Kinetic Analysis of the Motility of Giant Virus-Infected Amoebae Using Phase-Contrast Microscopic Images

Abstract

Tracking cell motility is a useful tool for the study of cell physiology and microbiology. Although phase-contrast microscopy is commonly used, the existence of optical artifacts called "halo" and "shade-off" have inhibited image analysis of moving cells. Here we show kinetic image analysis of Acanthamoeba motility using a newly developed computer program named "Phase-contrast-based Kinetic Analysis Algorithm for Amoebae (PKA3)," which revealed giant-virus-infected amoebae-specific motilities and aggregation profiles using time-lapse phase-contrast microscopic images. This program quantitatively detected the time-dependent, sequential changes in cellular number, size, shape, and direction and distance of cell motility. This method expands the potential of kinetic analysis of cultured cells using versatile phase-contrast images. Furthermore, this program could be a useful tool for investigating detailed kinetic mechanisms of cell motility, not only in virus-infected amoebae but also in other cells, including cancer cells, immune response cells, and neurons.

Continuous Real-Time Motility Analysis of Acanthamoeba Reveals Sustained Movement in Absence of Nutrients

“…However, very little is currently known about real-time Acanthamoeba behavior and how that behavior may translate to contact lens systems. Thus, in this study, we expanded a recently-described method [ 11 , 12 , 13 ] with the goal of providing specific answers as to how Acanthamoeba behave for future extrapolation into Acanthamoeba activity in the context of a potential contamination event in a contact lens care system, which could lead to an ocular infection. The present study takes the first steps in this endeavor: understanding how these amoeba behave in a potential overnight period, using several different types of commonly used “control” settings in ocular Acanthamoeba research, with and without nutrients provided.…”

“…These new tools have been used to successfully track and quantify the movements of many different microscopic populations, such as human neural stem cells, breast adenocarcinoma cells, and Drosophila [ 8 , 9 , 10 ]. These techniques have also recently been used to quantify the cytopathic effects on Acanthamoeba motility [ 11 ] after infection with a giant virus [ 12 ] and during exposure to an electric field [ 13 ]. However, to our knowledge, this technology has never been used to examine multiple Acanthamoeba strains for movements on various surfaces and in varying nutritional states for a period as long as 12 h.…”

<i>Marseilleviridae</i> Lineage B Diversity and Bunch Formation Inhibited by Galactose

“…(b) Kinetic image analysis of hokutovirus-infected amoeba cells in the presence or absence of monosaccharides. Time-lapse images of hokutovirus-infected or healthy amoeba cells cultured with media supplemented with or without monosaccharides (glucose, mannose, or galactose) were captured using a BZ-X800/X810 all-in-one fluorescent microscope and kinetic image analyses of cells were performed using the PKA3 algorithm developed in our laboratory, as previously described (Fukaya et al, 2020).…”

“…According to our PKA3 algorithm-based kinetic image analysis of amoeba cells cultured in media containing sugars (Fukaya et al, 2020), it was clear that the addition of galactose inhibited the increase in average "particle size" in both single and aggregated cells (Fig. 2b, Movie S1, and S2).…”

“…Briefly, 45-sec time-lapse images of virus-infected amoeba cells cultured with monosaccharides were captured using all-in-one fluorescent microscopy with a BZ-X800/X810 microscope (Keyence). Kinetic analyses of cells were performed as previously described using a phase-contrast-based kinetic analysis algorithm for amoebae (PKA3) developed in our laboratory (Fukaya et al, 2020).…”