Super-resolved 3D Tracking of Cargo Transport Through Nuclear Pore Complexes by Astigmatism Imaging

This protocol describes a two-color astigmatic imaging approach that enables direct 3D visualization of cargo transport trajectories relative to a super-resolved octagonal double-ring scaffold structure of the nuclear pore complex (NPC). Though astigmatism imaging is commonly achieved via a cylindrical lens, this protocol utilizes an adaptive optics (AO) system, which enables optimization of the astigmatism for the precision needs of the experiment as well as correction of the focal mismatch arising from chromatic aberrations in multi-color applications. With this approach, single particle spatial precision values in x, y, and z are typically 5-20 nm, and these depend on astigmatism, photon level and position in z. The method enables resolution of transport conduits through the ~60 nm diameter pore of NPCs by particle tracking on the millisecond timescale. The success of this approach is enabled by the high rigidity of fully active NPCs within the nuclear envelope of permeabilized cells. For a detailed application of this protocol, please refer to https://www.nature.com/articles/s41556-021-00815-6. The figure and table numbers in this protocol that are indicated with an “NCB” prefix (e.g., NCB Figure X) refer to the figures and table in this reference paper.

Growth Kinetics of Single Polymer Particles in Solution via Active-Feedback 3D Tracking

The ability to directly observe chemical reactions at the single-molecule and single-particle level has enabled the discovery of behaviors otherwise obscured by the ensemble averaging in bulk measurements. However powerful, a common restriction of these studies to date has been the absolute requirement to surface tether or otherwise immobilize the chemical reagent/reaction of interest. This constraint arose from a fundamental limitation of conventional microscopy techniques, which could not track molecules or particles rapidly diffusing in three dimensions, as occurs in solution. However, much chemistry occurs in the solution phase, leaving single-particle/-molecule analysis of this critical area of science beyond the scope of available technology. Here we report the first solution-phase studies and measurements of any chemical reaction at single-particle/-molecule level in freely diffusing solution. During chemical reaction, freely diffusing polymer particles (D ~ 10-12 m2/s) yielded single-particle 3D trajectories and real-time volumetric images that were analyzed to extract the growth rates of individual particles. These volumetric images show that the average growth rate is a poor representation of the true underlying variability in polymer-particle growth behavior. These data revealed statistically significant populations of faster- and slower-growing particles at different depths in the sample, showing emergent heterogeneity while particles are still in the solution phase. These results go against the prevailing premise that chemical processes freely diffusing in solution will exhibit uniform kinetics. These new understandings of mechanisms behind polymer growth variations bring about an exciting opportunity to control particle-size and plausibly molecular weight polydispersity by the rational design of conditions to dictate spatial growth gradients. We anticipate that these studies will launch a new field of solution-phase, nonensemble-averaged measurements of chemical reactions.

Pixel chamber: a solid-state active-target for 3D imaging of charm and beauty

The aim of the pixel chamber project is to develop the first “solid-state bubble chamber” for high precision measurement of charm and beauty. In this paper we will describe the idea for the first silicon active target conceived as an ultra-high granular stack of hundreds of very thin monolithic active pixel sensors (MAPS), which provides continuous, high-resolution 3D tracking of all of the particles produced in proton-silicon interactions occurring inside the detector volume, including open charm and beauty. We will also discuss the high-precision tracking and vertexing performances, showing that the vertex resolution can be up to one order of magnitude better than state-of-the-art detectors like the LHCb one.

Real-Time 3D Tracking of Laparoscopy Training Instruments for Assessment and Feedback

Assessment of minimally invasive surgical skills is a non-trivial task, usually requiring the presence and time of expert observers, including subjectivity and requiring special and expensive equipment and software. Although there are virtual simulators that provide self-assessment features, they are limited as the trainee loses the immediate feedback from realistic physical interaction. The physical training boxes, on the other hand, preserve the immediate physical feedback, but lack the automated self-assessment facilities. This study develops an algorithm for real-time tracking of laparoscopy instruments in the video cues of a standard physical laparoscopy training box with a single fisheye camera. The developed visual tracking algorithm recovers the 3D positions of the laparoscopic instrument tips, to which simple colored tapes (markers) are attached. With such system, the extracted instrument trajectories can be digitally processed, and automated self-assessment feedback can be provided. In this way, both the physical interaction feedback would be preserved and the need for the observance of an expert would be overcome. Real-time instrument tracking with a suitable assessment criterion would constitute a significant step towards provision of real-time (immediate) feedback to correct trainee actions and show them how the action should be performed. This study is a step towards achieving this with a low cost, automated, and widely applicable laparoscopy training and assessment system using a standard physical training box equipped with a fisheye camera.

