Stimulated emission double depletion nanoscopy with background correction at the single-pixel level.

Fluorescence microscopy images are inevitably tainted by background contributions including emission from out-of-focus planes, scattered light, and detector noise. In stimulated emission depletion (STED) nanoscopy, an additional, method-specific background arises from incomplete depletion and re-excitation by the depletion beam. Various approaches have been proposed to remove the background from a STED image, some of which rely on the acquisition of a separate background image that is subtracted from the STED image with a weighting factor. Using stimulated emission double depletion (STEDD) nanoscopy, we observed that the weighting factor varies locally in densely labeled samples, so that background removal with a single (global) weighting factor generates local image artifacts due to incorrect background subtraction. Here we present an algorithm that computes the optimal weighting factor at the single-pixel level, yielding a difference image with excellent suppression of low-frequency background.

Fast-exchanging spirocyclic rhodamine probes for aptamer-based super-resolution RNA imaging.

Live-cell RNA imaging with high spatial and temporal resolution remains a major challenge. Here we report the development of RhoBAST:SpyRho, a fluorescent light-up aptamer (FLAP) system ideally suited for visualizing RNAs in live or fixed cells with various advanced fluorescence microscopy modalities. Overcoming problems associated with low cell permeability, brightness, fluorogenicity, and signal-to-background ratio of previous fluorophores, we design a novel probe, SpyRho (Spirocyclic Rhodamine), which tightly binds to the RhoBAST aptamer. High brightness and fluorogenicity is achieved by shifting the equilibrium between spirolactam and quinoid. With its high affinity and fast ligand exchange, RhoBAST:SpyRho is a superb system for both super-resolution SMLM and STED imaging. Its excellent performance in SMLM and the first reported super-resolved STED images of specifically labeled RNA in live mammalian cells represent significant advances over other FLAPs. The versatility of RhoBAST:SpyRho is further demonstrated by imaging endogenous chromosomal loci and proteins.

A chemical probe for BAG1 targets androgen receptor-positive prostate cancer through oxidative stress signaling pathway.

BAG1 is a family of polypeptides with a conserved C-terminal BAG domain that functions as a nucleotide exchange factor for the molecular chaperone HSP70. BAG1 proteins also control several signaling processes including proteostasis, apoptosis, and transcription. The largest isoform, BAG1L, controls the activity of the androgen receptor (AR) and is upregulated in prostate cancer. Here, we show that BAG1L regulates AR dynamics in the nucleus and its ablation attenuates AR target gene expression especially those involved in oxidative stress and metabolism. We show that a small molecule, A4B17, that targets the BAG domain downregulates AR target genes similar to a complete BAG1L knockout and upregulates the expression of oxidative stress-induced genes involved in cell death. Furthermore, A4B17 outperformed the clinically approved antagonist enzalutamide in inhibiting cell proliferation and prostate tumor development in a mouse xenograft model. BAG1 inhibitors therefore offer unique opportunities for antagonizing AR action and prostate cancer growth.

Two plus one is almost three: a fast approximation for multi-view deconvolution.

Multi-view deconvolution is a powerful image-processing tool for light sheet fluorescence microscopy, providing isotropic resolution and enhancing the image content. However, performing these calculations on large datasets is computationally demanding and time-consuming even on high-end workstations. Especially in long-time measurements on developing animals, huge amounts of image data are acquired. To keep them manageable, redundancies should be removed right after image acquisition. To this end, we report a fast approximation to three-dimensional multi-view deconvolution, denoted 2D+1D multi-view deconvolution, which is able to keep up with the data flow. It first operates on the two dimensions perpendicular and subsequently on the one parallel to the rotation axis, exploiting the rotational symmetry of the point spread function along the rotation axis. We validated our algorithm and evaluated it quantitatively against two-dimensional and three-dimensional multi-view deconvolution using simulated and real image data. 2D+1D multi-view deconvolution takes similar computation time but performs markedly better than the two-dimensional approximation only. Therefore, it will be most useful for image processing in time-critical applications, where the full 3D multi-view deconvolution cannot keep up with the data flow.

Allele-specific endogenous tagging and quantitative analysis of ß-catenin in colorectal cancer cells.

