A robust and versatile platform for image scanning microscopy enabling super-resolution FLIM.

Image scanning microscopy (ISM) can improve the effective spatial resolution of confocal microscopy to its theoretical limit. However, current implementations are not robust or versatile, and are incompatible with fluorescence lifetime imaging (FLIM). We describe an implementation of ISM based on a single-photon detector array that enables super-resolution FLIM and improves multicolor, live-cell and in-depth imaging, thereby paving the way for a massive transition from confocal microscopy to ISM.

Nanoscale Distribution of Nuclear Sites by Super-Resolved Image Cross-Correlation Spectroscopy.

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.

Hypericin-Apomyoglobin: An Enhanced Photosensitizer Complex for the Treatment of Tumor Cells.

Bioavailability of photosensitizers for cancer photodynamic therapy is often hampered by their low solubility in water. Here, we overcome this issue by using the water-soluble protein apomyoglobin (apoMb) as a carrier for the photosensitizer hypericin (Hyp). The Hyp-apoMb complex is quickly uptaken by HeLa and PC3 cells at submicromolar concentrations. Fluorescence emission of Hyp-apoMb is exploited to localize the cellular distribution of the photosensitizer. The plasma membrane is rapidly and efficiently loaded, and fluorescence is observed in the cytoplasm only at later times and to a lesser extent. Comparison with cells loaded with Hyp alone demonstrates that the uptake of the photosensitizer without the protein carrier is a slower, less efficient process, that involves the whole cell structure without preferential accumulation at the plasma membrane. Cell viability assays demonstrate that the Hyp-apoMb exhibits superior performance over Hyp. Similar results were obtained using tumor spheroids as three-dimensional cell culture models.

Labeling and Selective Inactivation of Gram-Positive Bacteria Employing Bimodal Photoprobes with Dual Readouts.

Carbohydrate-conjugated silicon(IV) phthalocyanines with bimodal photoactivity were developed as probes with both fluorescent labeling and photosensitizing capabilities, and the concomitant fluorescent labeling and photoinduced inactivation of Gram-positive and Gram-negative models was explored. The maltohexaose-conjugated photoprobe provides a dual readout to distinguish between both groups of pathogens, as only the Gram-positive species was inactivated, even though both appeared labeled with near-infrared luminescence. Antibiotic resistance did not hinder the phototoxic effect, as even the methicillin-resistant pathogen Staphylococcus aureus (MRSA) was completely photoinactivated. Time-resolved confocal fluorescence microscopy analysis suggests that the photoprobe sticks onto the outer rim of the microorganisms, explaining the resistance of Gram-negative species on the basis of their membrane constitution. The mannose-conjugated photoprobe yields a different readout because it is able to label and to inactivate only the Gram-positive strain.

STED nanoscopy: a glimpse into the future.

The well-known saying of "Seeing is believing" became even more apt in biology when stimulated emission depletion (STED) nanoscopy was introduced in 1994 by the Nobel laureate S. Hell and coworkers. We presently stand at a juncture. Nanoscopy represented a revolution in fluorescence microscopy but now is a mature technique applied to many branches of biology, physics, chemistry, and materials science. We are currently looking ahead to the next generation of optical nanoscopes, to the new key player that will arise in the forthcoming years. This article gives an overview of the various cutting-edge implementations of STED nanoscopy and tries to shine a light into the future: imaging everything faster with unprecedented sensitivity and label-free.

New findings in ATP supply in rod outer segments: insights for retinopathies.

