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In vivo RESOLFT imaging of intact living Drosophila melanogaster larvae. (A) Living second instar larva expressing rsEGFP2-a-tubulin were placed under a coverslip between two spacers in Schneider's medium. (B) Confocal overview imaged through the cuticle of the larva. (C) Confocal (top) Figure 3 continued on next page
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Despite remarkable developments in diffraction unlimited super-resolution microscopy, in vivo nanoscopy of tissues and model organisms is still not satisfactorily established and rarely realized. RESOLFT nanoscopy is particularly suited for live cell imaging because it requires relatively low light levels to overcome the diffraction barrier. Previo...
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... in samples that express the fusion proteins in all cells, very pronounced out-of-focus fluorescence may occur. To enable RESOLFT microscopy in intact larvae, living rsEGFP2-a-tubulin expressing sec- ond instar larvae were placed between two spacers that separated the object slide and the cover glass, forming a cavity filled with Schneider's cell culture media ( Figure 3A). By this, the overall movements of the larvae were largely restrained, although movements of body muscles still occurred. ...
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... the occasional movements of the larvae, we could reliably record RESOLFT images of the microtubule network in these intact animals. Co-aligned microtubules that were fully blurred in the corresponding confocal images could be resolved ( Figure 3B-D). As in resected tissues, the measured resolution in the intact larvae was also 60 nm ( Figure 3E; Fig- ure 3-figure supplement 1), despite the fact that we were focusing through the opaque cuticle into tissue. ...
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Super-resolution light microscopy (SRM) offers a unique opportunity for diffraction-unlimited imaging of biomolecular activities in living cells. To realize such potential, genetically encoded indicators were developed recently from fluorescent proteins (FPs) that exhibit phototransformation behaviors including photoactivation, photoconversion, and...
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... So far, the majority of these experiments were carried out either in cell cultures or thin tissue sections. In contrast, there are only a handful of examples of super-resolution imaging in thick specimens [11][12][13][14], as imaging deep tissue is challenging for most super-resolution techniques. The coordinate-stochastic approaches (e.g. ...
Since its invention in 1994, super-resolution microscopy has become a popular tool for advanced imaging of biological structures, allowing visualisation of subcellular structures at a spatial scale below the diffraction limit. Thus, it is not surprising that recently, different super-resolution techniques are being applied in neuroscience, e.g. to resolve the clustering of neurotransmitter receptors and protein complex composition in presynaptic terminals. Still, the vast majority of these experiments were carried out either in cell cultures or very thin tissue sections, while there are only a few examples of super-resolution imaging in thick (> ~50 um) biological samples. In that context, the mammalian whole-mount retina has rarely been studied with super-resolution microscopy. Here, we aimed at establishing a stimulated-emission-depletion (STED) microscopy protocol for imaging whole-mount retina. To this end, we developed sample preparation including horizontal slicing of retinal tissue, an immunolabeling protocol with STED-compatible fluorophores and optimised the STED microscope's settings. We labelled subcellular structures in somata, dendrites, and axons of retinal ganglion cells in the inner mouse retina. Under optimal conditions, we achieved a mean lateral spatial resolution of ~120 nm (using the full width of half-maximum as a proxy) for the thinnest filamentous structures in our preparation and a resolution enhancement of two or higher compared to conventional confocal images. When combined with horizontal slicing of the retina, these settings allowed us visualisation of putative GABAergic horizontal cell synapses in the outer retina with a similar resolution. Taken together, we successfully established a STED protocol for reliable super-resolution imaging in the whole-mount mouse retina, which enables investigating, for instance, protein complex composition and cytoskeletal ultrastructure at retinal synapses in health and disease.
... September 2021 | Volume 9 | Article 746900 applied the heterozygous TgN (Thy1-EYFP) mice expressing enhanced yellow fluorescent protein (EYFP) or GFP, EGFP, which is under the control of the regulatory element from the thy1 gene ( Figure 1A) (Berning et al., 2012;Bethge et al., 2013). Similarly, zebrafish and Drosophila melanogaster are the commonly used animals for in vivo studies, such as the Isl1: GFP zebrafish larvae, in which Islet-1 promoter enabled the expression of GFP in all postmitotic motor neurons ( Figure 6A) , and the rsEGFP2 transfected Drosophila melanogaster, expressing rsEGFP2 a-tubulin through a standard phiC31 integrase based germ line transformation procedure ( Figure 6B) (Schnorrenberg et al., 2016). The other is to transfer the FPs into animal body to induce the expression of fused FPs or tags through recombinant adenoassociated virus (rAAV, serotype 2) infection method. ...
