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Single molecule localization microscopy (SMLM) allows the imaging of cellular structures with resolutions five to ten times below the diffraction limit of optical microscopy. It was originally introduced as a two-dimensional technique based on the localization of single emitters as projection onto the x-y imaging plane. The determination of the axial position of a fluorescent emitter is only possible by additional information. Here we report a method (spatial filter SMLM (SFSMLM)) that allows to determine the axial positions of fluorescent molecules and nanoparticles on the nanometer scale by the usage of two spatial filters, which are placed in two otherwise identical emission detection channels. SFSMLM allows axial localization in a range of ca. 1.5 μm with a localization precision of 15 - 30 nm in axial direction. The technique was utilized for localizing and imaging small cellular structures - e.g. actin filaments, vesicles and mitochondria - in three dimensions.

The chloride channel (CLC) protein family comprises ion channels and proton-coupled anion transporters with fundamental physiological roles in humans. Several properties of CLC proteins defy the rigid dichotomy between ion channels and transporters as these opposite thermodynamic mechanisms of transport are implemented in a very similar structural architecture. All the CLC transporters are expressed in intracellular organelles where they are somehow important for the ionic homeostasis of these compartments. However, their specific physiological role is still unclear. This chapter focuses on the biophysical properties and physiological role of the endosomal Cl−/H+ antiporter ClC-5 mutated in Dent’s disease.