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.

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.

Pulses of Ca2+ coordinate actin assembly and exocytosis for stepwise cell extension.

Many eukaryotic cells grow by extending their cell periphery in pulses. The molecular mechanisms underlying this process are not yet fully understood. Here we present a comprehensive model of stepwise cell extension by using the unique tip growth system of filamentous fungi. Live-cell imaging analysis, including superresolution microscopy, revealed that the fungus Aspergillus nidulans extends the hyphal tip in an oscillatory manner. The amount of F-actin and secretory vesicles (SV) accumulating at the hyphal tip oscillated with a positive temporal correlation, whereas vesicle amounts were negatively correlated to the growth rate. The intracellular Ca2+ level also pulsed with a positive temporal correlation to the amount of F-actin and SV at the hyphal tip. Two Ca2+ channels, MidA and CchA, were needed for proper tip growth and the oscillations of actin polymerization, exocytosis, and the growth rate. The data indicate a model in which transient Ca2+ pluses cause depolymerization of F-actin at the cortex and promote SV fusion with the plasma membrane, thereby extending the cell tip. Over time, Ca2+ diffuses away and F-actin and SV accumulate again at the hyphal tip. Our data provide evidence that temporally controlled actin polymerization and exocytosis are coordinated by pulsed Ca2+ influx, resulting in stepwise cell extension.

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.

Single-molecule FRET reveals the energy landscape of the full-length SAM-I riboswitch.

S-adenosyl-L-methionine (SAM) ligand binding induces major structural changes in SAM-I riboswitches, through which gene expression is regulated via transcription termination. Little is known about the conformations and motions governing the function of the full-length Bacillus subtilis yitJ SAM-I riboswitch. Therefore, we have explored its conformational energy landscape as a function of Mg2+ and SAM ligand concentrations using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling analysis. We resolved four conformational states both in the presence and the absence of SAM and determined their Mg2+-dependent fractional populations and conformational dynamics, including state lifetimes, interconversion rate coefficients and equilibration timescales. Riboswitches with terminator and antiterminator folds coexist, and SAM binding only gradually shifts the populations toward terminator states. We observed a pronounced acceleration of conformational transitions upon SAM binding, which may be crucial for off-switching during the brief decision window before expression of the downstream gene.

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.

In Situ Characterization of Protein Adsorption onto Nanoparticles by Fluorescence Correlation Spectroscopy.

Nanotechnology holds great promise for applications in many fields including biology and medicine. Unfortunately, the processes occurring at the interface between nanomaterials and living systems are exceedingly complex and not yet well understood, which has significantly hampered the realization of many nanobiotechnology applications. Whenever nanoparticles (NPs) are incorporated by a living organism, a protein adsorption layer, also known as the "protein corona", forms on the NP surface. Accordingly, living organisms interact with protein-coated rather than bare NPs, and their biological responses depend on the nature of the protein corona. In recent years, a wide variety of biophysical techniques have been employed to elucidate mechanistic aspects of NP-protein interactions. In most studies, NPs are immersed in protein or biofluid (e.g., blood serum) solutions and then separated from the liquid for analysis. Because this approach may modify the composition and structure of the protein corona, our group has pioneered the use of fluorescence correlation spectroscopy (FCS) as an in situ technique, capable of examining NP-protein interactions while the NPs are suspended in biological fluids. FCS allows us to measure, with subnanometer precision and as a function of protein concentration, the increase in hydrodynamic radius of the NPs due to protein adsorption. This Account aims at reviewing recent progress in the exploration of NP-protein interactions by using FCS. In vitro FCS studies of the adsorption of important serum proteins onto water-solubilized luminescent NPs always showed a stepwise increase of the NP radius upon protein binding in the form of a binding isotherm, regardless of the type of NP and its specific surface functionalization. This observation indicates formation of a protein monolayer on the NP. Structure-based calculations of protein surface potentials revealed that positively charged patches on the proteins interact electrostatically with negatively charged NP surfaces, and the observed protein layer thickness always matched the known molecular dimensions of the proteins binding in certain orientations. Temperature and NP surface functionalization have also been identified as important parameters controlling protein corona formation. Notably, while the corona formed from a single type of serum protein was reversible, protein adsorption from complex biological media such as blood serum was entirely irreversible. These quantitative in vitro studies are of great relevance to the bio-nano community and especially to researchers developing engineered nanomaterials for biological and biomedical applications. Future efforts will be directed toward elucidating kinetic aspects of protein corona formation and the detailed structure of the adsorbed proteins at the molecular level. To better appreciate the biological responses triggered by NP exposure, more efforts will be devoted to the exploration of the biomolecular corona as it forms on NPs in contact with living cells, tissues, and even entire model organisms. These studies are challenging when performed in a well-controlled and quantitative fashion and rely on the availability of sophisticated analytical tools, particularly, quantitative optical imaging techniques including FCS and related fluctuation methods.