... Reprinted fromLi et al. (2020) with permission from American Association for the Advancement of Science. (B) Drosophila melanogaster ubiquitously expressing rsEGFP2-a-tubulin(Schnorrenberg et al., 2016). Scale bar, 1 μm. ...
... Scale bar, 1 μm. Reprinted fromSchnorrenberg et al. (2016) with permission from eLife Sciences. (C) Neurons expressing Lifeact-EYFP by recombinant adeno-associated virus (AAV) infection for STED microscopy (Willig et al., 2014). ...
Over the past two decades, super-resolution microscopy (SRM), which offered a significant improvement in resolution over conventional light microscopy, has become a powerful tool to visualize biological activities in both fixed and living cells. However, completely understanding biological processes requires studying cells in a physiological context at high spatiotemporal resolution. Recently, SRM has showcased its ability to observe the detailed structures and dynamics in living species. Here we summarized recent technical advancements in SRM that have been successfully applied to in vivo imaging. Then, improvements in the labeling strategies are discussed together with the spectroscopic and chemical demands of the fluorophores. Finally, we broadly reviewed the current applications for super-resolution techniques in living species and highlighted some inherent challenges faced in this emerging field. We hope that this review could serve as an ideal reference for researchers as well as beginners in the relevant field of in vivo super resolution imaging.
... RESOLFT microscopy relies on reversibly photoswitchable fluorescent proteins (RSFPs) and has already been successfully applied to Drosophila larval and adult tissue. The previously reported rsEGFP2 was used as a transgenic fusion with α-tubulin in a still rare in vivo proof of concept of time-lapse super-resolution imaging [121]. rsEGFP2 can be switched from a non-fluorescent "OFF" state to a green fluorescent "ON" state by irradiation with 405 nm, while 488 nm are used for fluorescence excitation and to simul-taneously switch rsEGFP2 back to the "OFF" state. ...
The Drosophila eye has been used extensively to study numerous aspects of biological systems, for example, spatio-temporal regulation of differentiation, visual signal transduction, protein trafficking and neurodegeneration. Right from the advent of fluorescent proteins (FPs) near the end of the millennium, heterologously expressed fusion proteins comprising FPs have been applied in Drosophila vision research not only for subcellular localization of proteins but also for genetic screens and analysis of photoreceptor function. Here, we summarize applications for FPs used in the Drosophila eye as part of genetic screens, to study rhodopsin expression patterns, subcellular protein localization, membrane protein transport or as genetically encoded biosensors for Ca2+ and phospholipids in vivo. We also discuss recently developed FPs that are suitable for super-resolution or correlative light and electron microscopy (CLEM) approaches. Illustrating the possibilities provided by using FPs in Drosophila photoreceptors may aid research in other sensory or neuronal systems that have not yet been studied as well as the Drosophila eye.
... In the one-step RESOLFT approach utilized in this study, the 488 nm light dose applied was about ten times lower than typically used in sequential mode RESOLFT microscopy with rsEGFP2. 11,46 On the contrary, the 405 nm light dose was about five times higher than typically used in sequential mode RESOLFT microscopy with negative switching RSFPs. We were able to record consecutive images of living cells. ...
Reversibly switchable fluorescent proteins (RSFPs) can be repeatedly transferred between a fluorescent on- and a nonfluorescent off-state by illumination with light of different wavelengths. Negative switching RSFPs are switched from the on- to the off-state with the same wavelength that also excites fluorescence. Positive switching RSFPs have a reversed light response, where the fluorescence excitation wavelength induces the transition from the off- to the on-state. Reversible saturable optical linear (fluorescence) transitions (RESOLFT) nanoscopy utilizes these switching states to achieve diffraction-unlimited resolution but so far has primarily relied on negative switching RSFPs by using time sequential switching schemes. On the basis of the green fluorescent RSFP Padron, we engineered the positive switching RSFP Padron2. Compared to its predecessor, it can undergo 50-fold more switching cycles while displaying a contrast ratio between the on- and the off-states of more than 100:1. Because of its robust switching behavior, Padron2 supports a RESOLFT imaging scheme that entirely refrains from sequential switching as it only requires beam scanning of two spatially overlaid light distributions. Using Padron2, we demonstrate live-cell RESOLFT nanoscopy without sequential illumination steps.
