Lagrangian 3D tracking of fluorescent microscopic objects in motion

Darnige, Thierry; Figueroa-Morales, Nuris; Bohec, P.; Lindner, Anke; Clément, Éric

doi:10.1063/1.4982820

Cited by 24 publications

(22 citation statements)

References 12 publications

(28 reference statements)

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“…Using a syringe pump (dosing unit: Low Pressure Syringe Pump neMESYS 290N and base: Module BASE 120N) we flow the suspension inside the channel at different flow rates (1 1,88 4,5 9 18 50 nL.s −1 ), corresponding to wall shear rates of 1-50 s −1 . To have access to the 3D trajectories of single bacteria under flow we use a 3D Lagrangian tracker [41] which is based on real-time image processing, determining the displacement of a xy mechanical stage to keep the chosen object at a fixed position in the observation frame. The z displacement is based on the refocusing of the fluorescent object, keeping the moving object in focus with a precision of a few microns in z.…”

Section: Experimental Details a 3d Tracking Experimentsmentioning

confidence: 99%

Oscillatory surface rheotaxis of swimming E. coli bacteria

et al. 2019

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Bacterial contamination of biological channels, catheters or water resources is a major threat to public health, which can be amplified by the ability of bacteria to swim upstream. The mechanisms of this ‘rheotaxis’, the reorientation with respect to flow gradients, are still poorly understood. Here, we follow individual E. coli bacteria swimming at surfaces under shear flow using 3D Lagrangian tracking and fluorescent flagellar labelling. Three transitions are identified with increasing shear rate: Above a first critical shear rate, bacteria shift to swimming upstream. After a second threshold, we report the discovery of an oscillatory rheotaxis. Beyond a third transition, we further observe coexistence of rheotaxis along the positive and negative vorticity directions. A theoretical analysis explains these rheotaxis regimes and predicts the corresponding critical shear rates. Our results shed light on bacterial transport and reveal strategies for contamination prevention, rheotactic cell sorting, and microswimmer navigation in complex flow environments.

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Section: Experimental Details a 3d Tracking Experimentsmentioning

confidence: 99%

Oscillatory surface rheotaxis of swimming E. coli bacteria

et al. 2019

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“…We developed a device for keeping individual microscopic objects -as swimming bacteria-in focus, as they move in microfluidic chambers [27]. The system is based on real-time image processing, determining the displacement of the stage to keep the chosen object at a fixed position in the observation frame.…”

Section: The 3d Lagrangian Trackermentioning

confidence: 99%

“…In the x-y plane, the spatial limitations are virtually nonexistent, since the stage displacement can be as long as 15 cm, which is much bigger than the typical sizes of the sample (a few millimeters). Details of the apparatus are given in [27], as well as an exhaustive explanation of a method for correcting the mechanical backlash typically affecting these systems and a discussion of the device's performance and limitations.…”

Section: The 3d Lagrangian Trackermentioning

confidence: 99%

3D Spatial Exploration by E. coli Echoes Motor Temporal Variability

et al. 2020

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Unraveling bacterial strategies for spatial exploration is crucial for understanding the complexity in the organization of life. Bacterial motility determines the spatio-temporal structure of microbial communities, controls infection spreading and the microbiota organization in guts or in soils. Most theoretical approaches for modeling bacterial transport rely on their run-and-tumble motion. For Escherichia coli, the run time distribution was reported to follow a Poisson process with a single characteristic time related to the rotational switching of the flagellar motors. However, direct measurements on flagellar motors show heavy-tailed distributions of rotation times stemming from the intrinsic noise in the chemotactic mechanism. Currently, there is no direct experimental evidence that the stochasticity in the chemotactic machinery affect the macroscopic motility of bacteria. In stark contrast with the accepted vision of run-and-tumble, here we report a large behavioral variability of wild-type E. coli, revealed in their three-dimensional trajectories. At short observation times, a large distribution of run times is measured on a population and attributed to the slow fluctuations of a signaling protein triggering the flagellar motor reversal. Over long times, individual bacteria undergo significant changes in motility. We demonstrate that such a large distribution of run times introduces measurement biases in most practical situations. Our results reconcile the notorious conundrum between run time observations and motor switching statistics. We finally propose that statistical modeling of transport properties currently undertaken in the emerging framework of active matter studies, should be reconsidered under the scope of this large variability of motility features.