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.

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.

Genetic evidence for a microtubule-capture mechanism during polarised growth of Aspergillus nidulans.

The cellular switch from symmetry to polarity in eukaryotes depends on the microtubule (MT) and actin cytoskeletons. In fungi such as Schizosaccharomyces pombe or Aspergillus nidulans, the MT cytoskeleton determines the sites of actin polymerization through cortical cell-end marker proteins. Here we describe A. nidulans MT guidance protein A (MigA) as the first ortholog of the karyogamy protein Kar9 from Saccharomyces cerevisiae in filamentous fungi. A. nidulans MigA interacts with the cortical ApsA protein and is involved in spindle positioning during mitosis. MigA is also associated with septal and nuclear MT organizing centers (MTOCs). Super-resolution photoactivated localization microscopy (PALM) analyses revealed that MigA is recruited to assembling and retracting MT plus ends in an EbA-dependent manner. MigA is required for MT convergence in hyphal tips and plays a role in correct localization of the cell-end markers TeaA and TeaR. In addition, MigA interacts with a class-V myosin, suggesting that an active mechanism exists to capture MTs and to pull the ends along actin filaments. Hence, the organization of MTs and actin depend on each other, and positive feedback loops ensure robust polar growth.

Substrate binding in human indoleamine 2,3-dioxygenase 1: A spectroscopic analysis.

Human indoleamine 2,3-dioxygenase (hIDO1) is a heme enzyme that catalyzes the oxidative cleavage of the L-tryptophan indole ring. As increased levels of hIDO1 expression in tumor cells correlate with a poor prognosis for surviving several cancer types, hIDO1 has become an appealing drug target for cancer therapy. However, detailed structural knowledge of the catalytically active complex is necessary to eb able to design de novo inhibitors selective for hIDO1. Here we have applied Fourier transform infrared (FTIR) and nanosecond time-resolved optical spectroscopy to hIDO1 variants with modified heme pocket structures to identify important amino acid residues that stabilize the substrate in the active site. A cluster of small side chain residues at positions 260-265 ensures structural flexibility of the binding site. Thr379 and Arg231 are key residues acting in concert to bind the substrate. Thr379 is the final residue of a disordered loop; the neighboring Gly380, however, is still visible in the X-ray structure of the substrate-free protein, 20Å away from the heme iron. Therefore, large-scale conformational changes are necessary to bring Thr379 close to the substrate. The use of substrate analogs further reveals that an indole-like side chain with two aromatic rings and L-stereoisomery at the Cα are required for high affinity binding.

Precise background subtraction in stimulated emission double depletion nanoscopy.