... Enfin, une méthode récente appelée RESOLFT (Introduction, (Ha et al., 2016)) serait, d'après moi, un outil adapté pour étudier la dynamique des IPPs lors de leur fixation à une séquence cible, à l'échelle super-résolutive. Cependant, cette méthode ne permet pas de compter le nombre exact de complexes sur la séquence cible. ...
La régulation transcriptionnelle est le sujet principal de nombreuses recherches et est un mécanisme indispensable pour assurer les fonctions cellulaires de tout organisme. Les avancées technologiques dans le monde de la microscopie ouvrent de nouvelles opportunités pour visualiser différentes étapes de ce mécanisme. Notamment les dynamiques et la localisation d’un FT a l’échelle super-résolutive. Cependant, un unique FT n’est pas suffisant pour réguler finement l’activation ou la répression de la transcription d’un gène. En effet, différents complexes de FT coopèrent pour atteindre une telle précision de régulation. La visualisation de la fixation d’un complexe (binaire) sur sa séquence régulatrice cible serait donc un atout pour mieux déchiffrer la régulation transcription elle.La première partie de mon travail de Thèse a consisté à mettre en place des outils permettant de visualiser en microscopie confocale et super-résolution, la fixation de complexes Hox-cofacteur sur des séquences ADN cibles spécifiques. Ces outils ont été appliques pour quantifier l’enrichissement de différents complexes Hox/Exd au niveau d’un enhancer connu (appelé fkh250) du gène cible forkhead (fkh) dans les glandes salivaires de la larve de drosophile. J’ai combine la méthode de BiFC (confocale) ou BiFC-PALM (super-résolution) et le système ParB/INT pour visualiser simultanément les complexes Hox/Exd et l’enhancer fkh250, respectivement.Mes analyses confirment un enrichissement spécifique des complexes Hox/Exd sur les différents types d’enhancer fkh250. Surtout, des résultats préliminaires indiquent la possibilité de quantifier le nombre exact de complexes Hox/Exd fixes sur l’enhancer fkh250 a l’échelle super-résolutive.La deuxième partie de mon travail de thèse concerne l’analyse d’une nouvelle interaction entre les protéines Hox avec la Lamine C (LamC) pour une répression transcriptionnelle active des gènes lies a l’autophagie (atg) dans le corps gras de la larve de drosophile. Ce travail a permis de révéler un profil de co-expression typique des protéines Hox et de la LamC, au sein du noyau par imagerie confocale ≪ Lightning ≫ et l’importance de contrôler le positionnement des loci génomiques pour une régulation fine de la transcription.
... Optical Linear Fluorescence Transitions (RESOLFT) nanoscopy being the most prominent examples, are scanning approaches. Both methods have been previously used for the imaging of cellular dynamics in animal tissues (Berning, Willig, Steffens, Dibaj, & Hell, 2012;Dreier et al., 2019;Schnorrenberg et al., 2016); to the best of our knowledge they have not been used to image living plant cells or green tissues. For this study, we decided to evaluate RESOLFT nanoscopy (Brakemann et al., 2011;Grotjohann et al., 2011;Hell, Dyba, & Jakobs, 2004;Hofmann, Eggeling, Jakobs, & Hell, 2005) for the imaging of nanoscale structures in cells of intact plant tissues. ...
Subdiffraction super‐resolution fluorescence microscopy, or nanoscopy, has seen remarkable developments in the last two decades. Yet, for the visualization of plant cells, nanoscopy is still rarely used. In this study, we established RESOLFT nanoscopy on living green plant tissue. Live‐cell RESOLFT nanoscopy requires and utilizes comparatively low light doses and intensities to overcome the diffraction barrier. We generated a transgenic Arabidopsis thaliana plant line expressing the reversibly switchable fluorescent protein rsEGFP2 fused to the mammalian microtubule‐associated protein 4 (MAP4) in order to ubiquitously label the microtubule cytoskeleton. We demonstrate the use of RESOLFT nanoscopy for extended time‐lapse imaging of cortical microtubules in Arabidopsis leaf discs. By combining our approach with fluorescence lifetime gating, we were able to acquire live‐cell RESOLFT images even close to chloroplasts, which exhibit very strong autofluorescence. The data demonstrate the feasibility of subdiffraction resolution imaging in transgenic plant material with minimal requirements for sample preparation.