BACKGROUND INFORMATION: The rod outer segment (OS) is the specialised organelle where phototransduction takes place. Our previous proteomic and biochemical analyses on purified rod disks showed the functional expression of the respiratory chain complexes I-IV and F1 Fo -ATP synthase in OS disks, as well as active soluble tricarboxylic acid cycle enzymes. Here, we focussed our study on the whole OS that contains the cytosol and plasma membrane and disks as native flattened saccules, unlike spherical osmotically intact disks. RESULTS: OS were purified from bovine retinas and characterised for purity. Oximetry, ATP synthesis and cytochrome c oxidase (COX) assays were performed. The presence of COX and F1F0-ATP synthase (ATP synthase) was assessed by semi-quantitative Western blotting, immunofluorescence or confocal laser scanning microscopy on whole bovine retinas and bovine retinal sections and by immunogold transmission electron microscopy (TEM) of purified OS or bovine retinal sections. Both ATP synthase and COX are catalytically active in OS. These are able to consume oxygen (O2) in the presence of pyruvate and malate. CLSM analyses showed that rhodopsin autofluorescence and MitoTracker Deep Red 633 fluorescence co-localise on rod OS. Data are confirmed by co-localisation studies of ATP synthase with Rh in rod OS by immunofluorescence and TEM in bovine retinal sections. CONCLUSIONS: Our data confirm the expression and activity of COX and ATP synthase in OS, suggestive of the presence of an extra-mitochondrial oxidative phosphorylation in rod OS, meant to supply ATP for the visual transduction. In this respect, the membrane rich OS environment would be meant to absorb both light and O2. The ability of OS to manipulate O2 may shed light on the pathogenesis of many retinal degenerative diseases ascribed to oxidative stress, as well as on the efficacy of the treatment with dietary supplements, presently utilised as supporting therapies.

Functional expression of oxidative phosphorylation proteins in the rod outer segment disc.

The rod Outer Segment (OS) disc, an organelle devoid of mitochondria, is specialized in phototransduction, a process requiring a continual chemical energy supply. We have shown that OS discs express functional mitochondrial electron transport chains, Fo F1 -ATP synthase and the tricarboxylic acid cycle enzymes, all mitochondrial features. Here, we focus on oxygen consumption and adenosine triphosphate (ATP) synthesis by OS discs analysing electron transport chain I-III-IV and II-II-IV pathways, supported by reduced nicotinamide adenine dinucleotide and succinate, respectively. Interestingly, respiratory capacity of discs was measurable also in the presence of 3-hydroxy-butyrrate, a typical metabolic substrate for the brain. Data were supported by a two-dimensional electrophoresis analyses conducted as our previous one, but focused to those mitochondrial proteins that are involved in oxidative phosphorylation. Carbonic anhydrase was also found active in OS discs. Moreover, colocalization of Rhodopsin with respiratory complex I and ATP synthase seems a further step in the characterization of some proteins typical of the mitochondrial inner membranes that are expressed in the rod discs. The existence of oxygen utilization in the outer retina, likely supplying ATP for phototransduction, may shed light on some retinal pathologies related to oxidative stress of the outer retina.

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.

Assessing Differential Particle Deformability under Microfluidic Flow Conditions.

Assessing the mechanical behavior of nano- and micron-scale particles with complex shapes is fundamental in drug delivery. Although different techniques are available to quantify the bulk stiffness in static conditions, there is still uncertainty in assessing particle deformability in dynamic conditions. Here, a microfluidic chip is designed, engineered, and validated as a platform to assess the mechanical behavior of fluid-borne particles. Specifically, potassium hydroxide (KOH) wet etching was used to realize a channel incorporating a series of micropillars (filtering modules) with different geometries and openings, acting as microfilters in the direction of the flow. These filtering modules were designed with progressively decreasing openings, ranging in size from about 5 down to 1 µm. Discoidal polymeric nanoconstructs (DPNs), with a diameter of 5.5 µm and a height of 400 nm, were realized with different poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) ratios (PLGA/PEG), namely, 5:1 and 1:0, resulting in soft and rigid particles, respectively. Given the peculiar geometry of DPNs, the channel height was kept to 5 µm to limit particle tumbling or flipping along the flow. After thorough physicochemical and morphological characterization, DPNs were tested within the microfluidic chip to investigate their behavior under flow. As expected, most rigid DPNs were trapped in the first series of pillars, whereas soft DPNs were observed to cross multiple filtering modules and reach the micropillars with the smallest opening (1 µm). This experimental evidence was also supported by computational tools, where DPNs were modeled as a network of springs and beads immersed in a Newtonian fluid using the smoothed particle hydrodynamics (SPH) method. This preliminary study presents a combined experimental-computational framework to quantify, compare, and analyze the characteristics of particles having complex geometrical and mechanical attributes under flow conditions.