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“…Such truncation of track lengths also leads to a statistical bias since objects with small diffusivities have a greater contribution to the tracks [10]. Tracking microscopy offers a solution to this issue where the object is kept within the optical FOV using various closed-loop tracking methods [19, 20, 21, 22, 23]. However, such approaches still limit the objects movement to the maximum size of the chamber which is ∼ 100 mm [22], leading to a track length that is much smaller than ecological scales (> 10 m ).…”

Section: Life Under Gravitymentioning

confidence: 99%

Scale-free Vertical Tracking Microscopy: Towards Bridging Scales in Biological Oceanography

Krishnamurthy

et al. 2019

Preprint

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Understanding key biophysical phenomena in the ocean often requires one to simultaneously focus on microscale entities, such as motile plankton and sedimenting particles, while maintaining the macroscale context of vertical transport in a highly stratified environment. This poses a conundrum: How to measure single organisms, at microscale resolution, in the lab, while allowing them to freely move hundreds of meters in the vertical direction? We present a solution in the form of a scale-free, vertical tracking microscope based on a circular "hydrodynamic-treadmill".Our technology allows us to transcend physiological and ecological scales, tracking organisms from marine zooplankton to single-cells over vertical scales of meters while resolving microflows and behavioral processes. We demonstrate measurements of sinking particles, including marine snow as they sediment tens of meters while capturing sub-particle-scale phenomena. We also demonstrate depth-patterned virtual-reality environments for novel behavioral analyses of microscale plankton. This technique offers a new experimental paradigm in microscale ocean biophysics by combining physiological-scale imaging with free movement in an ecological-scale patterned environment.One sentence summary: Scale-free vertical tracking microscopy captures, for the first time, untethered behavioral dynamics at cellular resolution for marine plankton.Our oceans represent the largest habitable ecosystem on the planet. With an average 1 depth of 4 kilometers, this unique ecosystem is highly vertically stratified with physical pa-2 rameters such as light, temperature, salinity and pressure varying dramatically as a function 3 of depth [1]. For example, only the first 200 meters of the ocean receives all the sunlight, 4 while the deeper parts of the ocean are effectively dark. For every 10 meters in depth, the 5 pressure increases by 1 atmosphere. Despite being only a few hundredths of the biomass 6 of terrestrial ecosystems [2], the oceans are responsible for half of the carbon fixed on our 7 planet [3]. Remarkably, this primary production in the ocean comes mostly from minuscule 8 plankton [4], the majority of whom are invisible to the naked eye. Although we have known 9 since the work of Haeckel [5] that the ocean abounds with microscopic plankton, only re-10 cently have we begun to realize their critical role in our planetary cycles [4, 6]. Despite their 11 importance, understanding the key biophysical mechanisms at the scale of planktonic single 12 cells and organisms, which help them navigate the ocean's complex vertical landscape, and in 13 turn influence planetary-scale processes, remains a major hurdle in biological oceanography. 14 A significant challenge in studying these biophysical processes is bridging the vast length 15 scales (from microns to kilometers) and time scales (from millisecond to days). Conven-16 tionally, vertical fluxes in the ocean are measured using sedimentation traps and sampling 17 at different depths [7]. Although crucial, the data is expensive t...

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Lagrangian 3D tracking of fluorescent microscopic objects in motion

Cited by 24 publications

References 12 publications

Oscillatory surface rheotaxis of swimming E. coli bacteria

Oscillatory surface rheotaxis of swimming E. coli bacteria

3D Spatial Exploration by E. coli Echoes Motor Temporal Variability

Scale-free Vertical Tracking Microscopy: Towards Bridging Scales in Biological Oceanography

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