Dataset Information

Pre-gelation staining expansion microscopy for visualisation of the Plasmodium liver stage.

ABSTRACT: Fluorescence and light microscopy are important tools in the history of natural science. However, the resolution of microscopes is limited by the diffraction of light. One possible method to circumvent this physical restriction is the recently developed expansion microscopy (ExM). However, the original ultrastructure ExM (U-ExM) protocol is very time-consuming, and some epitopes are lost during the process. In this study, we developed a shortened pre-gelation staining ExM (PS-ExM) protocol and tested it to investigate the Plasmodium liver stage. The protocol presented in this study allows expanding of pre-stained samples, which results in shorter incubation times, better preservation of some epitopes and the advantage that non-expanded controls can be performed alongside using the same staining protocol. The protocol applicability was accessed throughout the Plasmodium liver stage, showing isotropic five-fold expansion. Furthermore, we used PS-ExM to visualise parasite mitochondria as well as the association of lysosomes to the parasitophorous vacuole membrane (PVM) as an example of visualising host-pathogen interaction. We are convinced that this new tool will be helpful for a deeper understanding of the biology of the Plasmodium liver stage.

SUBMITTER: Atchou K

PROVIDER: S-EPMC10729816 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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Pre-gelation staining expansion microscopy for visualisation of the Plasmodium liver stage.

Atchou Kodzo K Berger Bianca Manuela BM Heussler Volker V Ochsenreiter Torsten T

Journal of cell science 20231130 22

Fluorescence and light microscopy are important tools in the history of natural science. However, the resolution of microscopes is limited by the diffraction of light. One possible method to circumvent this physical restriction is the recently developed expansion microscopy (ExM). However, the original ultrastructure ExM (U-ExM) protocol is very time-consuming, and some epitopes are lost during the process. In this study, we developed a shortened pre-gelation staining ExM (PS-ExM) protocol and t ...[more]

PMID: 37942994

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Project description:The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite's life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luc(con), expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1-5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium.

Dataset Information

Pre-gelation staining expansion microscopy for visualisation of the Plasmodium liver stage.

Publications

Pre-gelation staining expansion microscopy for visualisation of the Plasmodium liver stage.

Similar Datasets

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