Click Chemistry with Cell-Permeable Fluorophores Expands the Choice of Bioorthogonal Markers for Two-Color Live-Cell STED Nanoscopy.

STED nanoscopy allows for the direct observation of dynamic processes in living cells and tissues with diffraction-unlimited resolution. Although fluorescent proteins can be used for STED imaging, these labels are often outperformed in photostability by organic fluorescent dyes. This feature is especially crucial for time-lapse imaging. Unlike fluorescent proteins, organic fluorophores cannot be genetically fused to a target protein but require different labeling strategies. To achieve simultaneous imaging of more than one protein in the interior of the cell with organic fluorophores, bioorthogonal labeling techniques and cell-permeable dyes are needed. In addition, the fluorophores should preferentially emit in the red spectral range to reduce the potential phototoxic effects that can be induced by the STED light, which further restricts the choice of suitable markers. In this work, we selected five different cell-permeable organic dyes that fulfill all of the above requirements and applied them for SPIEDAC click labeling inside living cells. By combining click-chemistry-based protein labeling with other orthogonal and highly specific labeling methods, we demonstrate two-color STED imaging of different target structures in living specimens using different dye pairs. The excellent photostability of the dyes enables STED imaging for up to 60 frames, allowing the observation of dynamic processes in living cells over extended time periods at super-resolution.

Choroid plexuses carry nodal-like cilia that undergo axoneme regression from early adult stage.

Choroid plexuses (ChPs) produce cerebrospinal fluid and sense non-cell-autonomous stimuli to control the homeostasis of the central nervous system. They are mainly composed of epithelial multiciliated cells, whose development and function are still controversial. We have thus characterized the stepwise order of mammalian ChP epithelia cilia formation using a combination of super-resolution-microscopy approaches and mouse genetics. We show that ChP ciliated cells are built embryonically on a treadmill of spatiotemporally regulated events, starting with atypical centriole amplification and ending with the construction of nodal-like 9+0 cilia, characterized by both primary and motile features. ChP cilia undergo axoneme resorption at early postnatal stages through a microtubule destabilization process controlled by the microtubule-severing enzyme spastin and mitigated by polyglutamylation levels. Notably, this phenotype is preserved in humans, suggesting a conserved ciliary resorption mechanism in mammals.

MINFLUX imaging of a bacterial molecular machine at nanometer resolution.

The resolution achievable with the established super-resolution fluorescence nanoscopy methods, such as STORM or STED, is in general not sufficient to resolve protein complexes or even individual proteins. Recently, minimal photon flux (MINFLUX) nanoscopy has been introduced that combines the strengths of STED and STORM nanoscopy and can achieve a localization precision of less than 5 nm. We established a generally applicable workflow for MINFLUX imaging and applied it for the first time to a bacterial molecular machinein situ, i.e., the injectisome of the enteropathogenY. enterocolitica. We demonstrate with a pore protein of the injectisome that MINFLUX can achieve a resolution down to the single molecule levelin situ. By imaging a sorting platform protein using 3D-MINFLUX, insights into the precise localization and distribution of an injectisome component in a bacterial cell could be accomplished. MINFLUX nanoscopy has the potential to revolutionize super-resolution imaging of dynamic molecular processes in bacteria and eukaryotes.

Resolving the molecular architecture of the photoreceptor active zone with 3D-MINFLUX.

Cells assemble macromolecular complexes into scaffoldings that serve as substrates for catalytic processes. Years of molecular neurobiology research indicate that neurotransmission depends on such optimization strategies. However, the molecular topography of the presynaptic active zone (AZ), where transmitter is released upon synaptic vesicle (SV) fusion, remains to be visualized. Therefore, we implemented MINFLUX optical nanoscopy to resolve the AZ of rod photoreceptors. This was facilitated by a novel sample immobilization technique that we name heat-assisted rapid dehydration (HARD), wherein a thin layer of rod synaptic terminals (spherules) was transferred onto glass coverslips from fresh retinal slices. Rod ribbon AZs were readily immunolabeled and imaged in 3D with a precision of a few nanometers. Our 3D-MINFLUX results indicate that the SV release site in rods is a molecular complex of bassoon-RIM2-ubMunc13-2-Cav1.4, which repeats longitudinally on both sides of the ribbon.