An interaction between PRRT2 and Na+/K+ ATPase contributes to the control of neuronal excitability.

Mutations in PRoline Rich Transmembrane protein 2 (PRRT2) cause pleiotropic syndromes including benign infantile epilepsy, paroxysmal kinesigenic dyskinesia, episodic ataxia, that share the paroxysmal character of the clinical manifestations. PRRT2 is a neuronal protein that plays multiple roles in the regulation of neuronal development, excitability, and neurotransmitter release. To better understand the physiopathology of these clinical phenotypes, we investigated PRRT2 interactome in mouse brain by a pulldown-based proteomic approach and identified α1 and α3 Na+/K+ ATPase (NKA) pumps as major PRRT2-binding proteins. We confirmed PRRT2 and NKA interaction by biochemical approaches and showed their colocalization at neuronal plasma membrane. The acute or constitutive inactivation of PRRT2 had a functional impact on NKA. While PRRT2-deficiency did not modify NKA expression and surface exposure, it caused an increased clustering of α3-NKA on the plasma membrane. Electrophysiological recordings showed that PRRT2-deficiency in primary neurons impaired NKA function during neuronal stimulation without affecting pump activity under resting conditions. Both phenotypes were fully normalized by re-expression of PRRT2 in PRRT2-deficient neurons. In addition, the NKA-dependent afterhyperpolarization that follows high-frequency firing was also reduced in PRRT2-silenced neurons. Taken together, these results demonstrate that PRRT2 is a physiological modulator of NKA function and suggest that an impaired NKA activity contributes to the hyperexcitability phenotype caused by PRRT2 deficiency.

Volumetric Lissajous confocal microscopy with tunable spatiotemporal resolution.

Dynamic biological systems present challenges to existing three-dimensional (3D) optical microscopes because of their continuous temporal and spatial changes. Most techniques are rigid in adapting the acquisition parameters over time, as in confocal microscopy, where a laser beam is sequentially scanned at a predefined spatial sampling rate and pixel dwell time. Such lack of tunability forces a user to provide scan parameters, which may not be optimal, based on the best assumption before an acquisition starts. Here, we developed volumetric Lissajous confocal microscopy to achieve unsurpassed 3D scanning speed with a tunable sampling rate. The system combines an acoustic liquid lens for continuous axial focus translation with a resonant scanning mirror. Accordingly, the excitation beam follows a dynamic Lissajous trajectory enabling sub-millisecond acquisitions of image series containing 3D information at a sub-Nyquist sampling rate. By temporal accumulation and/or advanced interpolation algorithms, the volumetric imaging rate is selectable using a post-processing step at the desired spatiotemporal resolution for events of interest. We demonstrate multicolor and calcium imaging over volumes of tens of cubic microns with 3D acquisition speeds of 30 Hz and frame rates up to 5 kHz.

Cellulose acetate - essential oil nanocapsules with antimicrobial activity for biomedical applications.

This study aimed to obtain bioactive nanosystems by combining cellulose acetate with three selected essential oils (EOs) to create spherical nanocapsules (NCs) using the solvent/anti-solvent technique. The biological activity of the obtained NCs was promoted by the use of some antimicrobial EOs: Peppermint, Cinnamon and lemongrass which were grafted on the cellulose acetate molecules. Due to their chemistry, such as long hydrocarbon tails and heads with functional groups these EOs were playing also the role of surfactant-like substance facilitating the formation of NCs. A dispersion of NCs was obtained in water and various spectroscopy techniques used to examine their size, morphology and chemistry. Dynamic light scattering calculate the size of the NCs whereas scanning electron microscopy showed their morphology. Fluorescent microscopy and Raman spectroscopy proved the attachment of the EOs in the cellulose acetate molecules. The antimicrobial activity of the obtained nanomaterials was tested against four microbial strains (bacteria: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and a yeast strain of Candida albicans). The obtained results demonstrated that such NCs can be used in a variety of applications including medical, pharmaceutical recipients and in household products for treating or preventing microbial colonization and biofilm development.

