Rotational Dynamics of Optically Trapped Human Spermatozoa

Introduction. Optical trapping is a laser-based method for probing the physiological and mechanical properties of cells in a noninvasive manner. As sperm motility is an important criterion for assessing the male fertility potential, this technique is used to study sperm cell motility behavior and rotational dynamics. Methods and Patients. An integrated optical system with near-infrared laser beam has been used to analyze rotational dynamics of live sperm cells from oligozoospermic and asthenozoospermic cases and compared with controls. Results. The linear, translational motion of the sperm is converted into rotational motion on being optically trapped, without causing any adverse effect on spermatozoa. The rotational speed of sperm cells from infertile men is observed to be significantly less as compared to controls. Conclusions. Distinguishing normal and abnormal sperm cells on the basis of beat frequency above 5.6 Hz may be an important step in modern reproductive biology to sort and select good quality spermatozoa. The application of laser-assisted technique in biology has the potential to be a valuable tool for assessment of sperm fertilization capacity for improving assisted reproductive technology

Assembling Neurospheres: Dynamics of Neural Progenitor/Stem Cell Aggregation Probed Using an Optical Trap

Optical trapping (tweezing) has been used in conjunction with fluid flow technology to dissect the mechanics and spatio-temporal dynamics of how neural progenitor/stem cells (NSCs) adhere and aggregate. Hitherto unavailable information has been obtained on the most probable minimum time (∼5 s) and most probable minimum distance of approach (4–6 µm) required for irreversible adhesion of proximate cells to occur. Our experiments also allow us to study and quantify the spatial characteristics of filopodial- and membrane-mediated adhesion, and to probe the functional dynamics of NSCs to quantify a lower limit of the adhesive force by which NSCs aggregate (∼18 pN). Our findings, which we also validate by computational modeling, have important implications for the neurosphere assay: once aggregated, neurospheres cannot disassemble merely by being subjected to shaking or by thermal effects. Our findings provide quantitative affirmation to the notion that the neurosphere assay may not be a valid measure of clonality and “stemness”. Post-adhesion dynamics were also studied and oscillatory motion in filopodia-mediated adhesion was observed. Furthermore, we have also explored the effect of the removal of calcium ions: both filopodia-mediated as well as membrane-membrane adhesion were inhibited. On the other hand, F-actin disrupted the dynamics of such adhesion events such that filopodia-mediated adhesion was inhibited but not membrane-membrane adhesion

Time-lapse images from real-time movies depicting the outcome of a 1-hour treatment of NSCs with EGTA, a calcium ion chelator.

<p>A) absence of membrane-mediated adhesion between the two EGTA-treated NSCs brought into close contact for ∼6 s (data extracted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038613#pone.0038613.s008" target="_blank">Movie S6</a>); B) filopodial contact between the two EGTA-treated NSCs results in adhesion at ∼5 s. Subsequently, the NSCs remain adhered when the trap focus is moved slightly, but they are eventually pulled apart ∼8 s later. The scale bar denotes 10 µm.</p

Time-lapse images of filopodia-mediated cell adhesion dynamics.

<p>A) – D) Time lapse images from a real-time movie (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038613#pone.0038613.s005" target="_blank">Movie S3</a>) showing adhesion of a neural progenitor cell to a neurosphere via filopodial interaction. The white cross denotes the position of the optically-trapped cell being brought towards the neurosphere. The single cell adheres to the neurosphere after approximately 5 seconds. Note the faintly visible filopodial bridge connecting the neural progenitor to the neurosphere in panel C). The scale bar denotes 10 µm.</p

Phase contrast images of neurospheres and single neural progenitor cells from dissociated neurospheres.

<p>A) Two fused neurospheres; B) Fused neurospheres with filopodia visible on the surface; C) Differential interference contrast image of a single neural progenitor cell with filopodia; and D) a single neural progenitor cell without filopodia. Filopodia are indicated by white arrows.</p

Histograms showing the percentage of cells that underwent irreversible adhesion as a function of contact time for filopodia-mediated adhesion (top panel) and membrane-mediated adhesion (lower panel).

<p>A minimum of 50 cells were used for each measurement. Time determination was by analyzing individual frames from real-time movies, with each frame being temporally separated from adjacent frames by 40 ms.</p

Histogram depicting the lower limit of the force required to separate two adhered neural progenitor cells.

<p>This corresponds to the force required by a cell to escape from the flow cell of the optical trap schematically depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038613#pone-0038613-g001" target="_blank">Fig 1B and 1C</a>. Data presented pertains to measurements made on 56 cells.</p

Differential effect of 0.1 µM Cytochalasin-D treatment on membrane and filopodial adhesion.

<p>A) Time-lapse images from a real-time movie (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038613#pone.0038613.s009" target="_blank">Movie S7</a>) showing a cell (initially at the trap focus, X) approaching a neurosphere. As the trap is moved away (panel IV), the cell is seen to remain adhered to the neurosphere. B) Time-lapse images from a real-time movie (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038613#pone.0038613.s010" target="_blank">Movie S8</a>) of a single cell approaching a filopodium on the neurosphere. Despite close proximity (for up to 7 seconds), no filopodial adhesion takes place: the cell remains within the trap focal volume, X, as it is moved away from the neurosphere. The cartoon below panel B is a chimera depicting both phenomena (as cells numbered 1 and 2, respectively). The scale bar denotes 10 µm.</p