For the improved understanding of biological systems on the nanoscale, it is necessary to enhance the resolution of light microscopy in the visible wavelength range beyond the limits of conventional epifluorescence microscopy (optical resolution of about 200 nm laterally, 600 nm axially). Recently, various far-field methods have been developed allowing a substantial increase of resolution ("superresolution microscopy", or "lightoptical nanoscopy"). This opens an avenue to 'nano-image' intact and even living cells, as well as other biostructures like viruses, down to the molecular detail. Thus, it is possible to combine light optical spatial nanoscale information with ultrastructure analyses and the molecular interaction information provided by molecular cell biology. In this review, we describe the principles of spectrally assigned localization microscopy (SALM) of biological nanostructures, focusing on a special SALM approach, spectral precision distance/position determination microscopy (SPDM) with physically modified fluorochromes (SPDM(Phymod) . Generally, this SPDM method is based on high-precision localization of fluorescent molecules, which can be discriminated using reversibly bleached states of the fluorophores for their optical isolation. A variety of application examples is presented, ranging from superresolution microscopy of membrane and cytoplasmic protein distribution to dual-color SPDM of nuclear proteins. At present, we can achieve an optical resolution of cellular structures down to the 20-nm range, with best values around 5 nm (∼1/100 of the exciting wavelength).
The development of safe engineered nanoparticles (NPs) requires a detailed understanding of their interaction mechanisms on a cellular level. Therefore, quantification of NP internalization is crucial to predict the potential impact of intracellular NP doses, providing essential information for risk assessment as well as for drug delivery applications. In this study, the internalization of 25 nm and 85 nm silica nanoparticles (SNPs) in alveolar type II cells (A549) was quantified by application of super-resolution STED (stimulated emission depletion) microscopy. Cells were exposed to equal particle number concentrations (9.2 × 1010 particles mL−1) of each particle size and the sedimentation of particles during exposure was taken into account. Microscopy images revealed that particles of both sizes entered the cells after 5 h incubation in serum supplemented and serum-free medium. According to the in vitro sedimentation, diffusion, and dosimetry (ISDD) model 20–27% of the particles sedimented. In comparison, 102-103 NPs per cell were detected intracellularly serum-containing medium. Furthermore, in the presence of serum, no cytotoxicity was induced by the SNPs. In serum-free medium, large agglomerates of both particle sizes covered the cells whereas only high concentrations (≥ 3.8 × 1012 particles mL−1) of the smaller particles induced cytotoxicity.
HepaRG is a bipotent stem cell line that can be differentiated towards hepatocyte-like and biliary-like cells. The entire cultivation process requires 1 month and relies on the addition of 2% dimethyl sulfoxide (DMSO) to the culture. Our motivation in this research is to differentiate HepaRG cells (progenitor cells and undifferentiated cells) towards hepatocyte-like cells by minimizing the cultivation time and without using DMSO treatment by instead using a microfluidic device combined with the following strategies: (a) comparison of extracellular matrices (matrigel and collagen I), (b) types of flow (one or both sides), and (c) effects of DMSO. Our results demonstrate that matrigel promotes the differentiation of progenitor cells towards hepatocytes and biliary-like cells. Moreover, the frequent formation of HepaRG cell clusters was observed by a supply of both sides of flow, and the cell viability and liver specific functions were influenced by DMSO. Finally, differentiated HepaRG progenitor cells cultured in a microfluidic device for 14 days without DMSO treatment yielded 70%of hepatocyte-like cells with a highly polarized organization that reacted to stimulation with IL-6 to produce C-reactive protein (CRP). This culture model has high potential for investigating cell differentiation and liver pathophysiology research.
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