Low-resolution background in stimulated emission depletion (STED) nanoscopy can arise from incomplete depletion or re-excitation by the STED beam. We have recently introduced stimulated emission double depletion (STEDD), a technique to efficiently suppress this background. In STEDD, the conventional, doughnut-shaped STED pulse, which depletes excited fluorophores outside the center of the focal region, is followed by a second Gaussian STED pulse, which specifically depletes the central region. The background is removed by calculating a weighted difference of photon events collected before and after the second STED pulse. Here, we present a simple, yet powerful, method to determine the weight factor, which depends on the fluorescence decay, from a direct analysis of the acquired data. We vary the weight factor to identify its optimal value as the one for which the weight of high-frequency components in the spectrum of the acquired STEDD image is maximized. This strategy is also applicable to other differential approaches for background suppression in imaging.

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.

Rac-mediated Stimulation of Phospholipase Cγ2 Amplifies B Cell Receptor-induced Calcium Signaling.

The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca(2+). The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ2 (PLCγ2), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ2 to functionally reconstitute cultured PLCγ2-deficient DT40 B cells and to examine the effects of the Rac-PLCγ2 interaction on BCR-mediated changes of intracellular Ca(2+) and regulation of Ca(2+)-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ2 interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca(2+) release from intracellular stores; (iii) Ca(2+) entry from the extracellular compartment; and (iv) nuclear translocation of the Ca(2+)-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ2 activity serves to amplify B cell receptor-induced Ca(2+) signaling.

Confocal laser scanning microscopy with spatiotemporal structured illumination.

Confocal laser scanning microscopy (CLSM), which is widely utilized in the biological and biomedical sciences, is limited in spatial resolution due to diffraction to about half the light wavelength. Here we have combined structured illumination with CLSM to enhance its spatial resolution. To this end, we have used a spatial light modulator (SLM) to generate fringe patterns of different orientations and phase shifts in the excitation spot without any mechanical movement. We have achieved 1.8 and 1.7 times enhanced lateral and axial resolutions, respectively, by synthesizing the object spectrum along different illumination directions. This technique is thus a promising tool for high-resolution morphological or fluorescence imaging, especially in deep tissue.

The cell-end marker TeaA and the microtubule polymerase AlpA contribute to microtubule guidance at the hyphal tip cortex of Aspergillus nidulans to provide polarity maintenance.

In the absence of landmark proteins, hyphae of Aspergillus nidulans lose their direction of growth and show a zigzag growth pattern. Here, we show that the cell-end marker protein TeaA is important for localizing the growth machinery at hyphal tips. The central position of TeaA at the tip correlated with the convergence of the microtubule (MT) ends to a single point. Conversely, in the absence of TeaA, the MTs often failed to converge to a single point at the cortex. Further analysis suggested a functional connection between TeaA and AlpA (an ortholog of the MT polymerase Dis1/CKAP5/XMAP215) for proper regulation of MT growth at hyphal tips. AlpA localized at MT plus-ends, and bimolecular fluorescence complementation assays suggested that it interacted with TeaA after MT plus-ends reached the tip cortex. In vitro MT polymerization assays showed that AlpA promoted MT growth up to sevenfold. Addition of the C-terminal region of TeaA increased the catastrophe frequency of the MTs. Thus, the control of the AlpA activity through TeaA might be a novel principle for MT growth regulation after reaching the cortex. In addition, we present evidence that the curvature of hyphal tips also could be involved in the control of MT growth at hyphal tips.

Unravelling RNA-substrate interactions in a ribozyme-catalysed reaction using fluorescent turn-on probes.

The Diels-Alder reaction is one of the most important C-C bond-forming reactions in organic chemistry, and much effort has been devoted to controlling its enantio- and diastereoselectivity. The Diels-Alderase ribozyme (DAse) catalyses the reaction between anthracene dienes and maleimide dienophiles with multiple-turnover, stereoselectivity, and up to 1100-fold rate acceleration. Here, a new generation of anthracene-BODIPY-based fluorescent probes was developed to monitor catalysis by the DAse. The brightness of these probes increases up to 93-fold upon reaction with N-pentylmaleimide (NPM), making these useful tools for investigating the stereochemistry of the ribozyme-catalysed reaction. With these probes, we observed that the DAse catalyses the reaction with >91% de and >99% ee. The stereochemistry of the major product was determined unambiguously by rotating-frame nuclear Overhauser NMR spectroscopy (ROESY-NMR) and is in agreement with crystallographic structure information. The pronounced fluorescence change of the probes furthermore allowed a complete kinetic analysis, which revealed an ordered bi uni type reaction mechanism, with the dienophile binding first.