Mitochondrial Protein Abundance Gradients Require the Distribution of Separated Mitochondria.

Mitochondria are highly dynamic organelles that interchange their contents mediated by fission and fusion. However, it has previously been shown that the mitochondria of cultured human epithelial cells exhibit a gradient in the relative abundance of several proteins, with the perinuclear mitochondria generally exhibiting a higher protein abundance than the peripheral mitochondria. The molecular mechanisms that are required for the establishment and the maintenance of such inner-cellular mitochondrial protein abundance gradients are unknown. We verified the existence of inner-cellular gradients in the abundance of clusters of the mitochondrial outer membrane protein Tom20 in the mitochondria of kidney epithelial cells from an African green monkey (Vero cells) using STED nanoscopy and confocal microscopy. We found that the Tom20 gradients are established immediately after cell division and require the presence of microtubules. Furthermore, the gradients are abrogated in hyperfused mitochondrial networks. Our results suggest that inner-cellular protein abundance gradients from the perinuclear to the peripheral mitochondria are established by the trafficking of individual mitochondria to their respective cellular destination.

Predicting the membrane permeability of organic fluorescent probes by the deep neural network based lipophilicity descriptor DeepFl-LogP.

Light microscopy has become an indispensable tool for the life sciences, as it enables the rapid acquisition of three-dimensional images from the interior of living cells/tissues. Over the last decades, super-resolution light microscopy techniques have been developed, which allow a resolution up to an order of magnitude higher than that of conventional light microscopy. Those techniques require labelling of cellular structures with fluorescent probes exhibiting specific properties, which are supplied from outside and therefore have to surpass cell membranes. Currently, major efforts are undertaken to develop probes which can surpass cell membranes and exhibit the photophysical properties required for super-resolution imaging. However, the process of probe development is still based on a tedious and time consuming manual screening. An accurate computer based model that enables the prediction of the cell permeability based on their chemical structure would therefore be an invaluable asset for the development of fluorescent probes. Unfortunately, current models, which are based on multiple molecular descriptors, are not well suited for this task as they require high effort in the usage and exhibit moderate accuracy in their prediction. Here, we present a novel fragment based lipophilicity descriptor DeepFL-LogP, which was developed on the basis of a deep neural network. DeepFL-LogP exhibits excellent correlation with the experimental partition coefficient reference data (R2 = 0.892 and MSE = 0.359) of drug-like substances. Further a simple threshold permeability model on the basis of this descriptor allows to categorize the permeability of fluorescent probes with 96% accuracy. This novel descriptor is expected to largely simplify and speed up the development process for novel cell permeable fluorophores.

MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope.

The recently introduced minimal photon fluxes (MINFLUX) concept pushed the resolution of fluorescence microscopy to molecular dimensions. Initial demonstrations relied on custom made, specialized microscopes, raising the question of the method's general availability. Here, we show that MINFLUX implemented with a standard microscope stand can attain 1-3 nm resolution in three dimensions, rendering fluorescence microscopy with molecule-scale resolution widely applicable. Advances, such as synchronized electro-optical and galvanometric beam steering and a stabilization that locks the sample position to sub-nanometer precision with respect to the stand, ensure nanometer-precise and accurate real-time localization of individually activated fluorophores. In our MINFLUX imaging of cell- and neurobiological samples, ~800 detected photons suffice to attain a localization precision of 2.2 nm, whereas ~2500 photons yield precisions <1 nm (standard deviation). We further demonstrate 3D imaging with localization precision of ~2.4 nm in the focal plane and ~1.9 nm along the optic axis. Localizing with a precision of <20 nm within ~100 µs, we establish this spatio-temporal resolution in single fluorophore tracking and apply it to the diffusion of single labeled lipids in lipid-bilayer model membranes.

