Abstract
We describe the development of a tracking device, mounted on an epi-fluorescent inverted microscope, suited to obtain time resolved 3D Lagrangian tracks of fluorescent passive or active micro-objects in microfluidic devices. The system is based on real-time image processing, determining the displacement of a x, y 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 determining the displacement of a piezo mover keeping the moving object in focus. Track coordinates of the object with respect to the microfluidic device as well as images of the object are obtained at a frequency of several tenths of Hertz. This device is particularly well adapted to obtain trajectories of motile micro-organisms in microfluidic devices with or without flow.
Highlights
In their seminal work, Berg and Brown have shown in 1972 that tracking individual micro-organisms in 3D was a determinant step to understand bacterial motility and chemotactic response to chemical gradients.1 At that time the device built by Berg, designed to perform the tracking and 3D trajectory reconstruction of bacteria, was conceptually and technically outstanding.2Since other techniques of 3D tracking have emerged
Using post-processing, these tracking methods lead to the 3D reconstruction of a few simple trajectories of objects as Brownian spherical colloids6 or bacteria
We describe the technical details of a Lagrangian 3D tracking method that extends the seminal work of Berg2 using modern visualization tools and data treatment possibilities
Summary
Berg and Brown have shown in 1972 that tracking individual micro-organisms in 3D was a determinant step to understand bacterial motility and chemotactic response to chemical gradients. At that time the device built by Berg, designed to perform the tracking and 3D trajectory reconstruction of bacteria, was conceptually and technically outstanding.. The development of fast piezo scanning has led to high speed image stack acquisition techniques These techniques were used to reconstruct, by post processing, the swimming trajectories of micro-organisms.. Using post-processing, these tracking methods lead to the 3D reconstruction of a few simple trajectories of objects as Brownian spherical colloids or bacteria.7 This method has recently led to important advances in the understanding of sperm swimming and chemotaxis.. A unique feature of the technique described here is the possibility to track individual bright spots in a crowded environment of opaque objects It can, for example, be used to follow a specific fluorescent bacterium in a suspension of non-fluorescent bacteria, in order to characterize the features of collective motion
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