Numerical study of transient absorption saturation in single-layer graphene for optical nanoscopy applications.

Transient absorption, or pump-probe microscopy is an absorption-based technique that can explore samples ultrafast dynamic properties and provide fluorescence-free contrast mechanisms. When applied to graphene and its derivatives, this technique exploits the graphene transient response caused by the ultrafast interband transition as the imaging contrast mechanism. The saturation of this transition is fundamental to allow for super-resolution optical far-field imaging, following the reversible saturable optical fluorescence transitions (RESOLFT) concept, although not involving fluorescence. With this aim, we propose a model to numerically compute the temporal evolution under saturation conditions of the single-layer graphene molecular states, which are involved in the transient absorption. Exploiting an algorithm based on the fourth order Runge-Kutta (RK4) method, and the density matrix approach, we numerically demonstrate that the transient absorption signal of single-layer graphene varies linearly as a function of excitation intensity until it reaches saturation. We experimentally verify this model using a custom pump-probe super-resolution microscope. The results define the intensities necessary to achieve super-resolution in a pump-probe nanoscope while studying graphene-based materials and open the possibility of predicting such a saturation process in other light-matter interactions that undergo the same transition.

DEK oncoprotein participates in heterochromatin replication via SUMO-dependent nuclear bodies.

The correct inheritance of chromatin structure is key for maintaining genome function and cell identity and preventing cellular transformation. DEK, a conserved non-histone 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 has remained elusive. Here, we report the identification of DEK bodies, focal assemblies of DEK that regularly occur at specific, yet unidentified, sites of heterochromatin replication exclusively in late S-phase. In these bodies, DEK localizes in direct proximity to active replisomes in agreement with a function in the early maturation of heterochromatin. A high-throughput siRNA screen, supported by mutational and biochemical analyses, identifies SUMO as one regulator of DEK body formation, linking DEK to the complex SUMO protein 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 the contribution of DEK to cancer development.

Super-resolution microscopy reveals the nanoscale cluster architecture of the DEK protein cancer biomarker.

DEK protein, a key chromatin regulator, is strongly overexpressed in various forms of cancer. While conventional microscopy revealed DEK as uniformly distributed within the cell nucleus, advanced super-resolution techniques uncovered cluster-like structures. However, a comprehensive understanding of DEK's cellular distribution and its implications in cancer and cell growth remained elusive. To bridge this gap, we employed single-molecule localization microscopy (SMLM) to dissect DEK's nanoscale organization in both normal-like and aggressive breast cancer cell lines. Our investigation included characteristics such as localizations per cluster, cluster areas, and intra-cluster localization densities (ICLDs). We elucidated how cluster features align with different breast cell types and how chromatin decompaction influences DEK clusters in these contexts. Our results indicate that DEK's intra-cluster localization density and nano-organization remain preserved and not significantly influenced by protein overexpression or chromatin compaction changes. This study advances the understanding of DEK's role in cancer and underscores its stable nanoscale behavior.

Image scanning microscopy with a doughnut beam: signal strength and integrated intensity.

We discuss the effects of image scanning microscopy using doughnut beam illumination on the properties of signal strength and integrated intensity. Doughnut beam illumination can give better optical sectioning and background rejection than Airy disk illumination. The outer pixels of a detector array give a signal from defocused regions, so digital processing of these (e.g., by simple subtraction) can further improve optical sectioning and background rejection from a single in-focus scan.

Guided Superficial Enhanced Fluid Fat Injection (SEFFI) Procedures for Facial Rejuvenation: An Italian Multicenter Retrospective Case Report.

BACKGROUND: The aging process starts in the center of the face, in the periocular region and around the mouth, with a combination of volume loss, tissue descent, deepened wrinkles, and the loss of skin structure and quality. Recently, several studies have demonstrated the efficacy of therapies based on autologous adipose tissue grafting, which 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 aims to verify the ability of guided superficial enhanced fluid fat injection (SEFFI) in the facial area to correct volume loss and skin aging, proving that this standardized procedure has a very low rate of complications. METHODS: We retrospectively collected data from 2365 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: guided SEFFI was used alone in more than 60% of the patients and in all facial areas. In about one-tenth of the patients, guided SEFFI was combined with a botulin toxin treatment or hyaluronic acid filling. Other procedures were used more rarely. Ecchymosis in the donor or injection sites was the most frequent adverse event but was only observed in 14.2% and 38.6% of the patients, respectively. CONCLUSIONS: The guided SEFFI technique is standardized and minimally invasive, leading to very few complications. It constitutes a promising antiaging medical treatment that combines effectiveness, safety, and simplicity.