Three dimensional live-cell STED microscopy at increased depth using a water immersion objective.

Modern fluorescence superresolution microscopes are capable of imaging living cells on the nanometer scale. One of those techniques is stimulated emission depletion (STED) which increases the microscope's resolution many times in the lateral and the axial directions. To achieve these high resolutions not only close to the coverslip but also at greater depths, the choice of objective becomes crucial. Oil immersion objectives have frequently been used for STED imaging since their high numerical aperture (NA) leads to high spatial resolutions. But during live-cell imaging, especially at great penetration depths, these objectives have a distinct disadvantage. The refractive index mismatch between the immersion oil and the usually aqueous embedding media of living specimens results in unwanted spherical aberrations. These aberrations distort the point spread functions (PSFs). Notably, during z- and 3D-STED imaging, the resolution increase along the optical axis is majorly hampered if at all possible. To overcome this limitation, we here use a water immersion objective in combination with a spatial light modulator for z-STED measurements of living samples at great depths. This compact design allows for switching between objectives without having to adapt the STED beam path and enables on the fly alterations of the STED PSF to correct for aberrations. Furthermore, we derive the influence of the NA on the axial STED resolution theoretically and experimentally. We show under live-cell imaging conditions that a water immersion objective leads to far superior results than an oil immersion objective at penetration depths of 5-180 µm.

Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis.

The Bcl-2 family proteins Bax and Bak are essential for the execution of many apoptotic programs. During apoptosis, Bax translocates to the mitochondria and mediates the permeabilization of the outer membrane, thereby facilitating the release of pro-apoptotic proteins. Yet the mechanistic details of the Bax-induced membrane permeabilization have so far remained elusive. Here, we demonstrate that activated Bax molecules, besides forming large and compact clusters, also assemble, potentially with other proteins including Bak, into ring-like structures in the mitochondrial outer membrane. STED nanoscopy indicates that the area enclosed by a Bax ring is devoid of mitochondrial outer membrane proteins such as Tom20, Tom22, and Sam50. This strongly supports the view that the Bax rings surround an opening required for mitochondrial outer membrane permeabilization (MOMP). Even though these Bax assemblies may be necessary for MOMP, we demonstrate that at least in Drp1 knockdown cells, these assemblies are not sufficient for full cytochrome c release. Together, our super-resolution data provide direct evidence in support of large Bax-delineated pores in the mitochondrial outer membrane as being crucial for Bax-mediated MOMP in cells.

Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid.

Mammalian mitochondrial DNA (mtDNA) is packaged by mitochondrial transcription factor A (TFAM) into mitochondrial nucleoids that are of key importance in controlling the transmission and expression of mtDNA. Nucleoid ultrastructure is poorly defined, and therefore we used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Rotary shadowing electron microscopy revealed that nucleoid formation in vitro is a multistep process initiated by TFAM aggregation and cross-strand binding. Superresolution microscopy of cultivated cells showed that increased mtDNA copy number increases nucleoid numbers without altering their sizes. Electron cryo-tomography visualized nucleoids at high resolution in isolated mammalian mitochondria and confirmed the sizes observed by superresolution microscopy of cell lines. We conclude that the fundamental organizational unit of the mitochondrial nucleoid is a single copy of mtDNA compacted by TFAM, and we suggest a packaging mechanism.

Far-Red Emitting Fluorescent Dyes for Optical Nanoscopy: Fluorinated Silicon-Rhodamines (SiRF Dyes) and Phosphorylated Oxazines.

