Achromatic light patterning and improved image reconstruction for parallelized RESOLFT nanoscopy.

Fluorescence microscopy is rapidly turning into nanoscopy. Among the various nanoscopy methods, the STED/RESOLFT super-resolution family has recently been expanded to image even large fields of view within a few seconds. This advance relies on using light patterns featuring substantial arrays of intensity minima for discerning features by switching their fluorophores between 'on' and 'off' states of fluorescence. Here we show that splitting the light with a grating and recombining it in the focal plane of the objective lens renders arrays of minima with wavelength-independent periodicity. This colour-independent creation of periodic patterns facilitates coaligned on- and off-switching and readout with combinations chosen from a range of wavelengths. Applying up to three such periodic patterns on the switchable fluorescent proteins Dreiklang and rsCherryRev1.4, we demonstrate highly parallelized, multicolour RESOLFT nanoscopy in living cells for ~100 × 100 µm2 fields of view. Individual keratin filaments were rendered at a FWHM of ~60-80 nm, with effective resolution for the filaments of ~80-100 nm. We discuss the impact of novel image reconstruction algorithms featuring background elimination by spatial bandpass filtering, as well as strategies that incorporate complete image formation models.

Probing the functional equivalence of otoferlin and synaptotagmin 1 in exocytosis.

Cochlear inner hair cells (IHCs) use Ca(2+)-dependent exocytosis of glutamate to signal sound information. Otoferlin (Otof), a C(2) domain protein essential for IHC exocytosis and hearing, may serve as a Ca(2+) sensor in vesicle fusion in IHCs that seem to lack the classical neuronal Ca(2+) sensors synaptotagmin 1 (Syt1) and Syt2. Support for the Ca(2+) sensor of fusion hypothesis for otoferlin function comes from biochemical experiments, but additional roles in late exocytosis upstream of fusion have been indicated by physiological studies. Here, we tested the functional equivalence of otoferlin and Syt1 in three neurosecretory model systems: auditory IHCs, adrenal chromaffin cells, and hippocampal neurons. Long-term and short-term ectopic expression of Syt1 in IHCs of Otof (-/-) mice by viral gene transfer in the embryonic inner ear and organotypic culture failed to rescue their Ca(2+) influx-triggered exocytosis. Conversely, virally mediated overexpression of otoferlin did not restore phasic exocytosis in Syt1-deficient chromaffin cells or neurons but enhanced asynchronous release in the latter. We further tested exocytosis in Otof (-/-) hippocampal neurons and in Syt1(-/-) IHCs but found no deficits in vesicle fusion. Expression analysis of different synaptotagmin isoforms indicated that Syt1 and Syt2 are absent from mature IHCs. Our data argue against a simple functional equivalence of the two C(2) domain proteins in exocytosis of IHC ribbon synapses, chromaffin cells, and hippocampal synapses.

Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses.

We describe a STED microscope optimized for colocalization experiments with up to three colors. Two fluorescence labels are separated by their fluorescence lifetime whereas a third channel is discriminated by the wavelength of fluorescence emission. Since it does not require a second STED beam, separating by lifetime is insensitive to drift and thus optimally suited for colocalization analyses. Furthermore, we propose a setup having a second STED beam for long duration multicolor recording.

Formation of a Bazooka-Stardust complex is essential for plasma membrane polarity in epithelia.

Apical-basal polarity in Drosophila melanogaster epithelia depends on several evolutionarily conserved proteins that have been assigned to two distinct protein complexes: the Bazooka (Baz)-PAR-6 (partitioning defective 6)-atypical protein kinase C (aPKC) complex and the Crumbs (Crb)-Stardust (Sdt) complex. These proteins operate in a functional hierarchy, in which Baz is required for the proper subcellular localization of all other proteins. We investigated how these proteins interact and how this interaction is regulated. We show that Baz recruits Sdt to the plasma membrane by direct interaction between the Postsynaptic density 95/Discs large/Zonula occludens 1 (PDZ) domain of Sdt and a region of Baz that contains a phosphorylation site for aPKC. Phosphorylation of Baz causes the dissociation of the Baz-Sdt complex. Overexpression of a nonphosphorylatable version of Baz blocks the dissociation of Sdt from Baz, causing phenotypes very similar to those of crb and sdt mutations. Our findings provide a molecular mechanism for the phosphorylation-dependent interaction between the Baz-PAR-3 and Crb complexes during the establishment of epithelial polarity.

Two-color STED microscopy reveals different degrees of colocalization between hexokinase-I and the three human VDAC isoforms.

