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Different labelling strategies adopted to mark α-synuclein affect differently the aggregation of this peptide in vitro. By using super-resolved correlative STED–AFM microscopy we discriminate between fluorescent and non fluorescent aggregates.

The aim of this study was to evaluate the use of autologous fat graft injection to correct lower eyelid position METHODS: A retrospective, observational, single blind, case-control study was carried out on 94 patients, presenting with lower eyelid retraction in 159 eyes. In the sub-population with monolateral eyelid retraction, the not affected site has been considered as a control and compared with the outcomes recorded after treatment of the contralateral side Follow-up at 12 months was performed with a subjective assessment carried out by a questionnaire administered to patients while objective result assessment was performed 12 months after surgery by two independent blind examiners.The eyelid upward reposition has improved one year after fat grafting in both bilateral (1,52 mm) and unilateral (1,7 mm) population: the latter allowed to statistically validate (P0.05) the result with respect to the not affected site.This is the first paper that highlights the outcomes of sole fat injection in the treatment of lower eyelid retraction: blind objective evaluation of surgical outcomes along with a patient assessment of both functional and aesthetic improvement one year after surgery confirm its efficacy and reliability along with the first case/control outcome evaluation of the technique carried on in the sub-population of 29 patients affected by unilateral lower eyelid retraction that validate the average improvement of the retracted eyelid one year after fat grafting as statistically significant. Nevertheless, longer follow-up periods and a larger sample size are needed to thoroughly confirm surgical outcomes and statistical results.This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Botulin toxin (BTX) is a potent neurotoxin produced by the bacterium Clostridium botulinum, since its approval by FDA in 2002 for cosmetic purpose has been widely used. Recent studies indicate that it exerts its activity on various type of skin cells and can be used in some dermatological disease.The aim of the study was to demonstrate how to use a peculiar dilution of botulinum toxin type A in the treatment of some dermatological disease like mild-to-moderate acne vulgaris and erythematotelangiectatic rosacea.50 patients were enrolled, 35 with mild-to-moderate acne and 15 with erythematotelangiectatic rosacea. Both group of patients were treated with a specific dilution of Onabotulinum toxin A called Microbotox. Patient images were taken before and 4 weeks after the treatment.The authors and patients were extremely satisfied with their treatments. There were no immediate or delayed complication in none of both group of patients.Botulinum toxin shows a great promise either in dermatological disease like mild-to-moderate acne vulgaris and erythematotelangiectatic rosacea. Microbotox appears to be a valid, long-lasting, and a standardized approach to treat these kind of two disease.

We report the development of a supramolecular structure endowed with photosensitizing properties and targeting capability for antimicrobial photodynamic inactivation. Our synthetic strategy uses the tetrameric bacterial protein streptavidin, labeled with the photosensitizer eosin, as the main building block. Biotinylated immunoglobulin G (IgG) from human serum, known to associate with

The goal of the present study was to comparatively analyze Social Cognition skills in a pediatric population diagnosed with Migraine or Epilepsy, compared to Typically Developing children (TD). The secondary aim was to relate Social Cognition skills with other migraine- or epilepsy-related variables and with executive and cognitive functions.In our cross-sectional observational study 119 children and adolescents (aged 6-16) with Migraine or Focal Epilepsy and 61 TD peers were recruited. Both the clinical groups and TD peers performed a neuropsychological evaluation through standardized test to assess Theory of Mind (TM), Emotion Recognition through facial expression (ER), executive function and non-verbal cognitive abilities.Children and adolescents with Migraine or Focal Epilepsy showed comparable scores between each other, however their scores were significantly lower than their TD peers, in both ER and TM. Social Cognition skills were significantly related to executive functions.Our study suggests that some chronic neurological conditions in childhood, such as Migraine and Epilepsy, may be associated with difficulties in Social Cognition skills, and that these difficulties may be related to a deficit in executive functions. The relationship between these two higher cognitive abilities should be further explored in future studies. Our results also suggest the importance of monitoring cognitive abilities in pediatric patients with Migraine or Epilepsy, in order to detect early impairment and ensure the necessary support.

