Abstract
Babesia microti causes “emergency” human babesiosis. However, little is known about the alterations in B. microti invaded red blood cells (Bm-RBCs) at the individual cell level. Through quantitative phase imaging techniques based on laser interferometry, we present the simultaneous measurements of structural, chemical, and mechanical modifications in individual mouse Bm-RBCs. 3-D refractive index maps of individual RBCs and in situ parasite vacuoles are imaged, from which total contents and concentration of dry mass are also precisely quantified. In addition, we examine the dynamic membrane fluctuation of Bm-RBCs, which provide information on cell membrane deformability.
Highlights
Tissues[11,12]
We show that invading B. microti parasites use the cytosolic ATP of host cells for their metabolism
3-D refractive index (RI) maps of Bm-red blood cells (RBCs) clearly reveal the shapes and locations of the PVs as well as overall shapes of host RBCs, whereas healthy RBCs exhibit characteristic donut shapes or discocytes. These PVs exhibit RI values lower than that of RBC cytosol, indicating that B. microti creates its own environment inside the cytosol of a RBC and depletes the hemoglobin (Hb) in PVs. 3-D RI maps of B. microti-invaded RBCs (Bm-RBCs) are measured in various infection stages depending on the number of PVs inside a host cell: single (Fig. 1B), double (Fig. 1C), quadruple (Fig. 1D), multiple (> 3) PVs (Fig. 1E) and without PVs (Fig. 1F), and a healthy RBC (Fig. 1A)
Summary
Sample preparation of mouse Bm-RBCs infection and determination of parasitemia. The animal care and use protocol adheres to Korean Laboratory Animal Act (Act No 902 & 93). Control and B. microti infected blood were 300 times diluted in DPBS buffer. To measure the morphological parameters, we used the reconstructed 3-D RI maps by the diffraction optical tomography algorithm from measured multiple optical phase maps corresponding to various illumination angles on the sample. The total dry mass of a RBC was obtained from the integrating 2-D optical phase over entire cell area with RI increment of proteins as[47],. All samples were measured using ATP bioluminescent somatic cell assay kit (Sigma-aldrich, Saint Louis, USA). To a separate vial containing 100 μ L of 1× Somatic Cell ATP Releasing Reagent, 50 μ L of ultrapure water was added, and 50 μ L of the cell sample to be assayed was added and immediately measure the light to be emitted IS.
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