Three-dimensional tracking of the ciliate Tetrahymena reveals the mechanism of ciliary stroke-driven helical swimming

Helical swimming in free-space is a common behavior among microorganisms, such as ciliates that are covered with thousands hair-like motile cilia, and is thought to be essential for cells to orient directly to an external stimulus. However, a direct quantification of their three-dimensional (3D) helical trajectories has not been reported, in part due to difficulty in tracking 3D swimming behavior of ciliates, especially Tetrahymena with a small, transparent cell body. Here, we conducted 3D tracking of fluorescent microbeads within a cell to directly visualize the helical swimming exhibited by Tetrahymena. Our technique showed that Tetrahymena swims along a right-handed helical path with right-handed rolling of its cell body. Using the Tetrahymena cell permeabilized with detergent treatment, we also observed that influx of Ca2+ into cilia changed the 3D-trajectory patterns of Tetrahymena swimming, indicating that the beating pattern of cilia is the determining factor in its swimming behavior.

Positive Effects of Fast Growth on Locomotor Performance in Pelagic Fish Juveniles: In Contrast to Existing Evidence

Many laboratory experiments on aquatic vertebrates that inhabit closed water or coastal areas have highlighted negative effects of fast growth on swimming performance. Nonetheless, field studies on pelagic fishes have provided evidence of survival advantages of faster growing individuals. To reconcile this contradiction, we examined the relationship between growth rate and swimming performance as a continuous function for juveniles of chub mackerel (scomber japonicus) using 3D tracking analysis. For experiments, 20, 24, 27 and 30 days-post-hatch individuals within the size range of 14.5–25.3 mm were used. We found that the growth–swimming (burst speed) relationship in chub mackerel was substantially positive and it was suggested to be supported by morphological traits such as muscle area, which also positively correlated with growth rate. This finding is consistent with field observations showing selective survival of fast-growing individuals of this species, reconciling the current contradiction between laboratory experiments and field observations. Growth was suggested to trade off with swimming performance, as reported in many previous studies, when it was extremely fast. Therefore, a dome-shaped quadratic curve described the relationship between growth rate and burst speed better than a linear or generalized linear model. These results, obtained from the rarely tested offshore species, strongly suggests the importance of experimental verification using animals that inhabit various types of habitats in understanding the principles underlying the evolution of growth–locomotor relationship.

Deep learning based behavioral profiling of rodent stroke recovery

Stroke research heavily relies on rodent behavior when assessing underlying disease mechanisms and treatment efficacy. Although functional motor recovery is considered the primary targeted outcome, tests in rodents are still poorly reproducible, and often unsuitable for unraveling the complex behavior after injury. Here, we provide a comprehensive 3D gait analysis of mice after focal cerebral ischemia based on the new deep learning-based software (DeepLabCut, DLC) that only requires basic behavioral equipment. We demonstrate a high precision 3D tracking of 10 body parts (including all relevant joints and reference landmarks) in several mouse strains with an accuracy of 99.4%. Building on this rigor motion tracking, a comprehensive post-analysis (with >100 parameters) unveils biologically relevant differences in locomotor profiles after a stroke over a time course of three weeks. We further refine the widely used ladder rung test using deep learning and compare its performance to human annotators. The generated DLC-assisted tests were then benchmarked to five widely used conventional behavioral set-ups (neurological scoring, rotarod, ladder rung walk, cylinder test, and single-pellet grasping) regarding sensitivity, accuracy, time use and costs. We conclude that deep learning-based motion tracking with comprehensive post-analysis provides accurate and sensitive data to describe the complex recovery of rodents following a stroke. The experimental set-up and analysis can also benefit a range of other neurological injuries that affect locomotion.