Significance: A single plant can produce from hundreds to millions of lateral roots (LRs), which facilitate water and mineral uptake and transport. LRs originate in a tissue called the pericycle, but the underlying mechanism is obscure. With the aid of clonal analysis, we deduced that LR initiation begins with a single pericycle founder cell (FC) which then recruits the neighboring pericycle cells to become FCs. A time-lapse analysis validated this conclusion and also showed that FC recruitment is an auxin-dependent and gradual process that takes place in an unpredictable pattern. Thus, LR initiation is not a one-step process, as previously considered.

Tracing tubular structures from biomedical images is important for a wide range of applications. Particularly, the spermatozoon is an essential cell whose flagella has a tubular form. Its main function is to fertilize the egg and the flagellum is fundamental to achieve this task which depends importantly on the dynamics of intracellular calcium ([Ca2+]i). Measuring [Ca2+]i along the flagellum in 3D is not a simple matter since it requires: (i) sophisticated fluorescence imaging techniques dealing with low intensity and signal to noise ratio (SNR) and (ii) tracing the flagellum’s centerline. Most of the algorithms proposed to trace tubular structures have been developed for multi-branch structures not being adequate for single tubular structures with low SNR. Taking into account the prior knowledge that the flagellum is constituted by a single tubular structure, we propose an automatic method to trace and track multiple single tubular structures from 3D+t images. First, an algorithm based on one-class classification allows enhancement of the flagellum. This enhanced 3D image permits guiding an iterative centerline algorithm towards the flagellum’s centerline. Each sperm is assigned an ID to keep track of it in 3D+t. Our algorithm was quantitatively evaluated using as ground truth 564 semi-manual traces (six 3D+t image stacks) comparing them to those obtained from state-of-the-art tubular structure centerline extraction algorithms. The qualitative and quantitative results show that our algorithm is extracting similar traces as compared with ground truth and it is more robust and accurate to trace the flagellum’s centerline than multi-branch algorithms.

Are there intracellular Ca²⁺ ([Ca²⁺]_i) oscillations correlated with flagellar beating in human sperm? The results reveal statistically significant [Ca²⁺]_i oscillations that are correlated with the human sperm flagellar beating frequency, when measured in three-dimensions (3D). Fast [Ca²⁺]_i oscillations that are correlated to the beating flagellar frequency of cells swimming in a restricted volume have been detected in hamster sperm. To date, such findings have not been confirmed in any other mammalian sperm species. An important question that has remained regarding these observations is whether the fast [Ca²⁺]_i oscillations are real or might they be due to remaining defocusing effects of the Z component arising from the 3D beating of the flagella. Healthy donors whose semen samples fulfill the WHO criteria between the age of 18–28 were selected. Cells from at least six different donors were utilized for analysis. Approximately the same number of experimental and control cells were analyzed. Motile cells were obtained by the swim-up technique and were loaded with Fluo-4 (Ca²⁺ sensitive dye) or with Calcein (Ca²⁺ insensitive dye). Ni²⁺ was used as a non-specific plasma membrane Ca²⁺ channel blocker. Fluorescence data and flagella position were acquired in 3D. Each cell was recorded for up to 5.6 s within a depth of 16 microns with a high speed camera (coupled to an image intensifier) acquiring at a rate of 3000 frames per second, while an oscillating objective vibrated at 90 Hz via a piezoelectric device. From these samples, eight experimental and nine control sperm cells were analyzed in both 2D and 3D. We have implemented a new system that allows [Ca²⁺]_i measurements of the human sperm flagellum beating in 3D. These measurements reveal statistically significant [Ca²⁺]_i oscillations that correlate with the flagellar beating frequency. These oscillations may arise from intracellular sources and/or Ca²⁺ transporters, as they were insensitive to external Ni²⁺, a non-specific plasma membrane Ca²⁺ channel blocker. N/A. Analysis in 3D needs a very fast image acquisition rate to correctly sample a volume containing swimming sperm. This condition requires a very short exposure time per image making it necessary to use an image intensifier which also increases noise. The lengthy analysis time required to obtain reliable results limited the number of cells that could be analyzed. The possibility of recording flagellar [Ca²⁺]_i oscillations described here may open a new avenue to better understand ciliary and flagellar beating that are fundamental for mucociliary clearance, oocyte transport, fertilization, cerebrospinal fluid pressure regulation and developmental left-right symmetry breaking in the embryonic node. This work was supported by Consejo Nacional de Ciencia y Tecnología (CONACyT) (grants 253952 to G.C.; 156667 to F.M.M. and Fronteras 71 39908-Q to A.D. and Post-doctoral scholarships 366844 to P.H.-H. and 291028 to F.M.) and the Dirección General de Asuntos del Personal Académico of the Universidad Nacional Autónoma de México (DGAPA-UNAM) (grants CJIC/CTIC/4898/2016 to F.M. and IN205516 to A.D.). There are no conflicts of interest to declare.

Human spermatozoa are the archetype of long-term self-organizing transport in nature and are critical for reproductive success. They utilize coordinated head and flagellar movements to swim long distances within the female reproductive tract in order to find and fertilize the egg. However, to date, long-term analysis of the sperm head–flagellar movements, or indeed those of other flagellated microorganisms, remains elusive due to limitations in microscopy and flagellar-tracking techniques. Here, we present a novel methodology based on local orientation and isotropy of bio-images to obtain long-term kinematic and physiological parameters of individual free-swimming spermatozoa without requiring image segmentation (thresholding). This computer-assisted segmentation-free method evaluates, for the first time, characteristics of the head movement and flagellar beating for up to 9.2 min. We demonstrate its powerful use by showing how releasing Ca2+ from internal stores significantly alters long-term sperm behavior. The method allows for straightforward generalization to other bio-imaging applications, such as studies of bull sperm and Trypanosoma, or indeed of other flagellated microorganisms – appealing to communities other than those investigating sperm biology.