Far-red emitting fluorescent dyes for optical microscopy, stimulated emission depletion (STED), and ground-state depletion (GSDIM) super-resolution microscopy are presented. Fluorinated silicon-rhodamines (SiRF dyes) and phosphorylated oxazines have absorption and emission maxima at about λ≈660 and 680 nm, respectively, possess high photostability, and large fluorescence quantum yields in water. A high-yielding synthetic path to introduce three aromatic fluorine atoms and unconventional conjugation/solubilization spacers into the scaffold of a silicon-rhodamine is described. The bathochromic shift in SiRF dyes is achieved without additional fused rings or double bonds. As a result, the molecular size and molecular mass stay quite small (<600 Da). The use of the λ=800 nm STED beam instead of the commonly used one at λ=750-775 nm provides excellent imaging performance and suppresses re-excitation of SiRF and the oxazine dyes. The photophysical properties and immunofluorescence imaging performance of these new far-red emitting dyes (photobleaching, optical resolution, and switch-off behavior) are discussed in detail and compared with those of some well-established fluorophores with similar spectral properties.

Organization of Mitochondrial Gene Expression in Two Distinct Ribosome-Containing Assemblies.

Mitochondria contain their own genetic system that provides subunits of the complexes driving oxidative phosphorylation. A quarter of the mitochondrial proteome participates in gene expression, but how all these factors are orchestrated and spatially organized is currently unknown. Here, we established a method to purify and analyze native and intact complexes of mitochondrial ribosomes. Quantitative mass spectrometry revealed extensive interactions of ribosomes with factors involved in all the steps of posttranscriptional gene expression. These interactions result in large expressosome-like assemblies that we termed mitochondrial organization of gene expression (MIOREX) complexes. Superresolution microscopy revealed that most MIOREX complexes are evenly distributed throughout the mitochondrial network, whereas a subset is present as nucleoid-MIOREX complexes that unite the whole spectrum of organellar gene expression. Our work therefore provides a conceptual framework for the spatial organization of mitochondrial protein synthesis that likely developed to facilitate gene expression in the organelle.

Masked rhodamine dyes of five principal colors revealed by photolysis of a 2-diazo-1-indanone caging group: synthesis, photophysics, and light microscopy applications.

Caged rhodamine dyes (Rhodamines NN) of five basic colors were synthesized and used as "hidden" markers in subdiffractional and conventional light microscopy. These masked fluorophores with a 2-diazo-1-indanone group can be irreversibly photoactivated, either by irradiation with UV- or violet light (one-photon process), or by exposure to intense red light (λ∼750 nm; two-photon mode). All dyes possess a very small 2-diazoketone caging group incorporated into the 2-diazo-1-indanone residue with a quaternary carbon atom (C-3) and a spiro-9H-xanthene fragment. Initially they are non-colored (pale yellow), non-fluorescent, and absorb at λ=330-350 nm (molar extinction coefficient (ε)≈10(4) M(-1) cm(-1)) with a band edge that extends to about λ=440 nm. The absorption and emission bands of the uncaged derivatives are tunable over a wide range (λ=511-633 and 525-653 nm, respectively). The unmasked dyes are highly colored and fluorescent (ε=3-8×10(4) M(-1) cm(-1) and fluorescence quantum yields (ϕ)=40-85% in the unbound state and in methanol). By stepwise and orthogonal protection of carboxylic and sulfonic acid groups a highly water-soluble caged red-emitting dye with two sulfonic acid residues was prepared. Rhodamines NN were decorated with amino-reactive N-hydroxysuccinimidyl ester groups, applied in aqueous buffers, easily conjugated with proteins, and readily photoactivated (uncaged) with λ=375-420 nm light or intense red light (λ=775 nm). Protein conjugates with optimal degrees of labeling (3-6) were prepared and uncaged with λ=405 nm light in aqueous buffer solutions (ϕ=20-38%). The photochemical cleavage of the masking group generates only molecular nitrogen. Some 10-40% of the non-fluorescent (dark) byproducts are also formed. However, they have low absorbance and do not quench the fluorescence of the uncaged dyes. Photoactivation of the individual molecules of Rhodamines NN (e.g., due to reversible or irreversible transition to a "dark" non-emitting state or photobleaching) provides multicolor images with subdiffractional optical resolution. The applicability of these novel caged fluorophores in super-resolution optical microscopy is exemplified.