Wnt signaling plays important roles in development, homeostasis, and tumorigenesis. Mutations in ß-catenin that activate Wnt signaling have been found in colorectal and hepatocellular carcinomas. However, the dynamics of wild-type and mutant forms of ß-catenin are not fully understood. Here, we genome-engineered fluorescently tagged alleles of endogenous ß-catenin in a colorectal cancer cell line. Wild-type and oncogenic mutant alleles were tagged with different fluorescent proteins, enabling the analysis of both variants in the same cell. We analyzed the properties of both ß-catenin alleles using immunoprecipitation, immunofluorescence, and fluorescence correlation spectroscopy approaches, revealing distinctly different biophysical properties. In addition, activation of Wnt signaling by treatment with a GSK3ß inhibitor or a truncating APC mutation modulated the wild-type allele to mimic the properties of the mutant ß-catenin allele. The one-step tagging strategy demonstrates how genome engineering can be employed for the parallel functional analysis of different genetic variants.

Axial line-scanning stimulated emission depletion fluorescence correlation spectroscopy.

Investigating the dynamics and interactions of biomolecules within or attached to membranes of living cells is crucial for understanding biology at the molecular level. In this pursuit, classical, diffraction-limited optical fluorescence microscopy is widely used, but it faces limitations due to (1) the heterogeneity of biomembranes on the nanoscale and (2) the intrinsic motion of membranes with respect to the focus. Here we introduce a new confocal microscopy-based fluctuation spectroscopy technique aimed at alleviating these two problems, called axial line-scanning stimulated emission depletion fluorescence correlation spectroscopy (axial ls-STED-FCS). Axial line scanning by means of a tunable acoustic gradient index of refraction lens provides a time resolution of a few microseconds, which is more than two orders of magnitude greater than that of conventional, lateral line-scanning fluorescence correlation spectroscopy (typically around 1 ms). Using STED excitation, the observation area on the membrane can be reduced 10-100 fold, resulting in sub-diffraction spatial resolution and the ability to study samples with densely labeled membranes. Due to these attractive properties, we expect that the axial ls-STED-FCS will find wide application, especially in the biomolecular sciences.

Wavelet-based background and noise subtraction for fluorescence microscopy images.

Fluorescence microscopy images are inevitably contaminated by background intensity contributions. Fluorescence from out-of-focus planes and scattered light are important sources of slowly varying, low spatial frequency background, whereas background varying from pixel to pixel (high frequency noise) is introduced by the detection system. Here we present a powerful, easy-to-use software, wavelet-based background and noise subtraction (WBNS), which effectively removes both of these components. To assess its performance, we apply WBNS to synthetic images and compare the results quantitatively with the ground truth and with images processed by other background removal algorithms. We further evaluate WBNS on real images taken with a light-sheet microscope and a super-resolution stimulated emission depletion microscope. For both cases, we compare the WBNS algorithm with hardware-based background removal techniques and present a quantitative assessment of the results. WBNS shows an excellent performance in all these applications and significantly enhances the visual appearance of fluorescence images. Moreover, it may serve as a pre-processing step for further quantitative analysis.

Super-resolution RNA imaging using a rhodamine-binding aptamer with fast exchange kinetics.

Overcoming limitations of previous fluorescent light-up RNA aptamers for super-resolution imaging, we present RhoBAST, an aptamer that binds a fluorogenic rhodamine dye with fast association and dissociation kinetics. Its intermittent fluorescence emission enables single-molecule localization microscopy with a resolution not limited by photobleaching. We use RhoBAST to image subcellular structures of RNA in live and fixed cells with about 10-nm localization precision and a high signal-to-noise ratio.

Confocal and Super-resolution Imaging of RNA in Live Bacteria Using a Fluorogenic Silicon Rhodamine-binding Aptamer.

Genetically encoded light-up RNA aptamers have been shown to be promising tools for the visualization of RNAs in living cells, helping us to advance our understanding of the broad and complex life of RNA. Although a handful of light-up aptamers spanning the visible wavelength region have been developed, none of them have yet been reported to be compatible with advanced super-resolution techniques, mainly due to poor photophysical properties of their small-molecule fluorogens. Here, we describe a detailed protocol for fluorescence microscopy of mRNA in live bacteria using the recently reported fluorogenic silicon rhodamine binding aptamer (SiRA) featuring excellent photophysical properties. Notably, with SiRA, we demonstrated the first aptamer-based RNA visualization using super-resolution (STED) microscopy. This imaging method can be especially valuable for visualization of RNA in prokaryotes since the size of a bacterium is only a few times greater than the optical resolution of a conventional microscope.

SiRA: A Silicon Rhodamine-Binding Aptamer for Live-Cell Super-Resolution RNA Imaging.