Exploiting the tunability of stimulated emission depletion microscopy for super-resolution imaging of nuclear structures.

Imaging of nuclear structures within intact eukaryotic nuclei is imperative to understand the effect of chromatin folding on genome function. Recent developments of super-resolution fluorescence microscopy techniques combine high specificity, sensitivity, and less-invasive sample preparation procedures with the sub-diffraction spatial resolution required to image chromatin at the nanoscale. Here, we present a method to enhance the spatial resolution of a stimulated-emission depletion (STED) microscope based only on the modulation of the STED intensity during the acquisition of a STED image. This modulation induces spatially encoded variations of the fluorescence emission that can be visualized in the phasor plot and used to improve and quantify the effective spatial resolution of the STED image. We show that the method can be used to remove direct excitation by the STED beam and perform dual color imaging. We apply this method to the visualization of transcription and replication foci within intact nuclei of eukaryotic cells.

MRCKα is activated by caspase cleavage to assemble an apical actin ring for epithelial cell extrusion.

Extrusion of apoptotic cells from epithelial tissues requires orchestrated morphological rearrangements of the apoptotic cell and its neighbors. However, the connections between the apoptotic cascade and events leading to extrusion are not fully understood. Here, we characterize an apoptotic extrusion apical actin ring (EAAR) that is assembled within the apoptotic cell and drives epithelial extrusion. Caspase-mediated cleavage of myotonic dystrophy kinase-related CDC42-binding kinase-α (MRCKα) triggers a signaling pathway that leads to the assembly of EAAR that pulls actin bundles, resulting in the compaction and removal of the cell body. We provide a detailed portrait of the EAAR including F-actin flow, the contribution of myosin contraction, and actin polymerization at bundles' terminals when the product of MRCKα cleavage is expressed. These results add to our understanding of the mechanisms controlling the process of epithelial extrusion by establishing a causal relationship between the triggering events of apoptosis, the activation of MRCKα, and its subsequent effects on the dynamics of actomyosin cytoskeleton rearrangement.

Effect of polyphenolic phytochemicals on ectopic oxidative phosphorylation in rod outer segments of bovine retina.

BACKGROUND AND PURPOSE: The rod outer segments (OS) of the retina are specialized organelles where phototransduction takes place. The mitochondrial electron transport complexes I-IV, cytochrome c and Fo F1 -ATP synthase are functionally expressed in the OS disks. Here, we have studied the effect of some polyphenolic compounds acting as inhibitors of mitochondrial ATPase/synthase activity on the OS ectopic Fo F1 - ATP synthase. The mechanism of apoptosis in the OS was also investigated studying the expression of cytochrome c, caspase 9 and 3 and Apaf-1. EXPERIMENTAL APPROACH: We prepared OS from fresh bovine retinae. Semi-quantitative Western blotting, confocal and electron microscopy, and cytofluorimetry were used along with biochemical analyses such as oximetry, ATP synthesis and hydrolysis. KEY RESULTS: Resveratrol and curcumin plus piperine inhibited ATP synthesis and oxygen consumption in the OS. Epigallocatechin gallate and quercetin inhibited ATP hydrolysis and oxygen consumption in the OS. Malondialdehyde and hydrogen peroxide were produced in respiring OS in the presence of substrates. Cytochrome c was located inside the disk membranes. Procaspase 9 and 3, as well as Apaf-1 were expressed in the OS. CONCLUSIONS AND IMPLICATIONS: These polyphenolic phytochemicals modulated the Fo F1 -ATP synthase activity of the the OS reducing production of reactive oxygen intermediates by the OS ectopic electron transport chain. Polyphenols decrease membrane peroxidation and cytochrome c release from disks, preventing the induction of caspase-dependent apoptosis in the OS Such effects are relevant in the design of protection against functional impairment of the OS following oxidative stress from exposure to intense illumination.