Phenotyping single-cell motility in microfluidic confinement

Abstract

At all scales, the movement patterns of organisms serve as dynamic read-outs of their behaviour and physiology. We devised a novel droplet microfluidics assay to encapsulate single algal microswimmers inside closed arenas, and comprehensively studied their roaming behaviour subject to a large number of environmental stimuli. We compared two model species, Chlamydomonas reinhardtii (freshwater alga, 2 cilia), and Pyramimonas octopus (marine alga, 8 cilia), and detailed their highly-stereotyped behaviours and the emergence of a trio of macroscopic swimming states (smooth-forward, quiescent, tumbling or excitable backward). Harnessing ultralong timeseries statistics, we reconstructed the species-dependent reaction network that underlies the choice of locomotor behaviour in these aneural organisms, and discovered the presence of macroscopic non-equilibrium probability fluxes in these active systems. We also revealed for the first time how microswimmer motility changes instantaneously when a chemical is added to their microhabitat, by inducing deterministic fusion between paired droplets - one containing a trapped cell, and the other, a pharmacological agent that perturbs cellular excitability. By coupling single-cell entrapment with unprecedented tracking resolution, speed and duration, our approach offers unique and potent opportunities for diagnostics, drug-screening, and for querying the genetic basis of micro-organismal behaviour.

Switching of behavioral modes and their modulation by a geometrical cue in the ciliate Stentor coeruleus

“…In Stentor , photoresponse ( Song et al, 1980a ; Song et al, 1980b ; Iwatsuki, 1992 ), external mechanical stimuli ( Wood, 1969b ) and chemical avoidance responses ( Jennings, 1906 ; Dexter et al, 2019 ; Trinh et al, 2019 ) have been reported previously. The behaviors of microorganisms are strongly influenced by extracellular geometries ( Kantsler et al, 2013 ; Kunita et al, 2014 ; Beppu et al, 2017 ; Beppu et al,2021 ; Ishikawa and Kikuchi, 2018 ; Nishigami et al, 2018 ; Ohmura et al, 2018 ; Ohmura et al, 2021 ; Ostapenko et al, 2018 ; Bhattacharjee and Datta, 2019 ; Bentley et al, 2021 ; Okuyama et al, 2021 ; Théry et al, 2021 ) corresponding to mud, dead leaves or other varieties of sediments in their habitats. In our study, we evaluated the effect of a structure on behavioral transition.…”

“…Even in bacteria and protists, a navigation ability is required to survive, and their habitats consist of diverse microscopic structures that affect their behaviors. Indeed, the behavioral responses of these organisms have been studied in various microspace geometries: porous media ( Bhattacharjee and Datta, 2019 ), rigid surfaces ( Kantsler et al, 2013 ; Ohmura et al, 2018 , 2021 ), dead ends ( Kunita et al, 2014 ), corners ( Théry et al, 2021 ), interstices between inclined plates ( Ishikawa and Kikuchi, 2018 ) and confined areas ( Beppu et al, 2017 , 2021 ; Ostapenko et al, 2018 ; Bentley et al, 2021 ). From these studies, it was determined that cell behaviors change in response to the geometrical structures of living spaces.…”

Distinct gaits of self-propelled quadriflagellate microswimmers

“…So far, we have focused solely on gaits that are stable in time, in which the swimmer dynamics are prescribed. In reality, long-time tracking of single flagellates has shown that gait and behaviour are both highly-dynamic, exhibiting stochastic and reversible fluctuations between distinct gaits or swimming modes [20]. Future work should seek to account for the statistics of such gait transitions as measured from experimental data to investigate their influence on the overall morphology of simulated trajectories.…”

“…We note that when path-sampling the trajectory history of live cells, stochastic transitions between different modes or gaits can occur due to biological noise and environmental perturbations, and this can significantly impact the overall swimming trajectory [20][21][22]. For simplicity, we shall neglect gaitswitching dynamics here and focus only on the consequences of a sustained, deterministic gait.…”