Although genetically encoded light-up RNA aptamers have become promising tools for visualizing and tracking RNAs in living cells, aptamer/ligand pairs that emit in the far-red and near-infrared (NIR) regions are still rare. In this work, we developed a light-up RNA aptamer that binds silicon rhodamines (SiRs). SiRs are photostable, NIR-emitting fluorophores that change their open-closed equilibrium between the noncolored spirolactone and the fluorescent zwitterion in response to their environment. This property is responsible for their high cell permeability and fluorogenic behavior. Aptamers binding to SiR were in vitro selected from a combinatorial RNA library. Sequencing, bioinformatic analysis, truncation, and mutational studies revealed a 50-nucleotide minimal aptamer, SiRA, which binds with nanomolar affinity to the target SiR. In addition to silicon rhodamines, SiRA binds structurally related rhodamines and carborhodamines, making it a versatile tool spanning the far-red region of the spectrum. Photophysical characterization showed that SiRA is remarkably resistant to photobleaching and constitutes the brightest far-red light-up aptamer system known to date owing to its favorable features: a fluorescence quantum yield of 0.98 and an extinction coefficient of 86 000 M-1cm-1. Using the SiRA system, we visualized the expression of RNAs in bacteria in no-wash live-cell imaging experiments and also report stimulated emission depletion (STED) super-resolution microscopy images of aptamer-based, fluorescently labeled mRNA in live cells. This work represents, to our knowledge, the first application of the popular SiR dyes and of intramolecular spirocyclization as a means of background reduction in the field of aptamer-based RNA imaging. We anticipate a high potential for this novel RNA labeling tool to address biological questions.

Highly Efficient One-Dimensional Triplet Exciton Transport in a Palladium-Porphyrin-Based Surface-Anchored Metal-Organic Framework.

Efficient photon-harvesting materials require easy-to-deposit materials exhibiting good absorption and excited-state transport properties. We demonstrate an organic thin-film material system, a palladium-porphyrin-based surface-anchored metal-organic framework (SURMOF) thin film that meets these requirements. Systematic investigations using transient absorption spectroscopy confirm that triplets are very mobile within single crystalline domains; a detailed analysis reveals a triplet transfer rate on the order of 1010 s-1. The crystalline nature of the SURMOFs also allows a thorough theoretical analysis using the density functional theory. The theoretical results reveal that the intermolecular exciton transfer can be described by a Dexter electron exchange mechanism that is considerably enhanced by virtual charge-transfer exciton intermediates. On the basis of the photophysical results, we predict exciton diffusion lengths on the order of several micrometers in perfectly ordered, single-crystalline SURMOFs. In the presently available samples, strong interactions of excitons with domain boundaries present in these metal-organic thin films limit the diffusion length to the diameter of these two-dimensional grains, which amount to about 100 nm. Our results demonstrate high potential of SURMOFs for light-harvesting applications.

Dual-mode phase and fluorescence imaging with a confocal laser scanning microscope.

We present dual-mode phase and fluorescence imaging in a confocal laser scanning microscopy (CLSM) system. For phase imaging, the depth of field of the CLSM system is extended by fast axial scanning with a tunable acoustic gradient index of refraction lens. Under transillumination, intensity images of the sample are recorded at a few different defocusing distances. The phase image is reconstructed from these intensity images by using the transport-of-intensity equation. The 3D fluorescence image is obtained by confocal scanning. The dual-mode images with pixel-to-pixel correspondence yield complementary quantitative structural and functional information. Combination of the two imaging modalities enables standalone determination of the refractive index of live cells.

Distinct amino acid motifs carrying multiple positive charges regulate membrane targeting of dysferlin and MG53.

Dysferlin (Dysf) and mitsugumin53 (MG53) are two key proteins involved in membrane repair of muscle cells which are efficiently recruited to the sarcolemma upon lesioning. Plasma membrane localization and recruitment of a Dysf fragment to membrane lesions in zebrafish myofibers relies on the presence of a short, polybasic amino acid motif, WRRFK. Here we show that the positive charges carried by this motif are responsible for this function. In mouse MG53, we have identified a similar motif with multiple basic residues, WKKMFR. A single amino acid replacement, K279A, leads to severe aggregation of MG53 in inclusion bodies in HeLa cells. This result is due to the loss of positive charge, as shown by studying the effects of other neutral amino acids at position 279. Consequently, our data suggest that positively charged amino acid stretches play an essential role in the localization and function of Dysf and MG53.