The voltage-dependent anion channel (VDAC, also known as mitochondrial porin) is the major transport channel mediating the transport of metabolites, including ATP, across the mitochondrial outer membrane. Biochemical data demonstrate the binding of the cytosolic protein hexokinase-I to VDAC, facilitating the direct access of hexokinase-I to the transported ATP. In human cells, three hVDAC isoforms have been identified. However, little is known on the distribution of these isoforms within the outer membrane of mitochondria and to what extent they colocalize with hexokinase-I. In this study we show that whereas hVDAC1 and hVDAC2 are localized predominantly within the same distinct domains in the outer membrane, hVDAC3 is mostly uniformly distributed over the surface of the mitochondrion. We used two-color stimulated emission depletion (STED) microscopy enabling a lateral resolution of ~40 nm to determine the detailed sub-mitochondrial distribution of the three hVDAC isoforms and hexokinase-I. Individual hVDAC and hexokinase-I clusters could thus be resolved which were concealed in the confocal images. Quantitative colocalization analysis of two-color STED images demonstrates that within the attained resolution, hexokinase-I and hVDAC3 exhibit a higher degree of colocalization than hexokinase-I with either hVDAC1 or hVDAC2. Furthermore, a substantial fraction of the mitochondria-bound hexokinase-I pool does not colocalize with any of the three hVDAC isoforms, suggesting a more complex interplay of these proteins than previously anticipated. This study demonstrates that two-color STED microscopy in conjunction with quantitative colocalization analysis is a powerful tool to study the complex distribution of membrane proteins in organelles such as mitochondria.PACS: 87.16.Tb, 87.85.Rs.

Limited intermixing of synaptic vesicle components upon vesicle recycling.

Synaptic vesicles recycle repeatedly in order to maintain synaptic transmission. We have previously proposed that upon exocytosis the vesicle components persist as clusters, which would be endocytosed as whole units. It has also been proposed that the vesicle components diffuse into the plasma membrane and are then randomly gathered into new vesicles. We found here that while strong stimulation (releasing the entire recycling pool) causes the diffusion of the vesicle marker synaptotagmin out of synaptic boutons, moderate stimulation (releasing approximately 19% of all vesicles) is followed by no measurable diffusion. In agreement with this observation, synaptotagmin molecules labeled with different fluorescently tagged antibodies did not appear to mix upon vesicle recycling, when investigated by subdiffraction resolution stimulated emission depletion (STED) microscopy. Finally, as protein diffusion from vesicles has been mainly observed using molecules tagged with pH-sensitive green fluorescent protein (pHluorin), we have also investigated the membrane patterning of several native and pHluorin-tagged proteins. While the native proteins had a clustered distribution, the GFP-tagged ones were diffused in the plasma membrane. We conclude that synaptic vesicle components intermix little, at least under moderate stimulation, possibly because of the formation of clusters in the plasma membrane. We suggest that several pHluorin-tagged vesicle proteins are less well integrated in clusters.

A STED microscope aligned by design.

STED microscopes are commonly built using separate optical paths for the excitation and the STED beam. As a result, the beams must be co-aligned and can be subject to mechanical drift. Here, we present a single-path STED microscope whose beams are aligned by design and hence is insensitive to mechanical drift. The design of a phase plate is described which selectively modulates the STED beam but leaves the excitation beam unaffected. The performance of the single-beam setup is on par with previous dual-beam designs.

STED microscopy with a supercontinuum laser source.

We report on a straightforward yet powerful implementation of stimulated emission depletion (STED) fluorescence microscopy providing subdiffraction resolution in the far-field. Utilizing the same super-continuum pulsed laser source both for excitation and STED, this implementation of STED microscopy avoids elaborate preparations of laser pulses and conveniently provides multicolor imaging. Operating at pulse repetition rates around 1 MHz, it also affords reduced photobleaching rates by allowing the fluorophore to relax from excitable metastable dark states involved in photodegradation. The imaging of dense nanoparticles and of the microtubular network of mammalian cells evidences a spatial resolution of 30-50 nm in the focal plane, i.e. by a factor of 8-9 beyond the diffraction barrier.

3D reconstruction of high-resolution STED microscope images.

Tackling biological problems often involves the imaging and localization of cellular structures on the nanometer scale. Although optical super-resolution below 100 nm can be readily attained with stimulated emission depletion (STED) and photoswitching microscopy methods, attaining an axial resolution <100 nm with focused light generally required the use of two lenses in a 4Pi configuration or exceptionally bright photochromic fluorophores. Here, we describe a simple technical solution for 3D nanoscopy of fixed samples: biological specimens are fluorescently labeled, embedded in a polymer resin, cut into thin sections, and then imaged via STED microscopy with nanoscale resolution. This approach allows a 3D image reconstruction with a resolution <80 nm in all directions using available state-of-the art STED microscopes.

Fluorescence fluctuation spectroscopy in subdiffraction focal volumes.

We establish fluorescence fluctuation spectroscopy (FFS) with nanoscale detection volumes generated by stimulated emission depletion. Our method applies fluorescence correlation spectroscopy and fluorescence intensity distribution analysis to extract molecular information about mobilities and fluorescence emission in solution. The combination of correlation analysis with that of photon intensity distributions reveals a fivefold squeezing of the detection volume over current diffraction-limited systems, which is in full agreement with the simultaneously demonstrated 25-fold reduction in (axial) focal transit time. Our method significantly extends the potential of far-field FFS, including for the noninvasive investigation of molecular reactions at higher concentrations.