Cellular membranes exhibit a multitude of highly curved morphologies such as buds, nanotubes, cisterna-like sheets defining the outlines of organelles. Here, we mimic cell compartmentation using an aqueous two-phase system of dextran and poly(ethylene glycol) encapsulated in giant vesicles. Upon osmotic deflation, the vesicle membrane forms nanotubes, which undergo surprising morphological transformations at the liquid-liquid interfaces inside the vesicles. At these interfaces, the nanotubes transform into cisterna-like double-membrane sheets (DMS) connected to the mother vesicle via short membrane necks. Using super-resolution (stimulated emission depletion) microscopy and theoretical considerations, we construct a morphology diagram predicting the tube-to-sheet transformation, which is driven by a decrease in the free energy. Nanotube knots can prohibit the tube-to-sheet transformation by blocking water influx into the tubes. Because both nanotubes and DMSs are frequently formed by cellular membranes, understanding the formation and transformation between these membrane morphologies provides insight into the origin and evolution of cellular organelles.

CTP synthase (CTPS) can form filamentous structures termed cytoophidia in cells in all three domains of life. In order to study the mesoscale structure of cytoophidia, we perform fluorescence recovery after photobleaching (FRAP) and stimulated emission depletion (STED) microscopy in human cells. By using an EGFP dimeric tag as a tool to explore the physical properties of cytoophidia, we find that cytoophidia are dynamic and reticular. The reticular structure of CTPS cytoophidia may provide space for other components, such as IMPDH. In addition, we observe CTPS granules with tentacles.

The autoimmune dermatosis pemphigus foliaceus (PF) is predominantly caused by IgG autoantibodies against the desmosomal cadherin desmoglein (Dsg) 1. The exact mechanisms that lead to the characteristic epidermal blistering are not yet fully understood. In the present study, we used a variety of biophysical methods to examine the fate of membrane-bound Dsg1 after incubation with PF patients\' IgG. Dispase-based dissociation assays confirmed that PF-IgG used for this study reduced intercellular adhesion in a manner dependent on phospholipase C (PLC)/Ca2+ and extracellular signal-regulated kinase (ERK) 1/2 signaling. Atomic force microscopy (AFM) revealed that Dsg1 binding on single molecule level paralleled effects on keratinocyte adhesion under the different conditions. Stimulated emission depletion (STED) super-resolution microscopy was used to investigate the localization of Dsg1 after PF-IgG incubation for 24 h. Under control conditions, Dsg1 was found to be in part co-localized with desmoplakin and thus inside of desmosomes as well as extra-desmosomal along the cell border. Incubation with PF-IgG reduced the extra-desmosomal Dsg1 fraction. In line with this, fluorescence recovery after photobleaching (FRAP) experiments demonstrated a strongly reduced mobility of Dsg1 in the cell membrane after PF-IgG treatment indicating remaining Dsg1 molecules were primarily located inside desmosomes. Mechanistically, experiments confirmed the involvement of PLC/Ca2+ since inhibition of PLC or 1,4,5-trisphosphate (IP3) receptor to reduce cytosolic Ca2+ reverted the effects of PF-IgG on Dsg1 intra-membrane mobility and localization. Taken together, our findings suggest that during the first 24 h PF-IgG induce redistribution predominantly of membrane-bound extradesmosomal Dsg1 in a PLC/Ca2+ dependent manner whereas Dsg1-containing desmosomes remain.

In spite of their value as genetically encodable reporters for imaging in living systems, fluorescent proteins have been used sporadically for stimulated emission depletion (STED) super-resolution imaging, owing to their moderate photophysical resistance, which does not enable reaching resolutions as high as for synthetic dyes. By a rational approach combining steady-state and ultrafast spectroscopy with gated STED imaging in living and fixed cells, we here demonstrate that F99S/M153T/V163A GFP (c3GFP) represents an efficient genetic reporter for STED, on account of no excited state absorption at depletion wavelengths <600 nm and a long emission lifetime. This makes c3GFP a valuable alternative to more common, but less photostable, EGFP and YFP/Citrine mutants for STED imaging studies targeting the green-yellow region of the optical spectrum.

Stimulated emission depletion (STED) microscopy is one of the optical superresolution microscopy (SRM) techniques, more recently also referred to as nanoscopy, that have risen to popularity among biologists during the past decade. These techniques keep pushing the physical boundaries of optical resolution toward the molecular scale. Thereby, they enable biologists to image cellular and tissue structures at a level of almost molecular detail that was previously only achievable using electron microscopy. All the while, they retain the advantages of light microscopy, in particular with regards to sample preparation and flexibility of imaging. Commercially available SRM setups have become more and more available and also increasingly sophisticated, both in terms of optical performance and, importantly, ease of use. Institutional microscopy core facilities now offer widespread access to this type of systems. However, the field has grown so rapidly, and keeps growing, that biologists can be easily overwhelmed by the multitude of available techniques and approaches. From this vast array of SRM modalities, STED stands out in one respect: it is essentially an extension to an advanced confocal microscope. Most experienced users of confocal microscopy will find the transition to STED microscopy relatively easy as compared with some other SRM techniques. This also applies to STED sample preparation. Nonetheless, because resolution in STED microscopy does not only depend on the wavelength of the incident light and the numerical aperture of the objective, but crucially also on the square root of the intensity of the depletion laser and, in general, on the photochemical interaction of the fluorophore with the depletion laser, some additional considerations are necessary in STED sample preparation. Here we describe the single color staining of the somatostatin receptor subtype 2A (SSTR2A) and dual color staining of the trans-Golgi-network protein TGN 38 and the t-SNARE syntaxin-6 for STED in the endocrine cell line AtT20 and STED imaging of the samples, providing the protocols in as general a form as possible. The protocols in this chapter are used in this way in an institutional microscopy core facility.