Imaging-based study demonstrates how the DEK nanoscale distribution differentially correlates with epigenetic marks in a breast cancer model.

Epigenetic dysregulation of chromatin is one of the hallmarks of cancer development and progression, and it is continuously investigated as a potential general bio-marker of this complex disease. One of the nuclear factors involved in gene regulation is the unique DEK protein-a histone chaperon modulating chromatin topology. DEK expression levels increase significantly from normal to cancer cells, hence raising the possibility of using DEK as a tumor marker. Although DEK is known to be implicated in epigenetic and transcriptional regulation, the details of these interactions and their relevance in cancer development remain largely elusive. In this work, we investigated the spatial correlation between the nuclear distribution of DEK and chromatin patterns-alongside breast cancer progression-leveraging image cross-correlation spectroscopy (ICCS) coupled with Proximity Ligation Assay (PLA) analysis. We performed our study on the model based on three well-established human breast cell lines to consider this tumor's heterogeneity (MCF10A, MCF7, and MDA-MB-231 cells). Our results show that overexpression of DEK correlates with the overall higher level of spatial proximity between DEK and histone marks corresponding to gene promoters regions (H3K9ac, H3K4me3), although it does not correlate with spatial proximity between DEK and gene enhancers (H3K27ac). Additionally, we observed that colocalizing fractions of DEK and histone marks are lower for the non-invasive cell subtype than for the highly invasive cell line (MDA-MB-231). Thus, this study suggests that the role of DEK on transcriptionally active chromatin regions varies depending on the subtype of the breast cancer cell line.

Photons Induce Vesicular Exocytotic Release of Glutamate in a Power-Dependent Way.

Increasing evidence indicates that photobiomodulation, based on tissue irradiation with photons in the red to near-infrared spectrum, may be an effective therapeutic approach to central nervous system disorders. Although nervous system functionality has been shown to be affected by photons in animal models, as well as in preliminary evidence in healthy subjects or in patients with neuropsychiatric disorders, the mechanisms involved in the photobiomodulation effects have not yet been clarified. We previously observed that photobiomodulation could stimulate glutamate release. Here, we investigate mechanisms potentially involved in the glutamate-releasing effect of photons from adult mouse cerebrocortical nerve terminals. We report evidence of photon ability to induce an exocytotic vesicular release of glutamate from the terminals of glutamatergic neurons in a power-dependent way. It can be hypothesized that photobiomodulation, depending on the potency, can release glutamate in a potentially neurotoxic or physiological range.

Nuclear and chromatin rearrangement associate to epigenome and gene expression changes in a model of in vitro adipogenesis and hypertrophy.

Hypertrophy of adipocytes represents the main cause of obesity. We investigated in vitro the changes associated with adipocyte differentiation and hypertrophy focusing on the nuclear morphometry and chromatin epigenetic remodelling. The 3 T3-L1 pre-adipocytes were firstly differentiated into mature adipocytes, then cultured with long-chain fatty acids to induce hypertrophy. Confocal and super-resolution stimulation emission depletion (STED) microscopy combined with ELISA assays allowed us to explore nuclear architecture, chromatin distribution and epigenetic modifications. In each condition, we quantified the triglyceride accumulation, the mRNA expression of adipogenesis and dysfunction markers, the release of five pro-inflammatory cytokines. Confocal microscopy revealed larger volume and less elongated shape of the nuclei in both mature and hypertrophic cells respect to pre-adipocytes, and a trend toward reduced chromatin compaction. Compared to mature adipocytes, the hypertrophic phenotype showed larger triglyceride content, increased PPARγ expression reduced IL-1a release, and up-regulation of a pool of genes markers for adipose tissue dysfunction. Moreover, a remodelling of both epigenome and chromatin organization was observed in hypertrophic adipocytes, with an increase in the average fluorescence of H3K9 acetylated domains in parallel with the increase in KAT2A expression, and a global hypomethylation of DNA. These findings making light on the nuclear changes during adipocyte differentiation and hypertrophy might help the strategies for treating obesity and metabolic complications.