... We illustrate the approach for RESOLFT nanoscopy imaging of Drosophila melanogaster larvae body wall muscles ubiquitously expressing rsEGFP2, a reversibly photoswitchable fluorescent protein (RSFP) [6], fused to α-tubulin. A commercial RESOLFT nanoscope was used for the recordings, and the preparation of the sample is described in [28]. Tubulin is known to form helices with a diameter of approximately 25 nm, each turn comprising 13 dimers of αand β-tubulin that are spaced 8 nm apart [29]. ...
Quantification of the numbers of molecules of interest in the specimen has emerged as a powerful capability of several fluorescence nanoscopy approaches. Carefully relating the measured signals from STED or RESOLFT scanning nanoscopy data to the contribution of a single molecule, reliable estimates of fluorescent molecule numbers can be obtained. To achieve this, higher-order signatures in the obtained photon statistics are analyzed, as arise from the antibunched nature of single-fluorophore emissions or in the signal variance among multiple on/off-switching cycles. In this chapter, we discuss the concepts and approaches demonstrated to date for counting molecules in STED/RESOLFT nanoscopy.
... The light intensities used are similar to those applied in live-cell confocal fluorescence microscopy and up to six orders of magnitude lower than those in STEDmicroscopy. The total light dose deposited on the sample is lower by 3-4 orders of magnitude compared to coordinate-stochastic nanoscopy [5,6]. As the light intensity is an important factor that determines phototoxicity [7], RESOLFT nanoscopy is particularly suitable for live-cell imaging approaches. ...
... While high quantum efficiencies for switching are beneficial for RESOLFT imaging by shortening the time consuming switching step, the connection between switching and fluorescence readout limits the number of collected photons in a single switching cycle. For point-scanning RESOLFT in living cells, the switching times are generally below 1 ms [5,6], while in parallelized RESOLFT schemes, switching times of tens of milliseconds are suitable [34,38]. ...
... Since the first proof of concept demonstration of RESOLFT nanoscopy using purified asFP595 [71], numerous RESOLFT live-cell applications have been reported [45]. It has been used in point-scanning (Fig. 9.5a) [5,6,14,23,25,42,73] as well as in various parallelized implementations ( Fig. 9.5b) [34,35,38,54,56,69,74,75]. ...
Diffraction-limited lens-based optical microscopy fails to discern fluorescent features closer than \(\sim \)200 nm. All super-resolution microscopy (nanoscopy) approaches that fundamentally overcome the diffraction barrier rely on fluorophores that can adopt different states, typically a fluorescent ‘on-’state and a dark, non-fluorescent ‘off-’state. In reversible saturable optical linear fluorescence transitions (RESOLFT) nanoscopy, light is applied to induce transitions between two states and to switch fluorophores on and off at defined spatial coordinates. RESOLFT nanoscopy relies on metastable reversibly switchable fluorophores. Thereby, it is particularly suited for live-cell imaging, because it requires relatively low light levels to overcome the diffraction barrier. Most implementations of RESOLFT nanoscopy utilize reversibly photoswitchable fluorescent proteins (RSFPs), which are derivatives of proteins from the green fluorescent protein (GFP) family. In recent years, analysis of the molecular mechanisms of the switching processes have paved the way to a rational design of new RSFPs with superior characteristics for super-resolution microscopy. In this chapter, we focus on the newly developed RSFPs, the light-driven switching mechanisms and the use of RSFPs for RESOLFT nanoscopy.
