METAPHOR: Metabolic evaluation through phasor-based hyperspectral imaging and organelle recognition for mouse blastocysts and oocytes.

Only 30% of embryos from in vitro fertilized oocytes successfully implant and develop to term, leading to repeated transfer cycles. To reduce time-to-pregnancy and stress for patients, there is a need for a diagnostic tool to better select embryos and oocytes based on their physiology. The current standard employs brightfield imaging, which provides limited physiological information. Here, we introduce METAPHOR: Metabolic Evaluation through Phasor-based Hyperspectral Imaging and Organelle Recognition. This non-invasive, label-free imaging method combines two-photon illumination and AI to deliver the metabolic profile of embryos and oocytes based on intrinsic autofluorescence signals. We used it to classify i) mouse blastocysts cultured under standard conditions or with depletion of selected metabolites (glucose, pyruvate, lactate); and ii) oocytes from young and old mouse females, or in vitro-aged oocytes. The imaging process was safe for blastocysts and oocytes. The METAPHOR classification of control vs. metabolites-depleted embryos reached an area under the ROC curve (AUC) of 93.7%, compared to 51% achieved for human grading using brightfield imaging. The binary classification of young vs. old/in vitro-aged oocytes and their blastulation prediction using METAPHOR reached an AUC of 96.2% and 82.2%, respectively. Finally, organelle recognition and segmentation based on the flavin adenine dinucleotide signal revealed that quantification of mitochondria size and distribution can be used as a biomarker to classify oocytes and embryos. The performance and safety of the method highlight the accuracy of noninvasive metabolic imaging as a complementary approach to evaluate oocytes and embryos based on their physiology.

From fibres to adhesives: evolution of spider capture threads from web anchors by radical changes in silk gland function.

Spider webs that serve as snares are one of the most fascinating and abundant type of animal architectures. In many cases they include an adhesive coating of silk lines-so-called viscid silk-for prey capture. The evolutionary switch from silk secretions forming solid fibres to soft aqueous adhesives remains an open question in the understanding of spider silk evolution. Here we functionally and chemically characterized the secretions of two types of silk glands and their behavioural use in the cellar spider, Pholcus phalangioides. Both being derived from the same ancestral gland type that produces fibres with a solidifying glue coat, the two types produce respectively a quickly solidifying glue applied in thread anchorages and prey wraps, or a permanently tacky glue deployed in snares. We found that the latter is characterized by a high concentration of organic salts and reduced spidroin content, showing up a possible pathway for the evolution of viscid properties by hygroscopic-salt-mediated hydration of solidifying adhesives. Understanding the underlying molecular basis for such radical switches in material properties not only helps to better understand the evolutionary origins and versatility of ecologically impactful spider web architectures, but also informs the bioengineering of spider silk-based products with tailored properties.

Controlling the non-linear emission of upconversion nanoparticles to enhance super-resolution imaging performance.

Upconversion nanoparticles (UCNPs) exhibit unique optical properties such as photo-emission stability, large anti-Stokes shift, and long excited-state lifetimes, allowing significant advances in a broad range of applications from biomedical sensing to super-resolution microscopy. In recent years, progress on nanoparticle synthesis led to the development of many strategies for enhancing their upconversion luminescence, focused in particular on heavy doping of lanthanide ions and core-shell structures. In this article, we investigate the non-linear emission properties of fully Yb-based core-shell UCNPs and their impact on the super-resolution performance of stimulated excitation-depletion (STED) microscopy and super-linear excitation-emission (uSEE) microscopy. Controlling the power-dependent emission curve enables us to relax constraints on the doping concentrations and to reduce the excitation power required for accessing sub-diffraction regimes. We take advantage of this feature to implement multiplexed super-resolution imaging of a two-sample mixture.

Simultaneous super-linear excitation-emission and emission depletion allows imaging of upconversion nanoparticles with higher sub-diffraction resolution.