Background: The ageing process starts in the center of the face, in the periocular region and around the mouth, with a combination of volume loss and tissue descent, wrinkles deepen, and loss of skin structure and quality. Recently, several studies proved the efficacy of therapies based on the autologous adipose tissue grafting that leverages the properties of stromal vascular fraction (SVF) and adipose derived mesenchymal stem cells (ADSCs) to accelerate the regenerative processes of the skin. This study aimed to verify the ability of the guided Superficial Enhanced Fluid Fat Injection (SEFFI) in the facial area to correct volume loss and skin aging and proved the very low rate of complications of this standardized procedure . Methods: we retrospectively collected data from 2,365 procedures performed in Italian centers between 2019 and 2021. Guided SEFFI was performed alone or combined with cosmetic treatments, including the use of hyaluronic acid filler, suspension threads, synthetic calcium hydroxylapatite, botulin toxin, and microneedling. Results: SEFFI was used alone in more than 60% of the patients, and in all face areas. In about one tenth of patients SEFFI was combined with botulin toxin treatment or hyaluronic acid filling. Other procedures were used more rarely. Ecchymosis in the donor or injection sites was the most frequent event, and yet observed in only 14.2% and 38.6% of patients, respectively. Conclusions: The SEFFI technique is standardized and minimally invasive, leading to very few complications. It can be a promising antiaging medical treatment that combines effectiveness, safety, and simplicity.

The correct inheritance of chromatin structure is key for maintaining genome function and preventing cellular transformation. DEK, a conserved chromatin protein, has recognized tumor-promoting properties, its overexpression being associated with poor prognosis in various cancer types. At the cellular level, DEK displays pleiotropic functions, influencing differentiation, apoptosis, and stemness, but a characteristic oncogenic mechanism remains elusive. Here, we report the identification of DEK bodies, focal assemblies of DEK occurring at specific, yet unidentified sites of heterochromatin replication. In these bodies, DEK localizes in direct proximity to active replisomes suggesting a function in the early maturation of heterochromatin. A high-throughput siRNA screen identifies SUMO as a major regulator of DEK body formation, linking DEK to the SUMO network that controls chromatin states and cell fate. This work combines and refines our previous data on DEK as a factor essential for heterochromatin integrity and facilitating replication under stress and delineates an avenue of further study for unraveling DEK’s contribution to cancer development.

The chromatin architectural factor DEK is involved in regulating the chromatin organization and is highly overexpressed in numerous forms of cancer. DEK is traditionally described as uniformly distributed within the nucleus by diffraction-limited microscopy studies, but super-resolution advent highlighted the formation of cluster-like DEK structures. Still, a characterization of the DEK protein cellular distribution and its role in cancer and cell proliferation is missing. In this work, we used single-molecule localization microscopy (SMLM) to investigate the nanoscale characteristics of DEK organization in normal-like and aggressive types of breast cancer cell lines, characterizing the number of localizations per cluster, as well as areas and density of clusters. We show how the cluster features correlate with the breast cell type and how the chromatin decompaction influences the DEK clusters in both cell lines. Our results suggest that the DEK density and nano-organization are preserved and are not influenced by protein overexpression itself or by chromatin compaction changes.

The study of the GABAA receptor itself and its pharmacology is of paramount importance for shedding light on the role of this receptor in the central nervous system. Caged compounds have emerged as powerful tools to support research in this field, as they allow to control, in space and time, the release of neurotransmitters enabling, for example, to map receptors’ distribution and dynamics. Here we focus on γ-aminobutyric acid (GABA)-caged compounds, particularly on a commercial complex called RuBi-GABA, which has high efficiency of uncaging upon irradiation at visible wavelengths. We characterized, by electrophysiological measurements, the effects of RuBi-GABA on GABAA receptors of rat cerebellar granule cells in vitro. In particular, we evaluated the effects of side products obtained after RuBi-GABA photolysis. For this purpose, we developed a procedure to separate the “RuBi-cage” from GABA after uncaging RuBi-GABA with a laser source; then, we compared electrophysiological measurements acquired with and without administering the RuBi-cage in the perfusing bath. In conclusion, to investigate the role of the “cage” molecules both near and far from the cell soma, we compared experiments performed changing the distance of the uncaging point from the cell.

