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We report a high-throughput and label-free computational imaging technique that simultaneously measures in three-dimensional (3D) space the locomotion and angular spin of the freely moving heads of microswimmers and the beating patterns of their flagella over a sample volume more than two orders-of-magnitude larger compared to existing optical modalities. Using this platform, we quantified the 3D locomotion of 2133 bovine sperms and determined the spin axis and the angular velocity of the sperm head, providing the perspective of an observer seated at the moving and spinning sperm head. In this constantly transforming perspective, flagellum-beating patterns are decoupled from both the 3D translation and spin of the head, which provides the opportunity to truly investigate the 3D spatio-temporal kinematics of the flagellum. In addition to providing unprecedented information on the 3D locomotion of microswimmers, this computational imaging technique could also be instrumental for micro-robotics and sensing research, enabling the high-throughput quantification of the impact of various stimuli and chemicals on the 3D swimming patterns of sperms, motile bacteria and other micro-organisms, generating new insights into taxis behaviors and the underlying biophysics.
We propose that this high-throughput and label-free computational microswimmer imaging platform not only provides unmatched capabilities for the measurement of 3D locomotion patterns of microswimmers, but also lays the foundation for new imaging tools and insights that can be transformative in micro-robotics and sensing-related research and applications. Furthermore, this imaging technique might provide a high-throughput tool to rapidly quantify the impact of various stimuli on the 3D swimming patterns of sperms and other motile micro-organisms, leading to new insights into 3D locomotion and taxis behaviors.
3D reconstruction of sperm locomotion. Step 1: Background-subtracted holograms resulting from dual-angle illumination undergo a holographic reconstruction process, which uses object support-based phase retrieval to mitigate the twin image artifact. Step 2: A two-dimensional tail fitting process is performed on these holographic reconstructions to establish the skeletons corresponding to both of the 2D projections of the sperm flagellum. These fitted skeletons are subsequently spatially smoothened and interpolated into 2D strands with a smaller node length. Step 3: 3D tracking and tail reconstruction. Based on the two illumination angles and corresponding projections, the height of each infinitesimal sub-section along the 3D strand is determined, and the 3D configuration of the entire strand, representing the flagellum, is reconstructed. This reconstruction process is also detailed in the Supplementary Information section and Figure 3. Step 4: Alternating phase-wrapping events between the two holographic reconstructions are used to determine the head spin direction and angular velocity (also detailed in the Results and Discussion section).
From the perspective of the global coordinate system of the image sensor chip in the present imaging technique or any microscopic imaging modality in general, the motion of the flagellum reflects the combination of the 3D translation, head spin and flagellum beating of the sperm; therefore, several different types of motion affect and directly determine the mathematical representation of the flagellar beating patterns when using such a global coordinate system. However, to better understand the flagellar kinematics of the sperm, it is desirable to isolate the 3D beating pattern that is only related to the flagellum itself, taking out the effects of head locomotion and spin. Obtaining the complete 3D information of freely moving sperm enables the decoupling of the flagellar beating patterns of the sperms from their head locomotion and spin, thereby enabling the observation of flagella beating under a local coordinate system that moves and spins together with the sperm head. Stated differently, we can obtain the perspective of an observer located on and moving with the sperm head, looking towards the flagellum, which isolates the sperm flagellar beating from other sources of motion (Figure 5).
Today, Muybridge is best known for his pioneering chronophotography of animal locomotion between 1878 and 1886, which used multiple cameras to capture the different positions in a stride, and for his zoopraxiscope, a device for projecting painted motion pictures from glass discs that pre-dated the flexible perforated film strip used in cinematography. From 1883 to 1886, he entered a very productive period at the University of Pennsylvania in Philadelphia, producing over 100,000 images of animals and humans in motion, occasionally capturing what the human eye could not distinguish as separate moments in time.
