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.

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.

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.

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.

Versatile Supramolecular Complex for Targeted Antimicrobial Photodynamic Inactivation.

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 Staphylococcus aureus protein A, was bound to the complex streptavidin-eosin. Fluorescence correlation spectroscopy and fluorescence microscopy demonstrate binding of the complex to S. aureus. Efficient photoinactivation is observed for S. aureus suspensions treated with IgG-streptavidin-eosin at concentrations higher than 0.5 µM and exposed to green light. The proposed strategy offers a flexible platform for targeting a variety of molecules and microbial species.

An Efficient Aequorea victoria Green Fluorescent Protein for Stimulated Emission Depletion Super-Resolution Microscopy.

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 wavelengths <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.

Phasor approach of Mueller matrix optical scanning microscopy for biological tissue imaging.

Mueller matrix microscopy is an advanced imaging technique providing a full characterization of the optical polarization fingerprint of a sample. The Lu-Chipman (LC) decomposition, a method based on the modeling of elementary polarimetric arrangements and matrix inversions, is the gold standard to extract each polarimetric component separately. However, this models the optical system as a small number of discrete optical elements and requires a priori knowledge of the order in which these elements occur. In stratified media or when the ordering is not known, the interpretation of the LC decomposition becomes difficult. In this work, we propose a new, to our knowledge, representation dedicated to the study of biological tissues that combines Mueller matrix microscopy with a phasor approach. We demonstrate that this method provides an easier and direct interpretation of the retardance images in any birefringent material without the use of mathematical assumptions regarding the structure of the sample and yields comparable contrast to the LC decomposition. By validating this approach through numerical simulations, we demonstrate that it is able to give access to localized structural information, resulting in a simple determination of the birefringent parameters at the microscopic level. We apply our novel, to our knowledge, method to typical biological tissues that are of interest in the field of biomedical diagnosis.

Chromatin investigation in the nucleus using a phasor approach to structured illumination microscopy.

Chromatin in the nucleus is organized in functional sites at variable level of compaction. Structured illumination microscopy (SIM) can be used to generate three-dimensional super-resolution (SR) imaging of chromatin by changing in phase and in orientation a periodic line illumination pattern. The spatial frequency domain is the natural choice to process SIM raw data and to reconstruct an SR image. Using an alternative approach, we demonstrate that the additional spatial information encoded in the knowledge of the position of the illumination pattern can be efficiently decoded using a generalized version of separation of photon by lifetime tuning (SPLIT) that does not require lifetime measurements. In the resulting SPLIT-SIM, the SR image is obtained by isolating a fraction of the intensity corresponding to the center of the diffraction-limited point spread function. This extends the use of the SPLIT approach from stimulated emission depletion microscopy to SIM. The SPLIT-SIM algorithm is based only on phasor analysis and does not require deconvolution. We show that SPLIT-SIM can be used to generate SR images of chromatin organizational motifs with tunable resolution and can be a valuable tool for the imaging of functional sites in the nucleus.

STED super-resolved microscopy.

Stimulated emission depletion (STED) microscopy provides subdiffraction resolution while preserving useful aspects of fluorescence microscopy, such as optical sectioning, and molecular specificity and sensitivity. However, sophisticated microscopy architectures and high illumination intensities have limited STED microscopy's widespread use in the past. Here we summarize the progress that is mitigating these problems and giving substantial momentum to STED microscopy applications. We discuss the future of this method in regard to spatiotemporal limits, live-cell imaging and combination with spectroscopy. Advances in these areas may elevate STED microscopy to a standard method for imaging in the life sciences.

Pixel reassignment in image scanning microscopy with a doughnut beam: example of maximum likelihood restoration.

In image scanning microscopy, the pinhole of a confocal microscope is replaced by a detector array. The point spread function for each detector element can be interpreted as the probability density function of the signal, the peak giving the most likely origin. This thus allows a form of maximum likelihood restoration, and compensation for aberrations, with similarities to adaptive optics. As an example of an aberration, we investigate theoretically and experimentally illumination with a vortex doughnut beam. After reassignment and summation over the detector array, the point spread function is compact, and the resolution and signal level higher than in a conventional microscope.

Combined approach using circular intensity differential scattering microscopy under phasor map data analysis.

Circular intensity differential scattering (CIDS) is based on the analysis of circular polarized light scattering and has been proven to be an interesting label-free microscopy technique sensitive to the chiral organization at the submicroscopic level. However, this approach averages the localized contrasts related to the sample polarimetric properties in the illumination volume. Additionally, the detection sensitivity suffers from the confinement of the mixture of structures, and it becomes an arduous task to discriminate the source of the signal. In this work, we show that a phasor map approach combined with CIDS microscopy has provided an intuitive view of the sample organization to recognize the presence of different molecular species in the illumination volume. The data represented in terms of polarization response mapped to a single point called a phasor also have the potential to pave the way for the analysis of large data sets. We validated this method by numerical simulations and compared the results with that of experimental data of optical devices of reference.

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.

Shape-Pure, Nearly Monodispersed CsPbBr3 Nanocubes Prepared Using Secondary Aliphatic Amines.