Upconversion nanoparticles (UCNPs) are becoming increasingly popular as biological markers as they offer photo-stable imaging in the near-infrared (NIR) biological transparency window. Imaging at NIR wavelengths benefits from low auto-fluorescence background and minimal photo-damage. However, as the diffraction limit increases with the wavelength, the imaging resolution deteriorates. To address this limitation, recently two independent approaches have been proposed for imaging UCNPs with sub-diffraction resolution, namely stimulated emission-depletion (STED) microscopy and super linear excitation-emission (uSEE) microscopy. Both methods are very sensitive to the UCNP composition and the imaging conditions, i.e. to the excitation and depletion power. Here, we demonstrate that the imaging conditions can be chosen in a way that activates both super-resolution regimes simultaneously when imaging NaYF4:Yb,Tm UCNPs. The combined uSEE-STED mode benefits from the advantages of both techniques, allowing for imaging with lateral resolution about six times better than the diffraction limit due to STED and simultaneous improvement of the axial resolution about twice over the diffraction limit due to uSEE. Conveniently, at certain imaging conditions, the uSEE-STED modality can achieve better resolution at four times lower laser power compared to STED mode, making the method appealing for biological applications. We illustrate this by imaging UCNPs functionalized by colominic acid in fixed neuronal phenotype cells.

3D sub-diffraction imaging in a conventional confocal configuration by exploiting super-linear emitters.

Sub-diffraction microscopy enables bio-imaging with unprecedented clarity. However, most super-resolution methods require complex, costly purpose-built systems, involve image post-processing and struggle with sub-diffraction imaging in 3D. Here, we realize a conceptually different super-resolution approach which circumvents these limitations and enables 3D sub-diffraction imaging on conventional confocal microscopes. We refer to it as super-linear excitation-emission (SEE) microscopy, as it relies on markers with super-linear dependence of the emission on the excitation power. Super-linear markers proposed here are upconversion nanoparticles of NaYF4, doped with 20% Yb and unconventionally high 8% Tm, which are conveniently excited in the near-infrared biological window. We develop a computational framework calculating the 3D resolution for any viable scanning beam shape and excitation-emission probe profile. Imaging of colominic acid-coated upconversion nanoparticles endocytosed by neuronal cells, at resolutions twice better than the diffraction limit both in lateral and axial directions, illustrates the applicability of SEE microscopy for sub-cellular biology.

Near-Field Mapping of Optical Fabry-Perot Modes in All-Dielectric Nanoantennas.

Subwavelength optical resonators and scatterers are dramatically expanding the toolset of the optical sciences and photonics engineering. By offering the opportunity to control and shape light waves in nanoscale volumes, recent developments using high-refractive-index dielectric scatterers gave rise to efficient flat-optical components such as lenses, polarizers, phase plates, color routers, and nonlinear elements with a subwavelength thickness. In this work, we take a deeper look into the unique interaction of light with rod-shaped amorphous silicon scatterers by tapping into their resonant modes with a localized subwavelength light source-an aperture scanning near-field probe. Our experimental configuration essentially constitutes a dielectric antenna that is locally driven by the aperture probe. We show how leaky transverse electric and magnetic modes can selectively be excited and form specific near-field distribution depending on wavelength and antenna dimensions. The probe's transmittance is furthermore enhanced upon coupling to the Fabry-Perot cavity modes, revealing all-dielectric nanorods as efficient transmitter antennas for the radiation of subwavelength emitters, in addition to constituting an elementary building block for all-dielectric metasurfaces and flat optics.

Development of Bright and Biocompatible Nanoruby and Its Application to Background-Free Time-Gated Imaging of G-Protein-Coupled Receptors.

