live cells activities, and conjugated with molecules such as lectins to label specific carbohydrates involved on the parasite-vector interaction. These QDs were successfully used for the study of in vitro and in vivo interaction of Trypanosoma cruzi and the triatomine Rhodnius prolixus. These QDs allowed us to acquire real time confocal images sequences of live T. cruzi-R. prolixus interactions for an extended period, causing no damage to the cells. By zooming to the region of interest, we have been able to acquire confocal images at the three to four frames per second rate. Our results show that QDs are physiological fluorescent markers capable to label living parasites and insect vector cells. QDs can be functionalized with lectins to specifically mark surface carbohydrates on perimicrovillar membrane of R. prolixus to follow, visualize, and understand interaction between vectors and its parasites in real-time.

Studying nanotoxic effects of CdTe quantum dots in Trypanosoma cruzi

“…In one study, mammalian (HeLa) cells or Dictyostelium discoideum cells did not present any alterations and continued the endocytic process of QDs after 20 days (Jaiswal et al 2003). Similar experiments performed by our group have demonstrated that insects fed QDs presented no significant apparent dysfunction or toxicity for up to 10 days (Feder et al 2009).…”

“…Parasites labelling -For the in vitro assay, 5 μL of a T. cruzi (1X107) suspension was incubated with 50 μL of diluted yellow-emitting CdSe QDs (10 μL CdSe to 90 μL of a 0.14 M PBS/0.01 M NaCl phosphate buffer, pH 7.2) and stabilized with MAA, for 60 min at 28°C (Feder et al 2009). The parasites were then observed by confocal microscopy.…”

“…CdTe QDs were built from the adaptation of two methods: Zhang et al (2000) and Gaponik et al (2002). For details, refer to the Materials and Methods section in the study by Feder et al (2009).…”

“…QDs are semiconductor nanocrystals used in the electronics industry and biomedical imaging. The main advantage of QDs in biomedical imaging is their high photostability, which allows long-term realtime study of in vitro, in vivo and ex vivo cell processes (Feder et al 2009, Kasemets et al 2009, Marquis et al 2009). However, this advantage would be overridden if QDs' toxicity to cells can change their functionality and cause artefacts.…”

II-VI Quantum Dots as Fluorescent Probes for Studying Trypanosomatides

“…CdTe/CdS-MAA and CdSe/CdS-MAA nanocrystals were also used by other research groups to study T. cruzi-insect vector interactions (Feder et al 2009). These QDs were synthesized by using mercaptoacetic acid (MAA).…”