Fully inorganic cesium lead halide perovskite (CsPbX3) nanocrystals (NCs) have been extensively studied due to their excellent optical properties, especially their high photoluminescence quantum yield (PLQY) and the ease with which the PL can be tuned across the visible spectrum. So far, most strategies for synthesizing CsPbX3 NCs are highly sensitive to the processing conditions and ligand combinations. For example, in the synthesis of nanocubes of different sizes, it is not uncommon to have samples that contain various other shapes, such as nanoplatelets and nanosheets. Here, we report a new colloidal synthesis method for preparing shape-pure and nearly monodispersed CsPbBr3 nanocubes using secondary amines. Regardless of the length of the alkyl chains, the oleic acid concentration, and the reaction temperature, only cube-shaped NCs were obtained. The shape purity and narrow size distribution of the nanocubes are evident from their sharp excitonic features and their ease of self-assembly in superlattices, reaching lateral dimensions of up to 50 µm. We attribute this excellent shape and phase purity to the inability of secondary amines to find the right steric conditions at the surface of the NCs, which consequently limits the formation of low-dimensional structures. Furthermore, no contamination from other phases was observed, not even from Cs4PbBr6, presumably due to the poor ability of secondary aliphatic amines to coordinate to PbBr2 and, hence, to provide a reaction environment that is depleted in Pb.

Spatial-domain filter enhanced subtraction microscopy and application to mid-IR imaging.

We have experimentally investigated the enhancement in spatial resolution by image subtraction in mid-infrared central solid-immersion lens (c-SIL) microscopy. The subtraction exploits a first image measured with the c-SIL point-spread function (PSF) realized with a Gaussian beam and a second image measured with the beam optically patterned by a silicon π-step phase plate, to realize a centrally hollow PSF. The intense sides lobes in both PSFs that are intrinsic to the SIL make the conventional weighted subtraction methods inadequate. A spatial-domain filter with a kernel optimized to match both experimental PSFs in their periphery was thus developed to modify the first image prior to subtraction, and this resulted in greatly improved performance, with polystyrene beads 1.4 ± 0.1 µm apart optically resolved with a mid-IR wavelength of 3.4 µm in water. Spatial-domain filtering is applicable to other PSF pairs, and simulations show that it also outperforms conventional subtraction methods for the Gaussian and doughnut beams widely used in visible and near-IR microscopy.

Assembly of Branched Colloidal Nanocrystals in Polymer Films Leads to Enhanced Viscous Deformation Resistance.

Progress in the integration of nanocrystals with polymers has enabled the creation of materials for applications ranging from photovoltaics to biosensing. However, controlling the nanocrystal segregation and aggregation in the polymer phase remains a challenging task, especially because nanocrystals tend to form amorphous clusters inside the polymer matrix. Here, we present the ability of octapod-shaped particles to overcome their strong entropy-driven tendency to aggregate disorderly and form instead centipede-like linear arrays that are randomly oriented and fully embedded in polystyrene films upon controlled solvent evaporation. This behavior cannot be entirely described by short-range van der Waals interactions between the octapods in the polymer solution. An important role here is played by the increment of the viscosity of the medium during the evaporation of the solvent, which prevents disaggregation of the chains once they are formed. We show that increasing the octapod loading in the blends does not impact the length of the linear arrays beyond a critical length, while it favors instead chain demixing to form self-segregated regions of parallel interlocked chains. Our experiments evidence that softening of the polymer matrix by ex situ heating of the films induces a tail-to-tail coupling of the preformed chains and leads to the formation of longer linear structures of octapods, up to 2 µm long. The presence of 1D arrays of octapods in free-standing polystyrene films improves the creep response by a remarkable 37%, owing to an octapod pinning effect of the polymer matrix.

Colloidal Synthesis of Quantum Confined Single Crystal CsPbBr3 Nanosheets with Lateral Size Control up to the Micrometer Range.

We report the nontemplated colloidal synthesis of single crystal CsPbBr3 perovskite nanosheets with lateral sizes up to a few micrometers and with thickness of just a few unit cells (i.e., below 5 nm), hence in the strong quantum confinement regime, by introducing short ligands (octanoic acid and octylamine) in the synthesis together with longer ones (oleic acid and oleylamine). The lateral size is tunable by varying the ratio of shorter ligands over longer ligands, while the thickness is mainly unaffected by this parameter and stays practically constant at 3 nm in all the syntheses conducted at short-to-long ligands volumetric ratio below 0.67. Beyond this ratio, control over the thickness is lost and a multimodal thickness distribution is observed.

Ultrastable Liquid-Liquid Interface as Viable Route for Controlled Deposition of Biodegradable Polymer Nanocapsules.

Liquid-liquid interfaces are highly dynamic and characterized by an elevated interfacial tension as compared to solid-liquid interfaces. Therefore, they are gaining an increasing interest as viable templates for ordered assembly of molecules and nanoparticles. However, liquid-liquid interfaces are more difficult to handle compared to solid-liquid interfaces; their intrinsic instability may affect the assembly process, especially in the case of multiple deposition. Indeed, some attempts have been made in the deposition of polymer multilayers at liquid-liquid interfaces, but with limited control over size and stability. This study reports on the preparation of an ultrastable liquid-liquid interface based on an O/W secondary miniemulsion and its possible use as a template for the self-assembly of polymeric multilayer nanocapsules. Such polymer nanocapsules are made of entirely biodegradable materials, with highly controlled size-well under 200 nm-and multi-compartment and multifunctional features enriching their field of application in drug delivery, as well as in other bionanotechnology fields.

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.

Gated CW-STED microscopy: a versatile tool for biological nanometer scale investigation.

Stimulation emission depletion (STED) microscopy breaks the spatial resolution limit of conventional light microscopy while retaining its major advantages, such as working under physiological conditions. These properties make STED microscopy a perfect tool for investigating dynamic sub-cellular processes in living organisms. However, up to now, the massive dissemination of STED microscopy has been hindered by the complexity and cost of its implementation. Gated CW-STED (gCW-STED) substantially helps solve this problem without sacrificing spatial resolution. Here, we describe a versatile gCW-STED microscope able to speedily image the specimen, at a resolution below 50 nm, with light intensities comparable to the more complicated all-pulsed STED system. We show this ability on calibration samples as well as on biological samples.