In point-scanning microscopy, optical sectioning is achieved using a small aperture placed in front of the detector, i.e. the detection pinhole, which rejects the out-of-focus background. The maximum level of optical sectioning is theoretically obtained for the minimum size of the pinhole aperture, but this is normally prevented by the dramatic reduction of the detected signal when the pinhole is closed, leading to a compromise between axial resolution and signal-to-noise ratio. We have recently demonstrated that, instead of closing the pinhole, one can reach a similar level of optical sectioning by tuning the pinhole size in a confocal microscope and by analyzing the resulting image series. The method, consisting in the application of the separation of photons by lifetime tuning (SPLIT) algorithm to series of images acquired with tunable pinhole size, is called SPLIT-pinhole (SPLIT-PIN). Here, we share and describe a SPLIT-PIN software for the processing of series of images acquired at tunable pinhole size, which generates images with reduced out-of-focus background. The software can be used on series of at least two images acquired on available commercial microscopes equipped with a tunable pinhole, including confocal and stimulated emission depletion (STED) microscopes. We demonstrate applicability on different types of imaging modalities: (1) confocal imaging of DNA in a non-adherent cell line; (2) removal of out-of-focus background in super-resolved STED microscopy; (3) imaging of live intestinal organoids stained with a membrane dye.

We present a novel approach to implement compressive sensing in laser scanning microscopes (LSM), specifically in image scanning microscopy (ISM), using a single-photon avalanche diode (SPAD) array detector. Our method addresses two significant limitations in applying compressive sensing to LSM: the time to compute the sampling matrix and the quality of reconstructed images. We employ a fixed sampling strategy, skipping alternate rows and columns during data acquisition, which reduces the number of points scanned by a factor of four and eliminates the need to compute different sampling matrices. By exploiting the parallel images generated by the SPAD array, we improve the quality of the reconstructed compressive-ISM images compared to standard compressive confocal LSM images. Our results demonstrate the effectiveness of our approach in producing higher-quality images with reduced data acquisition time and potential benefits in reducing photobleaching., Comment: Presented in ISCS23

The study of stoichiometry and supra-molecular organization of membrane (and membrane-associated) proteins plays a key role in understanding membrane structure and function. Single-molecule localization techniques (SML), besides providing imaging at unprecedented resolution, also offer quantitative tools such as stepwise photobleaching (SP) experiments and quantitative single-molecule localization (qSMLM). SML is becoming widely present in imaging core facilities but addressing biological problems by molecular counting experiments still remains not straightforward since experimental approaches for sample preparation require particular attention.We will focus on the experimental aspects that may prevent successful quantitative SML experiments of membrane-associated proteins.Depending on the specific experiment, to avoid artifacts and to miscount, fine-tuning of the expression levels and proper staining procedures are required, as well as optimized protocols and controls for counting.The work aims to highlight the crucial aspects that must be faced when quantitative single-molecule experiments are performed, helping to match the gap between sample preparation and the application of quantitative fluorescence microscopy techniques.

To date, the feasibility of super-resolution microscopy for imaging live and thick samples is still limited. Stimulated emission depletion (STED) microscopy requires high-intensity illumination to achieve sub-diffraction resolution, potentially introducing photodamage to live specimens. Moreover, the out-of-focus background may degrade the signal stemming from the focal plane. Here, we propose a new method to mitigate these limitations without drawbacks. First, we enhance a STED microscope with a detector array, enabling image scanning microscopy (ISM). Therefore, we implement STED-ISM, a method that exploits the working principle of ISM to reduce the depletion intensity and achieve a target resolution. Later, we develop Focus-ISM, a strategy to improve the optical sectioning and remove the background of any ISM-based imaging technique, with or without a STED beam. The proposed approach requires minimal architectural changes to a conventional microscope but provides substantial advantages for live and thick sample imaging.

