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- ItemKinetic and spectroscopic responses of pH-sensitive nanoparticles: Influence of the silica matrix(London : Royal Society of Chemistry, 2019) Clasen, A.; Wenderoth, S.; Tavernaro, I.; Fleddermann, J.; Kraegeloh, A.; Jung, G.Intracellular pH sensing with fluorescent nanoparticles is an emerging topic as pH plays several roles in physiology and pathologic processes. Here, nanoparticle-sized pH sensors (diameter far below 50 nm) for fluorescence imaging have been described. Consequently, a fluorescent derivative of pH-sensitive hydroxypyrene with pKa = 6.1 was synthesized and subsequently embedded in core and core-shell silica nanoparticles via a modified Stöber process. The detailed fluorescence spectroscopic characterization of the produced nanoparticles was carried out for retrieving information about the environment within the nanoparticle core. Several steady-state and time-resolved fluorescence spectroscopic methods hint to the screening of the probe molecule from the solvent, but it sustained interactions with hydrogen bonds similar to that of water. The incorporation of the indicator dye in the water-rich silica matrix neither changes the acidity constant nor dramatically slows down the protonation kinetics. However, cladding by another SiO2 shell leads to the partial substitution of water and decelerating the response of the probe molecule toward pH. The sensor is capable of monitoring pH changes in a physiological range by using ratiometric fluorescence excitation with λex = 405 nm and λex = 488 nm, as confirmed by the confocal fluorescence imaging of intracellular nanoparticle uptake.
- ItemLocal variations of HER2 dimerization in breast cancer cells discovered by correlative fluorescence and liquid electron microscopy(Washington, DC [u.a.] : Assoc., 2015) Peckys, Diana B.; Korf, Ulrike; de Jonge, NielsThe formation of HER2 homodimers plays an important role in breast cancer aggressiveness and progression; however, little is known about its localization. We have studied the intra- and intercellular variation of HER2 at the single-molecule level in intact SKBR3 breast cancer cells. Whole cells were visualized in hydrated state with correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM). The locations of individual HER2 receptors were detected using an anti-HER2 affibody in combination with a quantum dot (QD), a fluorescent nanoparticle. Fluorescence microscopy revealed considerable differences of HER2 membrane expression between individual cells and between different membrane regions of the same cell (that is, membrane ruffles and flat areas). Subsequent ESEM of the corresponding cellular regions provided images of individually labeled HER2 receptors. The high spatial resolution of 3 nm and the close proximity between the QD and the receptor allowed quantifying the stoichiometry of HER2 complexes, distinguishing between monomers, dimers, and higher-order clusters. Downstream data analysis based on calculating the pair correlation function from receptor positions showed that cellular regions exhibiting membrane ruffles contained a substantial fraction of HER2 in homodimeric state. Larger-order clusters were also present. Membrane areas with homogeneous membrane topography, on the contrary, displayed HER2 in random distribution. Second, HER2 homodimers appeared to be absent from a small subpopulation of cells exhibiting a flat membrane topography, possibly resting cells. Local differences in homodimer presence may point toward functional differences with possible relevance for studying metastasis and drug response.