Super-resolution microscopy of mitochondria.

Mitochondria, the powerhouses of the cell, are essential organelles in eukaryotic cells. With their complex inner architecture featuring a smooth outer and a highly convoluted inner membrane, they are challenging objects for microscopy. The diameter of mitochondria is generally close to the resolution limit of conventional light microscopy, rendering diffraction-unlimited super-resolution light microscopy (nanoscopy) for imaging submitochondrial protein distributions often mandatory. In this review, we discuss what can be expected when imaging mitochondria with conventional diffraction-limited and diffraction-unlimited microscopy. We provide an overview on recent studies using super-resolution microscopy to investigate mitochondria and discuss further developments and challenges in mitochondrial biology that might by addressed with these technologies in the future.

Polar red-emitting rhodamine dyes with reactive groups: synthesis, photophysical properties, and two-color STED nanoscopy applications.

The synthesis, reactivity, and photophysical properties of new rhodamines with intense red fluorescence, two polar residues (hydroxyls, primary phosphates, or sulfonic acid groups), and improved hydrolytic stability of the amino-reactive sites (NHS esters or mixed N-succinimidyl carbonates) are reported. All fluorophores contain an N-alkyl-1,2-dihydro-2,2,4-trimethylquinoline fragment, and most of them bear a fully substituted tetrafluoro phenyl ring with a secondary carboxamide group. The absorption and emission maxima in water are in the range of 635-639 and 655-659 nm, respectively. A vastly simplified approach to red-emitting rhodamines with two phosphate groups that are compatible with diverse functional linkers was developed. As an example, a phosphorylated dye with an azide residue was prepared and was used in a click reaction with a strained alkyne bearing an N-hydroxysuccinimid (NHS) ester group. This method bypasses the undesired activation of phosphate groups, and gives an amphiphilic amino-reactive dye, the solubility and distribution of which between aqueous and organic phases can be controlled by varying the pH. The presence of two hydroxyl groups and a phenyl ring with two carboxyl residues in the dyes with another substitution pattern is sufficient for providing the hydrophilic properties. Selective formation of a mono-N-hydroxysuccinimidyl ester from 5-carboxy isomer of this rhodamine is reported. The fluorescence quantum yields varied from 58 to 92% for free fluorophores, and amounted to 18-64% for antibody conjugates in aqueous buffers. The brightness and photostability of these fluorophores facilitated two-color stimulated emission depletion (STED) fluorescence nanoscopy of biological samples with high contrast and minimal background. Selecting a pair of fluorophores with absorption/emission bands at 579/609 and 635/655 nm enabled two-color channels with low cross-talk and negligible background at approximately 40 nm resolution.

SNAP-, CLIP- and Halo-tag labelling of budding yeast cells.

Fluorescence microscopy of the localization and the spatial and temporal dynamics of specifically labelled proteins is an indispensable tool in cell biology. Besides fluorescent proteins as tags, tag-mediated labelling utilizing self-labelling proteins as the SNAP-, CLIP-, or the Halo-tag are widely used, flexible labelling systems relying on exogenously supplied fluorophores. Unfortunately, labelling of live budding yeast cells proved to be challenging with these approaches because of the limited accessibility of the cell interior to the dyes. In this study we developed a fast and reliable electroporation-based labelling protocol for living budding yeast cells expressing SNAP-, CLIP-, or Halo-tagged fusion proteins. For the Halo-tag, we demonstrate that it is crucial to use the 6'-carboxy isomers and not the 5'-carboxy isomers of important dyes to ensure cell viability. We report on a simple rule for the analysis of ¹H NMR spectra to discriminate between 6'- and 5'-carboxy isomers of fluorescein and rhodamine derivatives. We demonstrate the usability of the labelling protocol by imaging yeast cells with STED super-resolution microscopy and dual colour live cell microscopy. The large number of available fluorophores for these self-labelling proteins and the simplicity of the protocol described here expands the available toolbox for the model organism Saccharomyces cerevisiae.

Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20 nm resolution.

We report on a fiber laser-based stimulated emission-depletion microscope providing down to ∼20 nm resolution in raw data images as well as 15-19 nm diameter probing areas in fluorescence correlation spectroscopy. Stimulated emission depletion pulses of nanosecond duration and 775 nm wavelength are used to silence two fluorophores simultaneously, ensuring offset-free colocalization analysis. The versatility of this superresolution method is exemplified by revealing the octameric arrangement of Xenopus nuclear pore complexes and by quantifying the diffusion of labeled lipid molecules in artificial and living cell membranes.

STED super-resolution microscopy reveals an array of MINOS clusters along human mitochondria.

The mitochondrial inner membrane organizing system (MINOS) is a conserved large hetero-oligomeric protein complex in the mitochondrial inner membrane, crucial for the maintenance of cristae morphology. MINOS has been suggested to represent the core of an extended protein network that controls mitochondrial function and structure, and has been linked to several human diseases. The spatial arrangement of MINOS within mitochondria is ill-defined, however. Using super-resolution stimulated emission depletion (STED) microscopy and immunogold electron microscopy, we determined the distribution of three known human MINOS subunits (mitofilin, MINOS1, and CHCHD3) in mammalian cells. Super-resolution microscopy revealed that all three subunits form similar clusters within mitochondria, and that MINOS is more abundant in mitochondria around the nucleus than in peripheral mitochondria. At the submitochondrial level, mitofilin, a core MINOS subunit, is preferentially localized at cristae junctions. In primary human fibroblasts, mitofilin labeling uncovered a regularly spaced pattern of clusters arranged in parallel to the cell growth surfaces. We suggest that this array of MINOS complexes might explain the observed phenomenon of largely horizontally arranged cristae junctions that connect the inner boundary membrane to lamellar cristae. The super-resolution images demonstrate an unexpectedly high level of regularity in the nanoscale distribution of the MINOS complex in human mitochondria, supporting an integrating role of MINOS in the structural organization of the organelle.

Carborhodol: a new hybrid fluorophore obtained by combination of fluorescein and carbopyronine dye cores.

Asymmetric hybrid fluorophores are built from the structural elements of two (or even more) symmetric dyes and can develop valuable new features which their parents do not possess. A new hybrid carborhodol dye was obtained by the combination of fluorescein and carbopyronine fluorophores. The brightly fluorescent hybrid dye with a linker and reactive group was prepared in 12 steps with overall yield of 1.6%. In aqueous solutions, it has absorption and emission maxima at 586 and 613 nm, respectively. Antibodies labeled with a carborhodol dye possess broad absorption and emission bands so that the effective Stokes shift is increased (compared with small Stokes shifts of the parent dyes) and the fluorescence quantum yield of 39% at a degree of labeling of 5.2. Two samples of secondary antibodies labeled with carborhodol and the benchmark red-emitting rhodamine dye (KK114) were used in two-color imaging experiments with excitation at 514-532 (carborhodol dye) and 633-640 nm (KK114). When emitted light was detected above 650 nm, the novel carborhodol dye provided a lower crosstalk than spectrally similar emitters (e. g., Atto594; crosstalk 40-60% with KK114 under the same conditions). The optical resolution of ca. 80 nm was attained using the new dye in stimulated emission depleted (STED) microscopy. The relatively short fluorescence lifetime in conjugates with antibodies (τ = 1.2-1.6 ns) suggests the possibility of dual FLIM with numerous dyes having τ values in the range of 3-5 ns. All of these features make the carborhodol fluorophore a valuable addition to the family of the red-emitting fluorescent dyes.