Stanford also wanted a proper picture of the horse at full speed, and was frustrated that the existing depictions and descriptions seemed incorrect. The human eye could not fully break down the action at the quick gaits of the trot and gallop. Up until this time, most artists painted horses at a trot with one foot always on the ground; and at a full gallop with the front legs extended forward and the hind legs extended to the rear, and all feet off the ground. There are stories that Stanford had made a $25,000 bet on his theories about horse locomotion, but no evidence has been found of such a wager. Instead, it has been estimated that he would spend a total of $50,000 over the next several years, to fund his investigations.
Muybridge had images from his motion studies hand-copied in the form of silhouettes or line drawings onto a disc, to be viewed in the machine he had invented, which he called a "zoopraxiscope". Later, his more-detailed images were hand-coloured and marketed commercially. A device he developed was later regarded as an early movie projector, and the process was an intermediate stage toward motion pictures or cinematography.
Muybridge and Stanford had a major falling-out concerning his research on equine locomotion. Stanford had asked his friend and horseman Dr JBD Stillman to write a book analysing The Horse in Motion, which was published in 1882. Stillman used Muybridge's photos as the basis for his 100 illustrations, and the photographer's research for the analysis, but he gave Muybridge no prominent credit. The historian Phillip Prodger later suggested that Stanford considered Muybridge as just one of his employees, and not deserving of special recognition. Stanford was quite proud of his role in creating the book, and commissioned a portrait of himself by Jean-Louis-Ernest Meissonier, in which a copy of the volume was visible under his arm.
To avoid ambiguity and inconsistency, it is critical to precisely define the criteria for scoring behavior both in the laboratory and in publications. For example, initiating backward locomotion is generally called a reversal. But, some researchers define a minimum distance the animal has to move backward to score as a bona fide reversal; some do not. Some researchers include omega turns as reversals. And, some researchers count increased backward locomotion as a reversal or response; most do not. A precise definition of the behavior to be scored is critical for analysis.
Harsh touch to the body is measured by prodding animals with a platinum wire in the midsection of the body (Chalfie and Sulston, 1981; Way and Chalfie, 1989). Nonmoving gravid adults are prodded at or just posterior to the vulva. Animals respond by initiating locomotion, usually by backing up. Animals to be assayed should be grown in the continuous presence of food. Animals who are starved and animals that have passed through the dauer stage often fail to respond to harsh touch regardless of the functional status of the PVD sensory neurons.
Nose touch is assayed by laying a hair on the surface of the plate in front of the animal. As an animal moves forward, it contacts the hair with the tip of the nose perpendicular to the direction of movement. Some practice is required to anticipate where the hair must be laid down for an animal to run into at 90 degrees. Normal animals immediately initiate backward locomotion. Defective animals either hurdle over the hair or slide their nose along the hair. An individual animal is tested no more than 10 times in a row to avoid inducing habituation. You can drop the assay plate to induce lethargic animals to move or reverse direction, but you cannot touch the animal to facilitate the assay.
Standard NGM agar plates (6 cm) are dried for 2 hours at 37°C without their lids. For quantitative locomotion assays, unseeded plates are preferable. Seeded plates should be used for egg-laying assays. For quantitative locomotion assays, the worms are first placed on unseeded plates containing no ethanol, so twice the number of plates to be used in the assay should be dried plus at least one more for use in calculating the volume of the media in each plate.
For locomotion assays, 10 worms of each genotype to be tested are transferred using minimal bacteria to one copper ring per genotype on unseeded plates containing no ethanol. The animals are allowed 30 minutes to become accustomed to a lack of food before being transferred without food to the assay plates. The plates are sealed with Parafilm and speed measurements are collected 10-50 minutes after the animals are transferred, typically after 20 minutes exposure. 3-4 genotypes (10 worms each) can be transferred in less than 2 minutes, which means that animals of different genotypes can be compared directly with minimal difference in the time of intoxication.
I prefer this measure of locomotion to others that have been used. The actual distance a worm moves per time may be influenced by the number of eggs it carries, the thickness of the bacterial lawn it is on, number of direction reversals the worm makes, etc. Counting the number of body bends seems to me to be a more direct measure of the effort the worm is making to move which should be less influenced by these factors. 2b1af7f3a8