Kinetic Study of Ligand Binding and Conformational Changes in Inducible Nitric Oxide Synthase.

Nitric oxide synthases (NOSs) are heme enzymes that generate highly reactive nitric oxide from l-arginine (l-Arg) in a complex mechanism that is still only partially understood. We have studied carbon monoxide (CO) binding to the oxygenase domain of murine inducible NOS (iNOS) by using flash photolysis. The P420 and P450 forms of the enzyme, assigned to a protonated and unprotonated proximal cysteine, through which the heme is anchored to the protein, show markedly different CO rebinding properties. The data suggest that P420 has a widely open distal pocket that admits water. CO rebinding to the P450 form strongly depends on the presence of the substrate l-Arg, the intermediate Nω-hydroxy-l-arginine, and the cofactor tetrahydrobiopterin. After adding these small molecules to the enzyme solution, the CO kinetics change slowly over the hours. This process can be described as a relaxation from a fast rebinding, metastable species to a slowly rebinding, thermodynamically stable species, which we associate with the enzymatically active form. Our results allow us to determine kinetic parameters of l-Arg binding to the ferrous deoxy iNOS protein for the first time and also provide clues regarding the nature of structural differences between the two interconverting species.

EmbryoMiner: A new framework for interactive knowledge discovery in large-scale cell tracking data of developing embryos.

State-of-the-art light-sheet and confocal microscopes allow recording of entire embryos in 3D and over time (3D+t) for many hours. Fluorescently labeled structures can be segmented and tracked automatically in these terabyte-scale 3D+t images, resulting in thousands of cell migration trajectories that provide detailed insights to large-scale tissue reorganization at the cellular level. Here we present EmbryoMiner, a new interactive open-source framework suitable for in-depth analyses and comparisons of entire embryos, including an extensive set of trajectory features. Starting at the whole-embryo level, the framework can be used to iteratively focus on a region of interest within the embryo, to investigate and test specific trajectory-based hypotheses and to extract quantitative features from the isolated trajectories. Thus, the new framework provides a valuable new way to quantitatively compare corresponding anatomical regions in different embryos that were manually selected based on biological prior knowledge. As a proof of concept, we analyzed 3D+t light-sheet microscopy images of zebrafish embryos, showcasing potential user applications that can be performed using the new framework.

The multi-state energy landscape of the SAM-I riboswitch: A single-molecule Förster resonance energy transfer spectroscopy study.

RNA (ribonucleic acid) molecules are highly flexible biopolymers fluctuating at physiological temperatures among many different conformations that are represented by minima in a hierarchical conformational free energy landscape. Here we have employed single-molecule FRET (smFRET) to explore the energy landscape of the B. subtilis yitJ SAM-I riboswitch (RS). In this small RNA molecule, specific binding of an S-adenosyl-L-methionine (SAM) ligand in the aptamer domain regulates gene expression by inducing structural changes in another domain, the expression platform, causing transcription termination by the RNA polymerase. We have measured smFRET histograms over wide ranges of Mg2+ concentration for three RS variants that were specifically labeled with fluorescent dyes on different sites. In the analysis, different conformations are associated with discrete Gaussian model distributions, which are typically fairly broad on the FRET efficiency scale and thus can be extremely challenging to unravel due to their mutual overlap. Our earlier work on two SAM-I RS variants revealed four major conformations. By introducing a global fitting procedure which models both the Mg2+ concentration dependencies of the fractional populations and the average FRET efficiencies of the individual FRET distributions according to Mg2+ binding isotherms, we were able to consistently describe the histogram data of both variants at all studied Mg2+ concentrations. With the third FRET-labeled variant, however, we found significant deviations when applying the four-state model to the data. This can arise because the different FRET labeling of the new variant allows two states to be distinguished that were previously not separable due to overlap. Indeed, the resulting five-state model presented here consistently describes the smFRET histograms of all three variants as well as their variations with Mg2+ concentration. We also performed a triangulation of the donor position for two of the constructs to explore how the expression platform is oriented with respect to the aptamer.

Cytoplasmic Transport Machinery of the SPF27 Homologue Num1 in Ustilago maydis.