This protocol describes the fluorescence in situ hybridization (FISH) of DNA probes on mitotic chromosome spreads optimized for two super-resolution microscopy approaches-structured illumination microscopy (SIM) and stimulated emission depletion (STED). It is based on traditional DNA FISH methods that can be combined with immunofluorescence labeling (Immuno-FISH). This technique previously allowed us to visualize ribosomal DNA linkages between human acrocentric chromosomes and provided information about the activity status of linked rDNA loci. Compared to the conventional wide-field and confocal microscopy, the quality of SIM and STED data depends a lot more on the optimal specimen preparation, choice of fluorophores, and quality of the fluorescent labeling. This protocol highlights details that make specimens suitable for super-resolution microscopy and tips for good imaging practices.

Molecular crowding is an inherent feature of cell interiors. Synthetic cells as provided by giant unilamellar vesicles (GUVs) encapsulating macromolecules (poly(ethylene glycol) and dextran) represent an excellent mimetic system to study membrane transformations associated with molecular crowding and protein condensation. Similarly to cells, such GUVs exhibit highly curved structures like nanotubes. Upon liquid-liquid phase separation their membrane deforms into apparent kinks at the contact line of the interface between the two aqueous phases. These structures, nanotubes, and kinks, have dimensions below optical resolution. Here, these are studied with super-resolution stimulated emission depletion (STED) microscopy facilitated by immobilization in a microfluidic device. The cylindrical nature of the nanotubes based on the superior resolution of STED and automated data analysis is demonstrated. The deduced membrane spontaneous curvature is in excellent agreement with theoretical predictions. Furthermore, the membrane kink-like structure is resolved as a smoothly curved membrane demonstrating the existence of the intrinsic contact angle, which describes the wettability contrast of the encapsulated phases to the membrane. Resolving these highly curved membrane structures with STED imaging provides important insights in the membrane properties and interactions underlying cellular activities.

The use of photoactivatable dyes in STED microscopy has so far been limited by two-photon activation through the STED beam and by the fact that photoactivatable dyes are poorly solvable in water. Herein, we report ONB-2SiR, a fluorophore that can be both photoactivated in the UV and specifically de-excited by STED at 775 nm. Likewise, we introduce a conjugation and purification protocol to effectively label primary and secondary antibodies with moderately water-soluble dyes. Greatly reducing dye aggregation, our technique provides a defined and tunable degree of labeling, and improves the imaging performance of dye conjugates in general.

C3 protein toxins produced by Clostridium (C.) botulinum and C. limosum are mono-ADP-ribosyltransferases, which specifically modify the GTPases Rho A/B/C in the cytosol of monocytic cells, thereby inhibiting Rho-mediated signal transduction in monocytes, macrophages, and osteoclasts. C3 toxins are selectively taken up into the cytosol of monocytic cells by endocytosis and translocate from acidic endosomes into the cytosol. The C3-catalyzed ADP-ribosylation of Rho proteins inhibits essential functions of these immune cells, such as migration and phagocytosis. Here, we demonstrate that C3 toxins enter and intoxicate dendritic cells in a time- and concentration-dependent manner. Both immature and mature human dendritic cells efficiently internalize C3 exoenzymes. These findings could also be extended to the chimeric fusion toxin C2IN-C3lim. Moreover, stimulated emission depletion (STED) microscopy revealed the localization of the internalized C3 protein in endosomes and emphasized its potential use as a carrier to deliver foreign proteins into dendritic cells. In contrast, the enzyme C2I from the binary C. botulinum C2 toxin was not taken up into dendritic cells, indicating the specific uptake of C3 toxins. Taken together, we identified human dendritic cells as novel target cells for clostridial C3 toxins and demonstrated the specific uptake of these toxins via endosomal vesicles.

Small molecules emitting in the NIR for tracking mitochondrial pH alteration with super-resolution are expected to play an essential role in biomedical applications. Herein, two small molecules based on pyridinium salt (P1 and P2) have been synthesized and systematically investigated. It was found that pyridinium salts P1 and P2 emitted in the NIR (about 610 nm), which could detect pH changes from 2.0 to 11.0 with good linearity and high sensitivity. Importantly, P2 could precisely target cellular mitochondria in a real-time manner under stimulated emission depletion (STED). These results implied a chemical strategy with a potential application in super-resolution imaging and mitochondrial pH determination.