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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.

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.

Photodynamic therapy (PDT) is a clinically approved procedure that can exert a curative action against malignant cells. The treatment implies the administration of a photoactive molecular species that, upon absorption of visible or near infrared light, sensitizes the formation of reactive oxygen species. These species are cytotoxic and lead to tumor cell death, damage vasculature, and induce inflammation. Clinical investigations demonstrated that PDT is curative and does not compromise other treatment options. One of the major limitations of the original method was the low selectivity of the photoactive compounds for malignant over healthy tissues. The development of conjugates with antibodies has endowed photosensitizing molecules with targeting capability, so that the compounds are delivered with unprecedented precision to the site of action. Given their fluorescence emission capability, these supramolecular species are intrinsically theranostic agents.

Optical microscopy demonstrated an incredible power and ability in producing large data set originated from biological samples by light interrogation and tunable in terms of spatial at temporal resolution down to the nano- and pico- scale, respectively. Such a data set is the core for developing an artificial microscope aiming to transform a label-free interrogation of the sample into a molecular-rich fluorescence-based image. The intelligent artificial microscope is AI-guided through a computational core based on three modules based on a convolutional neural network (CNN) and a tensor independent component analysis (tICA) un-supervised machine learning within a supervised deep learning strategy having the ambitious target to create a robust virtual environment "to see "what we could not perceive before". An interesting case study is related to understanding the visualisation of chromatin organisation.

Abstract Background Surface roughness is the main factor determining bacterial adhesion, biofilm growth and plaque formation on the dental surfaces in vivo. Air-polishing of dental surfaces removes biofilm but can also damage the surface by increasing its roughness. The purpose of this study was to investigate the surface damage of different conditions of air-polishing performed in vitro on a recently introduced dental restorative composite. Methods Abrasive powders of sodium bicarbonate and glycine, combined at different treatment times (5, 10 and 30 s) and distances (2 and 7 mm), have been tested. The resulting root mean square roughness of the surfaces has been measured by means of atomic force microscopy, and the data have been analyzed statistically to assess the significance. Additionally, a fractal analysis of the samples surfaces has been carried out. Results The minimum surface roughening was obtained by air-polishing with glycine powder for 5 s, at either of the considered distances, which resulted in a mean roughness of ~300 nm on a 30 × 30 μm2 surface area, whereas in the other cases it was in the range of 400-750 nm. Both untreated surfaces and surfaces treated with the maximum roughening conditions exhibited a fractal character, with comparable dimension in the 2.4-2.7 range, whereas this was not the case for the surfaces treated with the minimum roughening conditions. Conclusions For the dental practitioner it is of interest to learn that use of glycine in air polishing generates the least surface roughening on the considered restorative material, and thus is expected to provide the lowest rate of bacterial biofilm growth and dental plaque formation. Furthermore, the least roughening behaviour identified has been correlated with the disappearance of the surface fractal character, which could represent an integrative method for screening the air polishing treatment efficacy.

Abstract Multi-photon excitation (MPE) microscopy plays a growing role among microscopical techniques utilized for studying biological matter. In conjunction with confocal microscopy it can be considered the imaging workhorse of life science laboratories. Its roots can be found in a fundamental work written by Maria Goeppert Mayer more than 70 years ago. Nowadays, 2PE and MPE microscopes are expected to increase their impact in areas such biotechnology, neurobiology, embryology, tissue engineering, materials science where imaging can be coupled to the possibility of using the microscopes in an active way, too. As well, 2PE implementations in noninvasive optical bioscopy or laser-based treatments point out to the relevance in clinical applications. Here we report about some basic aspects related to the phenomenon, implications in three-dimensional imaging microscopy, practical aspects related to design and realization of MPE microscopes, and we only give a list of potential applications and variations on the theme in order to offer a starting point for advancing new applications and developments.

Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, super-resolution microscopy has gained considerable interest because it can be used to probe the spatial organization of functional sites in intact single-cell nuclei in the 20-250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a presegmentation of the image into objects and can be used to detect dynamic interactions. However, the combination of ICCS with super-resolution microscopy has not been explored yet. Here, we combine dual-color stimulated emission depletion (STED) nanoscopy with ICCS (STED-ICCS) to quantify the nanoscale distribution of functional nuclear sites. We show that super-resolved ICCS provides not only a value of the colocalized fraction but also the characteristic distances associated to correlated nuclear sites. As a validation, we quantify the nanoscale spatial distribution of three different pairs of functional nuclear sites in MCF10A cells. As expected, transcription foci and a transcriptionally repressive histone marker (H3K9me3) are not correlated. Conversely, nascent DNA replication foci and the proliferating cell nuclear antigen(PCNA) protein have a high level of proximity and are correlated at a nanometer distance scale that is close to the limit of our experimental approach. Finally, transcription foci are found at a distance of 130 nm from replication foci, indicating a spatial segregation at the nanoscale. Overall, our data demonstrate that STED-ICCS can be a powerful tool for the analysis of the nanoscale distribution of functional sites in the nucleus.

Super-resolution microscopy techniques can be challenging for live cells and thick samples. Here, the authors propose a method to reduce beam intensity and remove out-of-focus fluorescence background in image-scanning microscopy (ISM) and its combination with stimulated emission depletion (STED).

Abstract 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.

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 link between family violence, PTSD, and mother-child interaction patterns. Methods: Eighty-seven mother-child dyads from Mexico completed assessments for exposure to family violence and PTSD symptoms. We coded and analyzed observational tasks to assess prosocial parent-child interactions, such as 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.

Abstract Polarized light scanning microscopy is a non-invasive and contrast-enhancing technique to investigate anisotropic specimens and chiral organizations. However, such arrangements suffer from insensitivity to confined blend of structures at sub-diffraction level. Here for the first time, we present that the pixel-by-pixel polarization modulation converted to an image phasor approach issues an insightful view of cells to distinguish anomalous subcellular organizations. To this target, we propose an innovative robust way for identifying changes in the chromatin compaction and distortion of nucleus morphology induced by the activation of the lamin-A gene from Hutchinson–Gilford progeria syndrome that induces a strong polarization response. The phasor mapping is evaluated based on the modulation and phase image acquired from a scanning microscope compared to a confocal fluorescence modality of normal cell opposed to the progeria. The method is validated by characterizing polarization response of starch crystalline granules. Additionally, we show that the conversion of the polarization-resolved images into the phasor could further utilized for segmenting specific structures presenting various optical properties under the polarized light. In summary, image phasor analysis offers a distinctly sensitive fast and easy representation of the polarimetric contrast that can pave the way for remote diagnosis of pathological tissues in real-time.

The availability of the Airyscan detector in the Zeiss LSM 880 has made possible the development of a new concept in fluctuation correlation spectroscopy using super-resolution. The Airyscan unit acquires data simultaneously on 32 detectors arranged in a hexagonal array. This detector opens up the possibility to use fluctuation methods based on time correlation at single points or at a number of points simultaneously, as well as methods based on spatial correlation in the area covered by the detector. Given the frame rate of this detector, millions of frames can be acquired in seconds, providing a robust statistical basis for fluctuation data. We apply the comprehensive analysis to the molecular fluctuations of free GFP diffusing in live cells at different subcellular compartments to show that at the nanoscale different cell environments can be distinguished by the comprehensive fluctuation analysis.