Exploring color tuning strategies in red fluorescent proteins.

Red-emitting fluorescent proteins (RFPs) with fluorescence emission above 600 nm are advantageous for cell and tissue imaging applications for various reasons. Fluorescence from an RFP is well separated from cellular autofluorescence, which is in the green region of the spectrum, and red light is scattered less, which allows thicker specimens to be imaged. Moreover, the phototoxic response of cells is lower for red than blue or green light exposure. Further red-shifted FP variants can be obtained by genetic modifications causing an extension of the conjugated π-electron system of the chromophore, or by placing amino acids near the chromophore that stabilize its excited state or destabilize its ground state. We have selected the tetrameric RFP eqFP611 from Entacmaea quadricolor as a lead structure and discuss several rational design trials to generate RFP variants with improved photochemical properties.

Antiviral vaccines license T cell responses by suppressing granzyme B levels in human plasmacytoid dendritic cells.

Human plasmacytoid dendritic cells (pDC) are important modulators of adaptive T cell responses during viral infections. Recently, we found that human pDC produce the serine protease granzyme B (GrB), thereby regulating T cell proliferation in a GrB-dependent manner. In this study, we demonstrate that intrinsic GrB production by pDC is significantly inhibited in vitro and in vivo by clinically used vaccines against viral infections such as tick-borne encephalitis. We show that pDC GrB levels inversely correlate with the proliferative response of coincubated T cells and that GrB suppression by a specific Ab or a GrB substrate inhibitor results in enhanced T cell proliferation, suggesting a predominant role of GrB in pDC-dependent T cell licensing. Functionally, we demonstrate that GrB(high) but not GrB(low) pDC transfer GrB to T cells and may degrade the ζ-chain of the TCR in a GrB-dependent fashion, thereby providing a possible explanation for the observed T cell suppression by GrB-expressing pDC. Modulation of pDC-derived GrB activity represents a previously unknown mechanism by which both antiviral and vaccine-induced T cell responses may be regulated in vivo. Our results provide novel insights into pDC biology during vaccinations and may contribute to an improvement of prophylactic and therapeutic vaccines.

Fluorescent proteins for live-cell imaging with super-resolution.

Fluorescent proteins (FPs) from the GFP family have become indispensable as marker tools for imaging live cells, tissues and entire organisms. A wide variety of these proteins have been isolated from natural sources and engineered to optimize their properties as genetically encoded markers. Here we review recent developments in this field. A special focus is placed on photoactivatable FPs, for which the fluorescence emission can be controlled by light irradiation at specific wavelengths. They enable regional optical marking in pulse-chase experiments on live cells and tissues, and they are essential marker tools for live-cell optical imaging with super-resolution. Photoconvertible FPs, which can be activated irreversibly via a photo-induced chemical reaction that either turns on their emission or changes their emission wavelength, are excellent markers for localization-based super-resolution microscopy (e.g., PALM). Patterned illumination microscopy (e.g., RESOLFT), however, requires markers that can be reversibly photoactivated many times. Photoswitchable FPs can be toggled repeatedly between a fluorescent and a non-fluorescent state by means of a light-induced chromophore isomerization coupled to a protonation reaction. We discuss the mechanistic origins of the effect and illustrate how photoswitchable FPs are employed in RESOLFT imaging. For this purpose, special FP variants with low switching fatigue have been introduced in recent years. Despite nearly two decades of FP engineering by many laboratories, there is still room for further improvement of these important markers for live cell imaging.