... Advances in microscopy are furthermore expected to contribute to an improved molecular understanding of misfolded protein species in vivo. Super-resolution microscopy has revealed the morphology and the growth of fibrils at the nanometre scale in vitro and in cells (Kaminski Schierle et al., 2011b;Duim et al., 2014;Pinotsi et al., 2016), and continuing efforts to implement these technologies in vivo have already allowed the use of RESOLFT and STED nanoscopy to visualise organelles and the cytoskeleton in C. elegans (Dreier et al., 2019), Drosophila (Schnorrenberg et al., 2016) and the mouse brain (Berning et al., 2012). Furthermore, the use of biosensors in combination with fluorescence microscopy may shed more light on the conformational state of protein aggregates in vivo, as demonstrated e.g. by the changes in the fluorescence lifetime of protein tags upon amyloid formation (Kaminski Schierle et al., 2011a;Laine et al., 2019). ...
Neurodegenerative disorders, including Alzheimer's (AD) and Parkinson's diseases (PD), are characterised by the formation of aberrant assemblies of misfolded proteins. The discovery of disease-modifying drugs for these disorders is challenging, in part because we still have a limited understanding of their molecular origins. In this review, we discuss how biophysical approaches can help explain the formation of the aberrant conformational states of proteins whose neurotoxic effects underlie these diseases. We discuss in particular models based on the transgenic expression of amyloid-β (Aβ) and tau in AD, and α-synuclein in PD. Because biophysical methods have enabled an accurate quantification and a detailed understanding of the molecular mechanisms underlying protein misfolding and aggregation in vitro , we expect that the further development of these methods to probe directly the corresponding mechanisms in vivo will open effective routes for diagnostic and therapeutic interventions.
... Reversible saturable optical fluorescence transitions (RESOLFT) is a variant of STED, instead of a depletion laser being used for stimulated emission of fluorophores, it is used for photoswitching fluorophores to their dark state ('off state') (Hell et al., 2004). RESOLFT is compatible only with reversibly photoswitchable dyes or fluorescent proteins, but it comes with the advantage of using much lower depletion laser intensities (Huang et al., 2009;Schnorrenberg et al., 2016). ...
In my PhD, I set out to establish single molecule localization microscopy (SMLM) as a complementary technique to answer questions in structural cell biology. The strengths of SMLM are resolution in the nanometer regime, molecular specificity and the ability to record dynamic information. In this thesis, I report on two independent projects: 1. The use of nuclear pores as a versatile reference standard for quantitative superresolution microscopy. 2. Visualizing the self-assembly of alpha-synuclein fibril polymorphs. In the first project, we introduced to the field nuclear pore complexes (NPC) as a reference standard for quality control in superresolution microscopy. To this end we generated, four CRISPR cell lines with nucleoporin96 (Nup96) endogenously tagged with labels SNAP, Halo, mEGFP and mMaple. The success of NPCs as a reference standard is owed to its well characterized structural organization and composition. Nup96 is present as 32 copies divided equally over the cytoplasmic and nuclear ring of the NPC. It’s stereotypic arrangement facilitates the visualization of the NPC’s radial eightfold symmetry under SMLM. The overall dimensions of the NPC positions fluorophores at relevant distances for 2D and 3D resolution calibration and quality control. Knowledge of the absolute number of underlying labels present in each NPC allowed us to calculate the effective labeling efficiency of each labeling strategy. Having a defined number for fluorophores also allowed for counting of protein copy numbers within complexes using both diffraction-limited and superresolution microscopy. In the second project, I established a correlative transmission electron microscopy (TEM) and single molecule localization microscopy (SMLM) method to study the dynamic self-assembly of amyloid fibril polymorphs. Amyloid fibril polymorphism has been found in distinct neurodegenerative disease phenotypes. The ability to exist as different polymorphs has been a stumbling block towards understanding disease etiology. To address this need, I first established an imaging assay that enabled the visualization of the self-assembly process of single amyloid fibrils in real-time. To visualize individual fibrils, I used the point accumulation for imaging in nanoscale topography (PAINT) imaging strategy with fluorogenic amyloid binding dyes. This strategy allowed for imaging with unmodified protein monomers while achieving high labeling densities on fibrils permitting the characterization of respective fibril self-assembly. From my dynamic PAINT measurements, I have identified that fibrils exhibit growth characteristics specific to the solution conditions they are in. The unique opportunity of analyzing the polarization of the emitted fluorescence of each binding event enabled me to visualize fibril ultrastructure. To further validate observed structural features, I performed correlative TEM tomography and dynamic PAINT. Such multiparametric correlative imaging enables the description of fibril growth kinetics with respect to its underlying structure, which would otherwise not be possible.