Studying biomolecules in their native environment represents the ideal sample condition for structural biology investigations. Here we present a novel protocol which allows to delivery proteins into eukaryotic cells through a mild thermal stimulation. The data presented herein show the efficacy of this approach for delivering proteins in the intracellular environment of mammalian cells reaching a concentration range suitable for successfully applying biophysical methods, such as double electron electron resonance (DEER) measurements for characterising protein conformations.

Atomic force microscopy (AFM) is a nano‐mechanical tool uniquely suited for biological studies at the molecular scale. AFM operation is based on mechanical interaction between the tip and the sample, a mechanism of contrast capable of measuring different information, including surface topography, mechanical, and electrical properties. However, the lack of specificity highlights the need to integrate AFM data with other techniques providing compositional hints. In particular, optical microscopes based on fluorescence as a mechanism of contrast can access the local distribution of specific molecular species. The coupling between AFM and super‐resolved fluorescence microscopy solves the resolution mismatch between AFM and conventional fluorescence optical microscopy. Recent advances showed that also the inherently label‐free imaging capabilities of the AFM are fundamental to complement the fluorescence images. In this review, we have presented a brief historical view on correlative microscopy, and, finally, we have summarized the progress of correlative AFM‐super‐resolution microscopy in biological research., Correlative nanoscopy is a new field of application that implies integrating two or more microscopy techniques able to provide resolution at the nanoscale. In particular, in the last years, the coupling between the atomic force microscope (AFM) and optical super‐resolution microscopes gives rise to a new tool with the unique capability to investigate living/dynamic biological systems. Here, we presented the fundamental step in the development of this new technique. We decided to start from the origin of the correlative AFM‐optical microscopy, giving a historical overview on that, in our mind, could be helpful, especially for young researchers, to understand the needs that drive the efforts spent toward the development of optical nanoscopy.

As facial aging occurs, aesthetic changes of the nasal tip and the senile upper lip include tip ptosis, increase in skin length, loss of vermillion height and Cupid's bow, and vertical wrinkles.Regardless of the rejuvenation procedure, the lips are not to be considered as a single anatomical entity, instead they should be placed in a strong correlation with the nose. The aim of this study is therefore to demonstrate the effectiveness of the association of primary closed rhinoplasty together with indirect subnasal lip-lifting technique.A total of 45 patients were enrolled in this study and underwent primary closed rhinoplasty with indirect subnasal lip-lifting. The result showed an overall average nasolabial angle width reduction of 10.9% and lip length shortening of 23.5%, as assessed 1 year after the surgical procedure. When comparing before and after photographs and using the Subjective Global Aesthetic Improvement Scale (sGAIS), the overall satisfaction was rated 4.4 on 5, as “much improved.”The authors show that lips should not be considered as a single anatomical entity but that they indeed must be placed in a strong correlation with the nose since a droopy nasal tip is likely to be considered unattractive and is often associated with the appearance of a long nose with covered philtrum and upper lip.Tip rotation to a normal nasolabial angle width could grant a beneficial impact on overall nose aesthetics; however, it may not fully balance the midface profile as it is the “lip position-to-incisal show” ratio that defines beauty, whereas the balance among forehead, nasal tip, upper lip, and chin is what defines the contour of harmonious profiles.This is the first patient series to show that the combination of closed rhinoplasty and indirect lip-lifting is a highly effective, safe, and reliable procedure to address profile rejuvenation.

Programmed death ligand 1 (PD-L1) plays a key role in several human tumors, and it has recently become a crucial target for cancer therapy. Yet, its subtle regulatory roles beside the well characterized immunosuppression in PD-1/PD-L1 immune checkpoint are still obscure and should be strictly related to its molecular properties in different cell settings. In this study we targeted the plasma membrane organization of PD-L1 in a Non-Small Cell Lung Cancer (NSCLC) cell line by a multiscale fluorescence imaging toolbox that included super-resolution microscopy approaches to reach the nanoscale. Our findings revealed for the first time that PD-L1 is prevalently engaged in the cholesterol-enriched “raft” regions of the cell membrane. Clusterization and engagement in membrane rafts may afford novel targets for the immuno-oncology strategies in the framework of NSCLC and, possibly, other tumor types.

