Measuring Molecular Diffusion in Dynamic Subcellular Nanostructures by Fast Raster Image Correlation Spectroscopy and 3D Orbital Tracking.

Here we provide demonstration that fast fluorescence fluctuation spectroscopy is a fast and robust approach to extract information on the dynamics of molecules enclosed within subcellular nanostructures (e.g., organelles or vesicles) which are also moving in the complex cellular environment. In more detail, Raster Image Correlation Spectroscopy (RICS) performed at fast timescales (i.e., microseconds) reveals the fast motion of fluorescently labeled molecules within two exemplary dynamic subcellular nanostructures of biomedical interest, the lysosome and the insulin secretory granule (ISG). The measurement of molecular diffusion is then used to extract information on the average properties of subcellular nanostructures, such as macromolecular crowding or molecular aggregation. Concerning the lysosome, fast RICS on a fluorescent tracer allowed us to quantitatively assess the increase in organelle viscosity in the pathological condition of Krabbe disease. In the case of ISGs, fast RICS on two ISG-specific secreting peptides unveiled their differential aggregation propensity depending on intragranular concentration. Finally, a combination of fast RICS and feedback-based 3D orbital tracking was used to subtract the slow movement of subcellular nanostructures from the fast diffusion of molecules contained within them and independently validate the results. Results presented here not only demonstrate the acquired ability to address the dynamic behavior of molecules in moving, nanoscopic reference systems, but prove the relevance of this approach to advance our knowledge on cell function at the subcellular scale.

Osteoblast Responses to Titanium-Coated Subcellular Scaled Microgrooves.

Statistical data have consistently shown that implant loosening is a significant causative factor for revision surgeries. Both in vivo and in vitro studies have confirmed the positive influences of microgrooved titanium implant surfaces on improving orthopedic titanium implants compared with a smooth titanium surface. Complete cell-groove adhesion is a prerequisite for rapid and robust osseointegration. For the first time, this work has quantified the influence of the titanium groove width at the subcellular scale (5-20 µm) on osteoblast responses, using titanium-coated microgrooved silicon wafer specimens (surface roughness, Ra = ∼1.5 nm), which can avoid the latent influence of variations in surface roughness from the use of normal titanium substrates. The cell-groove adhesion increased from 53.07% to 98.55% with an increasing groove width from 5 to 20 µm. In addition, both the cell spreading area and cell width were proportional to groove width. However, no statistically significant influence (p > 0.05) of groove width was identified on cell proliferation and differentiation. An exponential model was proposed to predict the groove geometries that can facilitate complete cell-groove adhesion. The underlying mechanisms were discussed. The experimental findings of this study provide a unique basis for the design of titanium implant surfaces.