At the forefront of developing fluorescent probes for biological imaging applications are enhancements aimed at increasing their brightness, contrast, and photostability, especially toward demanding applications of single-molecule detection. In comparison with existing probes, nanorubies exhibit unlimited photostability and a long emission lifetime (∼4 ms), which enable continuous imaging at single-particle sensitivity in highly scattering and fluorescent biological specimens. However, their wide application as fluorescence probes has so far been hindered by the absence of facile methods for scaled-up high-volume production and molecularly specific targeting. The present work encompasses the large-scale production of colloidally stable nanoruby particles, the demonstration of their biofunctionality and negligible cytotoxicity, as well as the validation of its use for targeted biomolecular imaging. In addition, optical characteristics of nanorubies are found to be comparable or superior to those of state-of-the-art quantum dots. Protocols of reproducible and robust coupling of functional proteins to the nanoruby surface are also presented. As an example, NeutrAvidin-coupled nanoruby show excellent affinity and specificity to µ-opioid receptors in fixed and live cells, allowing wide-field imaging of G-protein coupled receptors with single-particle sensitivity.

Edge States and Topological Phase Transitions in Chains of Dielectric Nanoparticles.

Recently introduced field of topological photonics aims to explore the concepts of topological insulators for novel phenomena in optics. Here polymeric chains of subwavelength silicon nanodisks are studied and it is demonstrated that these chains can support two types of topological edge modes based on magnetic and electric Mie resonances, and their topological properties are fully dictated by the spatial arrangement of the nanoparticles in the chain. It is observed experimentally and described how theoretically topological phase transitions at the nanoscale define a change from trivial to nontrivial topological states when the edge mode is excited.

Mode parity-controlled Fano- and Lorentz-like line shapes arising in plasmonic nanorods.

We present the experimental observation of spectral lines of distinctly different shapes in the optical extinction cross-section of metallic nanorod antennas under near-normal plane wave illumination. Surface plasmon resonances of odd mode parity present Fano interference in the scattering cross-section, resulting in asymmetric spectral lines. Contrarily, modes with even parity appear as symmetric Lorentzian lines. Finite element simulations are used to verify the experimental results. The emergence of either constructive or destructive mode interference is explained with a semianalytical 1D line current model. This simple model directly explains the mode-parity dependence of the Fano-like interference. Plasmonic nanorods are widely used as half-wave optical dipole antennas. Our findings offer a perspective and theoretical framework for operating these antennas at higher-order modes.

Lateral magnetic near-field imaging of plasmonic nanoantennas with increasing complexity.

The design of many promising, newly emerging classes of photonic metamaterials and subwavelength confinement structures requires detailed knowledge and understanding of the electromagnetic near-field interactions between their building blocks. While the electric field distributions and, respectively, the electric interactions of different nanostructures can be routinely measured, for example, by scattering near-field microscopy, only recently experimental methods for imaging the magnetic field distributions became available. In this paper, we provide direct experimental maps of the lateral magnetic near-field distributions of variously shaped plasmonic nanoantennas by using hollow-pyramid aperture scanning near-field optical microscopy (SNOM). We study both simple plasmonic nanoresonators, such as bars, disks, rings and more complex antennas. For the studied structures, the magnetic near-field distributions of the complex resonators have been found to be a superposition of the magnetic near-fields of the individual constituting elements. These experimental results, explained and validated by numerical simulations, open new possibilities for engineering and characterization of complex plasmonic antennas with increased functionality.

Mapping magnetic near-field distributions of plasmonic nanoantennas.

We present direct experimental mapping of the lateral magnetic near-field distribution in plasmonic nanoantennas using aperture scanning near-field optical microscopy (SNOM). By means of full-field simulations it is demonstrated how the coupling of the hollow-pyramid aperture probe to the nanoantenna induces an effective magnetic dipole which efficiently excites surface plasmon resonances only at lateral magnetic field maxima. This excitation in turn affects the detected light intensity enabling the visualization of the lateral magnetic near-field distribution of multiple odd and even order plasmon modes with subwavelength spatial resolution.

Plasmon-enhanced sub-wavelength laser ablation: plasmonic nanojets.

In response to the incident light's electric field, the electron density oscillates in the plasmonic hotspots producing an electric current. Associated Ohmic losses raise the temperature of the material within the plasmonic hotspot above the melting point. A nanojet and nanosphere ejection can then be observed precisely from the plasmonic hotspots.