The antisecretory factor (AF) is an endogenous protein that counteracts intestinal hypersecretion and various inflammation conditions in vivo. It has been detected in many mammalian tissues and plasma, but its mechanisms of action are largely unknown. To study the pharmacological action of the AF on different GABA

This article deals with the developments of optical microscopy towards nanoscopy. Basic concepts of the methods implemented to obtain spatial super-resolution are described, along with concepts related to the study of biological systems at the molecular level. Fluorescence as a mechanism of contrast and spatial resolution will be the starting point to developing a multi-messenger optical microscope tunable down to the nanoscale in living systems. Moreover, the integration of optical nanoscopy with scanning probe microscopy and the charming possibility of using artificial intelligence approaches will be shortly outlined.

BACKGROUND: Family violence has been shown to have a dramatic impact on individual and family life in the United States and other countries. Numerous studies have assessed the influence that exposure to violence can have on family dynamics and parent-child relationships. However, less is known about the association between family violence and parent-child relationships with Mexican families. OBJECTIVE: Guided by social interaction learning theory, the purpose of this study was to explore the role of exposure to family violence on PTSD and mother-child interaction patterns. METHODS: Eighty-seven mother-child dyads from Mexico completed assessments for exposure to family violence, PTSD, and observational tasks were analyzed to assess prosocial parent-child interactions (i.e., positive communication and problem solving). We conducted an actor-partner independence model (APIM) to examine the association between exposure to family violence, PTSD and mother-child relationship dynamics. RESULTS: As expected, higher exposure to family violence was linked to higher PTSD symptoms for mothers. Unexpectedly, higher maternal PTSD symptoms were associated with better communication during dyadic interaction tasks with their children. CONCLUSIONS: The present study suggests that individuals from certain cultures (i.e., Mexico) may respond differently to experiencing family violence. The use of multiple measurement methods to assess the relational effects of trauma on family dynamics can advance the scientific understanding of trauma affected families.

The molecular mechanisms that underlie oncogene-induced genomic damage are still poorly understood. To understand how oncogenes affect chromatin architecture, it is important to visualize fundamental processes such as DNA replication and transcription in intact nuclei and quantify the alterations of their spatiotemporal organization induced by oncogenes. Here, we apply superresolution microscopy in combination with image cross correlation spectroscopy to the U937-PR9 cell line, an in vitro model of acute promyelocytic leukemia that allows us to activate the expression of the PML-RARα oncogene and analyze its effects on the spatiotemporal organization of functional nuclear processes. More specifically, we perform Tau-stimulated emission depletion imaging, a superresolution technique based on the concept of separation of photons by lifetime tuning. Tau-stimulated emission depletion imaging is combined with a robust image analysis protocol that quickly produces a value of colocalization fraction on several hundreds of single cells and allows observation of cell-to-cell variability. Upon activation of the oncogene, we detect a significant increase in the fraction of transcription sites colocalized with PML/PML-RARα. This increase of colocalization can be ascribed to oncogene-induced disruption of physiological PML bodies and the abnormal occurrence of a relatively large number of PML-RARα microspeckles. We also detect a significant cell-to-cell variability of this increase of colocalization, which can be ascribed, at least in part, to a heterogeneous response of the cells to the activation of the oncogene. These results prove that our method efficiently reveals oncogene-induced alterations in the spatial organization of nuclear processes and suggest that the abnormal localization of PML-RARα could interfere with the transcription machinery, potentially leading to DNA damage and genomic instability.