Optical-Quality Assessment of a Miniaturized Intraocular Telescope.

Age-related macular degeneration (AMD) causes severe vision impairments, including blindness. An option to improve vision in AMD patients is through intraocular lenses and optics. Among others, implantable miniaturized telescopes, which direct light to healthy lateral regions of the retina, can be highly effective in improving vision in AMD patients. Yet, the quality of the restored vision might be sensitive to the optical transmission and aberrations of the telescope. To shed light on these points, we studied the in vitro optical performance of an implantable miniaturized telescope, namely, the SING IMT™ (Samsara Vision Ltd., Far Hills, NJ, USA) designed to improve vision in patients affected by late-stage AMD. Specifically, we measured the optical transmission in the spectral range 350-750 nm of the implantable telescope with a fiber-optic spectrometer. Wavefront aberrations were studied by measuring the wavefront of a laser beam after passing through the telescope and expanding the measured wavefront into a Zernike polynomial basis. Wavefront concavity indicated that the SING IMT™ behaves as a diverging lens with a focal length of -111 mm. The device exhibited even optical transmission in the whole visible spectrum and effective curvature suitable for retinal images magnification with negligible geometrical aberrations. Optical spectrometry and in vitro wavefront analysis provide evidence supporting the feasibility of miniaturized telescopes as high-quality optical elements and a favorable option for AMD visual impairment treatments.

Thread Lifting of the Nose: Fully Comprehensive Approach to the Technique.

Nose thread lifting is a relatively recent technique. It offers a chance to approach nose morphology defects without turning to surgery to obtain a temporary improvement. Nevertheless, it lacks standardization and it can therefore offer quite variable results and short durability. The authors' experience is presented here along with a recommended methodological approach to deliver reliable techniques for predictable results. Thread lifting techniques for nose reshaping by means of insertion of poly-L-lactic/poly-caprolactone threads are presented, based on principles of graft-based techniques, to obtain temporary morphological correction of selected nose deformities. Methods: A total of 553 patients underwent nose reshaping using poly-L-lactic/poly-caprolactone threads. Of the procedures, 471 were primary treatment, and 82 were secondary treatment after a previous rhinoplasty. The mean follow-up period was carried out through patient photographs and lasted 33.4 months (range 2-60 months). Clinical examinations and patient satisfaction surveys were done at 6 months and 1 year from thread lifting. Results: The Freiburg questionnaire survey (subjective Global Aesthetic Improvement Scale) allowed the authors to confirm a 95% satisfaction rate after 6 months, and 62% at 1 year after treatment. A flow chart based on the recorded results is presented to support operators in selecting the proper correction method to adopt, based on the different listed indications. Conclusions: Nose reshaping techniques with poly-L-lactic/poly-caprolactone threads are presented together with patients' treatment satisfaction. Standardization is based upon the authors' experience. Contraindications and encountered complications are discussed, so as to provide the readers with a complete "state-of-the art" presentation of these techniques. In the authors' experience, this is a reliable and safe approach to obtain temporary amelioration of selected nose defects via a nonsurgical and minimally invasive method.

On the Advent of Super-Resolution Microscopy in the Realm of Polycomb Proteins.

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.

Letter in reply to Letter to the Editor: Fat injection as a valuable tool for lower eyelid retraction management: a retrospective, observational, single blind, case-control study.

DEAR EDITOR: We have read Dr Riccardo Fondrini and Colleagues commentary on our paper, and we are grateful for their remarks. We herewith would clarify the critical points mentioned about fat reabsorption and survival, along with the risk of periorbital chronic oedema as per the recorded outcomes in the presented case series. Lastly, we have unfortunately to disagree with the commentary's point related to recommended injection depth as it is mandatory to point out that in our experience the superficial injection of nano-fat is a crucial step for lower eyelid retraction management, otherwise a retraction may persist due to the untreated scar in the anterior lamella, thus missing the therapeutic goal that we aim to achieve. LEVEL OF EVIDENCE V: 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 .

