Stimulated emission depletion (STED) microscopy is routinely used to resolve the ultrastructure of cells with a ∼10-fold higher resolution compared to diffraction limited imaging. While STED microscopy is based on preparing the excited state of fluorescent probes with light, the recently developed expansion microscopy (ExM) provides subdiffraction resolution by physically enlarging the sample before microscopy. The expansion of the fixed cells by cross-linking and swelling of hydrogels easily enlarges the sample ∼4-fold and hence increases the effective optical resolution by this factor. To overcome the current limits of these complementary approaches, we combined ExM with STED (ExSTED) and demonstrated an increase in resolution of up to 30-fold compared to conventional microscopy (<10 nm lateral and ∼50 nm isotropic). While the increase in resolution is straightforward, we found that high-fidelity labeling via multi-epitopes is required to obtain emitter densities that allow ultrastructural details with ExSTED to be resolved. Our work provides a robust template for super-resolution microscopy of entire cells in the ten nanometer range.

Microtubule-crosslinking motor proteins, which slide antiparallel microtubules, are required for remodeling of microtubule networks. Hitherto, all microtubule-crosslinking motors have been shown to slide microtubules at constant velocity until no overlap between the microtubules remains, leading to breakdown of the initial microtubule geometry. Here, we show in vitro that the sliding velocity of microtubules, driven by human kinesin-14, HSET, decreases when microtubules start to slide apart, resulting in the maintenance of finite-length microtubule overlaps. We quantitatively explain this feedback by the local interaction kinetics of HSET with overlapping microtubules, causing retention of HSET in shortening overlaps. Consequently, the increased HSET density in the overlaps leads to a density-dependent decrease in sliding velocity and the generation of an entropic force antagonizing the force exerted by the motors. Our results demonstrate that a spatial arrangement of microtubules can regulate the collective action of molecular motors through local alteration of their individual interaction kinetics.

This study reports the in vitro and in vivo biological activities of recombinant human bone morphogenetic protein 2 (rhBMP-2) released from the core-shell structure of a nanofibrous barrier membrane as a sustained delivery model for bone regeneration. RhBMP-2 incorporating poly(ethylene glycol) was used as the core, and poly(caprolactone) was used as the shell surrounding the core. To determine its release profiles, the release solution was collected and the amount of rhBMP-2 was measured by ELlSA at different time points. In vitro rhBMP-2, released from the delivery system over at least 24 days, reached a stable rate of 500 pg per day and guided bone marrow mesenchymal stem cells (BMMSCs) to express osteogenic genes. The distribution and proliferation of BMMSCs in the nanofibrous barrier membrane was measured by laser confocal scanning microscopy (LCSM) and scanning electron microscopy (SEM). The biological activity of rhBMP-2 was tested in BMMSC/membrane culture in vitro and in a rabbit calvarium defect model in vivo. Osteogenic genes osteonectin (ON) and core binding factor-α1 (Cbf-α1) expression of the BMMSCs cultured on the BMP-2-PEG/PCL membrane were significantly higher than those of cells on the PEG/PCL membrane at the late time points using real-time PCR (p < 0.05). The membranes containing the rhBMP-2 group exhibited the fastest and most bone formation compared to others in rabbit cranial defect models (p < 0.05). This study revealed that rhBMP-2 could be incorporated into a core-shell electrospun membrane, and preserve sustained release capability and biological activity.

Stimulated emission depletion (STED) microscopy is routinely used to resolve the ultrastructure of cells with a ~10-fold higher resolution compared to diffraction limited imaging. While STED microscopy is based on preparing the excited state of fluorescent probes with light, the recently developed expansion microscopy (ExM) provides sub-diffraction resolution by physically enlarging the sample before microscopy. Expansion of fixed cells by crosslinking and swelling of hydrogels easily enlarges the sample ~4-fold and hence increases the effective optical resolution by this factor. To overcome the current limits of these complimentary approaches, we here combined ExM with STED (ExSTED) and demonstrate an increase in resolution of up to 30-fold compared to conventional microscopy (<10 nm lateral and ~50 nm isotropic). While the increase in resolution is straight forward, we found that high fidelity labelling via multi-epitopes is required to obtain emitter densities that allow to resolve ultra-structural details with ExSTED. Our work provides a robust template for super resolution microscopy of entire cells in the ten nanometer range.