The study of the GABA

Hypericin is a photosensitizing drug that is active against membrane-enveloped viruses and therefore constitutes a promising candidate for the treatment of SARS-CoV-2 infections. The antiviral efficacy of hypericin is largely determined by its affinity toward viral components and by the number of active molecules loaded on single viruses. Here we use an experimental approach to follow the interaction of hypericin with SARS-CoV-2, and we evaluate its antiviral efficacy, both in the dark and upon photoactivation. Binding to viral particles is directly visualized with fluorescence microscopy, and a strong affinity for the viral particles, most likely for the viral envelope, is measured spectroscopically. The loading of a maximum of approximately 30 molecules per viral particle is estimated, despite with marked heterogeneity among particles. Because of this interaction, nanomolar concentrations of photoactivated hypericin substantially reduce virus infectivity on Vero E6 cells, but a partial effect is also observed in dark conditions, suggesting multiple mechanisms of action for this drug.

Graphical abstract, We exploited a multi-scale microscopy imaging toolbox to address some major issues related to SARS-CoV-2 interactions with host cells. Our approach harnesses both conventional and super-resolution fluorescence microscopy and easily matches the spatial scale of single-virus/cell checkpoints. After its validation through the characterization of infected cells and virus morphology, we leveraged this toolbox to reveal subtle issues related to the entry phase of SARS-CoV-2 variants in Vero E6 cells. Our results show that in Vero E6 cells the B.1.1.7 strain (aka Alpha Variant of Concern) is associated with much faster kinetics of endocytic uptake compared to its ancestor B.1.177. Given the cell-entry scenario dominated by the endosomal “late pathway”, the faster internalization of B.1.1.7 could be directly related to the N501Y mutation in the S protein, which is known to strengthen the binding of Spike receptor binding domain with ACE2. Remarkably, we also directly observed the central role of clathrin as a mediator of endocytosis in the late pathway of entry. In keeping with the clathrin-mediated endocytosis, we highlighted the non-raft membrane localization of ACE2. Overall, we believe that our fluorescence microscopy-based approach represents a fertile strategy to investigate the molecular features of SARS-CoV-2 interactions with cells.

The photodynamic treatment for antimicrobial applications or anticancer therapy relies on reactive oxygen species generated by photosensitizing molecules after absorption of visible or near-infrared light. If the photosensitizing molecule is in close vicinity of the microorganism or the malignant cell, a photocytotoxic action is exerted. Therefore, the effectiveness of photosensitizing compounds strongly depends on their capability to target microbial or cancer-specific proteins. In this study, we report on the preparation and preliminary characterization of human recombinant myoglobin fused to the vasoactive intestinal peptide to target vasoactive intestinal peptide receptor (VPAC) receptors. Fe-protoporphyrin IX was replaced by the photosensitizing compound Zn-protoporphyrin IX. Taking advantage of the fluorescence emission by Zn-protoporphyrin IX, we show that the construct can bind prostate cancer cells where the VPAC receptors are expressed.

Super-resolution microscopy is routinely used for fixed and thin samples, while its feasibility for imaging live and thick samples is still limited. In the case of stimulated emission depletion (STED) microscopy, the high-intensity illumination required to achieve effective sub-diffraction resolution can introduce photo-damage, thus reducing the compatibility of the technique with live-cell imaging. Moreover, the out-of-focus fluorescence background may overcome the often faint signal stemming from the focal point, thus constraining imaging to thin samples. Here, we combined STED microscopy with image-scanning microscopy (ISM) to mitigate these limitations without any practical disadvantages. We first enhanced a laser scanning microscope (LSM) by introducing a detector array, hence providing access to a set of additional spatial information that is not available with a typical single-element detector. Then, we exploited this extended dataset to implement focus-ISM, a novel method that relaxes the high-intensity requirement of STED microscopy and removes the out-of-focus background. Additionally, we generalized the focus-ISM method to conventional LSM, namely without a STED beam. The proposed approach requires minimal architectural changes compared with conventional STED microscopes but provides substantial advantages for live and thick sample imaging while maintaining all compatibility with all recent advances in STED and confocal microscopy. As such, focus-ISM represents an essential step towards a universal super-resolved LSM technique for subcellular imaging.