SPLIT-PIN software enabling confocal and super-resolution imaging with a virtually closed pinhole.

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.

Adipocyte-hepatocyte crosstalk in cellular models of obesity: Role of soluble factors.

Hepatic steatosis is often a consequence of obesity. Adipose tissue is an important endocrine regulator of metabolic homeostasis in the body. In obesity, adipocytes become hypertrophic and develop an inflammatory phenotype, altering the panel of secreted adipokines. Moreover, excess fatty acids are, in part, released by adipocytes and delivered to the liver. These multiple pathways of adipose-liver crosstalk contribute to the development and progression of liver disease: TNFα induces hepatocyte dysfunction, excess of circulating fatty acids promotes hepatic steatosis and inflammation, whilst adipokines mediate and exacerbate liver injury. In this study, we investigated in vitro the effects and mechanisms of the crosstalk between adipocytes and hepatocytes, as a function of the different adipocyte status (mature vs hypertrophic) being mediated by soluble factors. We employed the conditioned medium method to test how mature and hypertrophic adipocytes distinctively affect the liver, leading to metabolic dysfunction. The media collected from adipocytes were characterized by high triglyceride content and led to lipid accumulation and fat-dependent dysfunction in hepatocytes. The present findings seem to suggest that, in addition to triglycerides, other soluble mediators, cytokines, are released by mature and hypertrophic adipocytes and influence the metabolic status of liver cells. Understanding the precise factors involved in the pathogenesis and pathophysiology of NAFLD in obesity will provide important insights into the mechanisms responsible for the metabolic complications of obesity, paving the way for new possible approaches.

Signal strength and integrated intensity in confocal and image scanning microscopy.

The properties of signal strength and integrated intensity in a scanned imaging system are reviewed. These properties are especially applied to confocal imaging systems, including image scanning microscopy. The integrated intensity, equal to the image of a uniform planar (sheet) object, rather than the peak of the point spread function, is a measure of the flux in an image. Analytic expressions are presented for the intensity in the detector plane for a uniform volume object, and for the resulting background. The variation in the integrated intensity with defocus for an offset point detector is presented. This axial fingerprint is independent of any pixel reassignment. The intensity in the detector plane is shown to contain the defocus information, and simple processing of the recorded data can improve optical sectioning and background rejection.

Piwil2 (Mili) sustains neurogenesis and prevents cellular senescence in the postnatal hippocampus.

Adult neural progenitor cells (aNPCs) ensure lifelong neurogenesis in the mammalian hippocampus. Proper regulation of aNPC fate has thus important implications for brain plasticity and healthy aging. Piwi proteins and the small noncoding RNAs interacting with them (piRNAs) have been proposed to control memory and anxiety, but the mechanism remains elusive. Here, we show that Piwil2 (Mili) is essential for proper neurogenesis in the postnatal mouse hippocampus. RNA sequencing of aNPCs and their differentiated progeny reveal that Mili and piRNAs are dynamically expressed in neurogenesis. Depletion of Mili and piRNAs in the adult hippocampus impairs aNPC differentiation toward a neural fate, induces senescence, and generates reactive glia. Transcripts modulated upon Mili depletion bear sequences complementary or homologous to piRNAs and include repetitive elements and mRNAs encoding essential proteins for proper neurogenesis. Our results provide evidence of a critical role for Mili in maintaining fitness and proper fate of aNPCs, underpinning a possible involvement of the piRNA pathway in brain plasticity and successful aging.

Fat Injection as a Valuable Tool for Lower Eyelid Retraction Management: A Retrospective, Observational, Single Blind and Case-Control Study.

BACKGROUND: 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. RESULTS: 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 (P<0.05) the result with respect to the not affected site. CONCLUSIONS: 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. LEVEL OF EVIDENCE IV: 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.

Focus image scanning microscopy for sharp and gentle super-resolved microscopy.

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.

Protein delivery to living cells by thermal stimulation for biophysical investigation.

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.

Alterations induced by the PML-RARα oncogene revealed by image cross correlation spectroscopy.

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.