In the phytopathogenic basidiomycete Ustilago maydis, the Num1 protein has a pivotal function in hyphal morphogenesis. Num1 functions as a core component of the spliceosome-associated Prp19/CDC5 complex (NTC). The interaction of Num1 with the kinesin motor Kin1 suggests a connection between a component of the splicing machinery and cytoplasmic trafficking processes. Previously it was shown that Num1 localizes predominantly in the nucleus; however, due to the diffraction-limited spatial resolution of conventional optical microscopy, it was not possible to attribute the localization to specific structures within the cytoplasm. We have now employed super-resolution localization microscopy to visualize Num1 in the cytoplasm by fusing it to a tandem dimeric Eos fluorescent protein (tdEosFP). The Num1 protein is localized within the cytoplasm with an enhanced density in the vicinity of microtubules. Num1 movement is found predominantly close to the nucleus. Movement is dependent on its interaction partner Kin1, but independent of Kin3. Our results provide strong evidence that, in addition to its involvement in splicing in the nucleus, Num1 has an additional functional role in the cytosol connected to the Kin1 motor protein.

Superresolution and pulse-chase imaging reveal the role of vesicle transport in polar growth of fungal cells.

Polarized growth of filamentous fungi requires continuous transport of biomolecules to the hyphal tip. To this end, construction materials are packaged in vesicles and transported by motor proteins along microtubules and actin filaments. We have studied these processes with quantitative superresolution localization microscopy of live Aspergillus nidulans cells expressing the photoconvertible protein mEosFPthermo fused to the chitin synthase ChsB. ChsB is mainly located at the Spitzenkörper near the hyphal tip and produces chitin, a key component of the cell wall. We have visualized the pulsatory dynamics of the Spitzenkörper, reflecting vesicle accumulation before exocytosis and their subsequent fusion with the apical plasma membrane. Furthermore, high-speed pulse-chase imaging after photoconversion of mEosFPthermo in a tightly focused spot revealed that ChsB is transported with two different speeds from the cell body to the hyphal tip and vice versa. Comparative analysis using motor protein deletion mutants allowed us to assign the fast movements (7 to 10 µm s-1) to transport of secretory vesicles by kinesin-1, and the slower ones (2 to 7 µm s-1) to transport by kinesin-3 on early endosomes. Our results show how motor proteins ensure the supply of vesicles to the hyphal tip, where temporally regulated exocytosis results in stepwise tip extension.

The protein corona on nanoparticles as viewed from a nanoparticle-sizing perspective.

Most surfaces of engineered nanoparticles (NPs) are reactive toward biomolecules. Therefore, whenever NPs become immersed in biological fluids, proteins and other biomolecules bind to the NP surface, forming an adsorption layer (biomolecular corona) that modifies the NPs' physicochemical properties and subsequent interactions with living systems. Its exploration is a formidable endeavor owing to the enormous diversity of engineered NPs in terms of their physicochemical properties and the vast number of biomolecules available in biofluids that may bind to NPs with widely different strengths. Significant progress has been made in our understanding of the biomolecular corona, but even very basic issues are still controversially debated. In fact, there are divergent views of its microscopic structure and dynamics, even on physical properties, such as its thickness. As an example, there is no agreement on whether proteins form monolayers or multilayers upon adsorption. In our quantitative studies of NP-protein interactions by in situ fluorescence correlation spectroscopy (FCS) with highly defined model NPs and important serum proteins, we have universally observed protein monolayer formation around NPs under saturation or even oversaturation conditions. Here, we critically discuss biomolecular corona characterization using FCS and dynamic light scattering and identify challenges and future opportunities. Further careful, quantitative experiments are needed to elucidate the mechanisms of biomolecular corona formation and to characterize its structure. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Refractive index measurement of suspended cells using opposed-view digital holographic microscopy.

Opposed-view digital holographic microscopy (OV-DHM) with autofocusing and out-of-focus background suppression was demonstrated and applied to measure the refractive index (RI) of suspended HeLa cells. In OV-DHM, a specimen is illuminated from two sides in a 4π-like configuration. The generated two opposite-view object waves, which have orthogonal polarization orientations, interfere with a common reference wave, and the generated holograms are recorded by a CMOS camera. The image plane of the sample was determined by finding the minimal variation between the two object waves. The out-of-focus background was suppressed by averaging the two object waves. Simultaneous determination of both the cell thickness and the phase retardation was avoided by using a spheroidal model for the detached cell obtained from confocal microscopy. Thus, the RI of suspended HeLa cells was measured from phase images of OV-DHM, with the thickness of the cells estimated by using a constant axial-to-lateral ratio. This measurement strategy reveals the RI with an accuracy of ∼10% of the RI difference between cells and surrounding medium.