The retina is a thin neuronal multilayer responsible for the detection of visual information. The first step in visual transduction occurs in the photoreceptor outer segment. The studies on photoreception and visual biochemistry have often utilized rod outer segments (OS) or OS disks purified from mammalian eyes. Literature reports several OS and disk purification procedures that rarely specify the procedure utilized to collect the retina from the eye. Some reports suggest the use of scissors, while others do not mention the issue as they declare to utilize frozen retinas. Because the OS are deeply embedded in the retinal pigmented epithelium (RPE), the detachment of the retina by a harsh pull-out can cause the fracture of the photoreceptor cilium. Here, we present a protocol maximizing OS yield. Eye semi-cups, obtained by hemisecting the eyeball and discarding the anterior chamber structures and the vitreous, are filled with Mammalian Ringer. After 10–15 min of incubation, the retinas spontaneously detach with their wealth of OS almost intact. The impressive ability of the present protocol to minimize the number of OS stuck inside the RPE, and therefore lost, compared with the classic procedure, is shown by confocal laser scanning microscopy analysis of samples stained ex vivo with a dye (MitoTracker deep red) that stains both retinal mitochondria and OS. Total protein assay of OS disks purified by either procedure also shows a 300% total protein yield improvement. The advantage of the protocol presented is its higher yield of photoreceptor OS for subsequent purification procedures, while maintaining the physiological features of the retina.

In spite of their value as genetically encodable reporters for imaging in living systems, fluorescent proteins have been used sporadically for stimulated emission depletion (STED) super-resolution imaging, owing to their moderate photophysical resistance, which does not enable reaching resolutions as high as for synthetic dyes. By a rational approach combining steady-state and ultrafast spectroscopy with gated STED imaging in living and fixed cells, we here demonstrate that F99S/M153T/V163A GFP (c3GFP) represents an efficient genetic reporter for STED, on account of no excited state absorption at depletion wavelengthslt;600 nm and a long emission lifetime. This makes c3GFP a valuable alternative to more common, but less photostable, EGFP and YFP/Citrine mutants for STED imaging studies targeting the green-yellow region of the optical spectrum.

Hyaluronic acid (HA) gel injections were first used to treat the tear trough in 2005 and since then it has been a mainstay of the approach to lower eyelid deformities.The authors present this retrospective multicentric observational study based on single-blind objective and subjective evaluation and patient satisfaction in relation to the aesthetic improvement of a large group of patients treated.Between January 2016 and December 2019, 600 patients (468 women and 132 men), were enrolled in this study, and 1200 tear trough deformities were treated with both needle and cannula techniques.Average follow-up time was 12 ± 1 months, and the outcomes were assessed both objectively and subjectively with respect to Hirmand's classification. Statistical analysis shows an inverse correlation between age and class amelioration.HA injection of the tear trough is most effective in patients between 30 and 40 years of age, while its benefits extend to up to 50 years old; afterward, it should no longer be the treatment of choice. This confirms that correction of tear trough with hyaluronic acid injections may provide an option to achieve immediate and durable results for up to one year after the injection.This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

The genomes of metazoans are organized at multiple spatial scales, ranging from the double helix of DNA to whole chromosomes. The intermediate genomic scale of kilobases to megabases, which corresponds to the 50–300 nm spatial scale, is particularly interesting, as the 3D arrangement of chromatin is implicated in multiple regulatory mechanisms. In this context, polycomb group (PcG) proteins stand as major epigenetic modulators of chromatin function, acting prevalently as repressors of gene transcription by combining chemical modifications of target histones with physical crosslinking of distal genomic regions and phase separation. The recent development of super-resolution microscopy (SRM) has strongly contributed to improving our comprehension of several aspects of nano-/mesoscale (10–200 nm) chromatin domains. Here, we review the current state-of-the-art SRM applied to PcG proteins, showing that the application of SRM to PcG activity and organization is still quite limited and mainly focused on the 3D assembly of PcG-controlled genomic loci. In this context, SRM approaches have mostly been applied to multilabel fluorescence in situ hybridization (FISH). However, SRM data have complemented the maps obtained from chromosome capture experiments and have opened a new window to observe how 3D chromatin topology is modulated by PcGs.