Measurement of membrane rigidity on trapped unilamellar phospholipid vesicles by using differential confocal microscopy.

We use optical tweezers to trap a unilamellar phospholipid vesicle and measure the out-of-plane thermal fluctuations by using differential confocal microscopy. Bending moduli of the lipid membranes are calculated directly from the mean-square values of the fluctuation amplitudes. Owing to the refractive index contrast between the inner and outer solutions of the vesicle, optical tweezers trap the vesicle laterally and improve the reliability of the measured fluctuation amplitudes along the optical axis. Bending moduli of membranes in gel or fluid phases obtained by the combination of differential confocal microscopy and optical tweezers are close to those reported previously. We also obtain the bending modulus of sphingomyelin membranes in the gel phase, which was not reported previously.

High aspect ratio nanoimprinted grooves of poly(lactic-co-glycolic acid) control the length and direction of retraction fibers during fibroblast cell division.

Retraction fibers (RFs) determine orientation of the cell division axis and guide the spreading of daughter cells. Long and unidirectional RFs, which are especially apparent during mitosis of cells in three-dimensional (3D) environments, enable improved control over cell fate, following division. However, 3D gel environments lack the cues necessary for predetermining the orientation of RFs to direct tissue architecture. While patterning of focal adhesion regions by microcontact printing can determine orientation of the RFs through enhancing focal adhesion numbers along particular directions, the RFs remain short due to the two-dimensional culture environment. Herein, the authors demonstrate that nanoimprinted grooves of polylactic acid glycolic acid (PLGA) with a high aspect ratio (A.R. of 2.0) can provide the cues necessary to control the direction of RFs, as well as enable the maintenance of long and unidirectional RFs as observed within 3D cultures, while the same is not possible with PLGA grooves of lower A.R. (1.0 or lower). Based on enhanced levels of contact guidance of premitotic fibroblast protrusions at high A.R. grooves and deeper levels of focal adhesion due to filopodia extensions into these grooves, it is suggested that submicron (800 nm width) PLGA grooves with A.R. of 2 are capable of supporting mechanical forces from cell protrusions to a greater depth, thereby enabling the maintenance of the protrusions as long and unidirectional RFs during cell division. Given the scalability and versatility of nanoimprint techniques, the authors envision a platform for designing nanostructures to direct tissue regeneration and developmental biology.

Three-dimensional tracking and temporal analysis of liposomal transport in live cells using bright-field imaging.

Gold nanoparticles (AuNPs) confined in liposomes of diameters around 200 nm produce strong scattering signal owing to surface plasmon resonance, and therefore bright-field optical tracking of the AuNP-encapsulating liposomes can be conducted in living cells. Using an optical profiling technique called noninterferometric wide-field optical profilometry and a bright-field tracking algorithm, the polynomial-fit Gaussian weight method, we analyze three-dimensional (3D) motion of such liposomes in living fibroblasts. The positioning accuracy in three dimensions is nearly 20 nm. We tag the liposome membranes with fibroblast growth factor-1 and reveal the intracellular transportation processes toward or away from the nucleus. On the basis of a temporal analysis of the intracellular 3D trajectories of AuNP-encapsulating liposomes, we identify directed and diffusive motions in the transportation processes.